Antihypertensive, cardio- and neuro-protective effects of Tenebrio molitor (Coleoptera: Tenebrionidae) defatted larvae in spontaneously hypertensive rats

Federica Pessina; Maria Frosini; Paola Marcolongo; Fabio Fusi; Simona Saponara; Alessandra Gamberucci; Massimo Valoti; Daniela Giustarini; Paolo Fiorenzani; Beatrice Gorelli; Valeria Francardi; Maurizio Botta; Elena Dreassi

doi:10.1371/journal.pone.0233788

. 2020 May 29;15(5):e0233788. doi: 10.1371/journal.pone.0233788

Antihypertensive, cardio- and neuro-protective effects of Tenebrio molitor (Coleoptera: Tenebrionidae) defatted larvae in spontaneously hypertensive rats

Federica Pessina ^1,^#, Maria Frosini ^2,^*,^#, Paola Marcolongo ¹, Fabio Fusi ³, Simona Saponara ², Alessandra Gamberucci ¹, Massimo Valoti ², Daniela Giustarini ³, Paolo Fiorenzani ⁴, Beatrice Gorelli ², Valeria Francardi ⁵, Maurizio Botta ^3,⁶, Elena Dreassi ³

Editor: Luis Eduardo M Quintas⁷

¹Dipartimento di Medicina Molecolare e dello Sviluppo, Università degli Studi di Siena, Siena, Italy

²Dipartimento di Scienze della Vita, Università degli Studi di Siena, Siena, Italy

³Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Siena, Italy

⁴Dipartimento di Scienze Mediche, Chirurgiche e Neuroscienze, Università degli Studi di Siena, Siena, Italy

⁵Consiglio per la Ricerca in Agricoltura e l’Analisi dell’Economia Agraria, Centro di ricerca Difesa e Certificazione (CREA-DC), Impruneta (Firenze), Italy

⁶Lead Discovery Siena Srl, Castelnuovo Berardenga, Siena, Italy

⁷Universidade Federal do Rio de Janeiro, BRAZIL

Competing Interests: The authors have read the journal's policies and have the following competing interests to declare: Lead Discovery Siena Srl provided support in the form of research materials. MB is the CEO of Lead Discovery Siena Srl, but did not receive salary from Lead Discovery Siena Srl. There are no patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

^✉

* E-mail: maria.frosini@unisi.it

Contributed equally.

Roles

Federica Pessina: Conceptualization, Data curation, Formal analysis, Methodology, Writing – review & editing

Maria Frosini: Conceptualization, Data curation, Investigation, Methodology, Software, Supervision, Validation, Writing – original draft, Writing – review & editing

Paola Marcolongo: Conceptualization, Data curation, Investigation, Writing – review & editing

Fabio Fusi: Conceptualization, Data curation, Formal analysis, Investigation, Writing – original draft, Writing – review & editing

Simona Saponara: Conceptualization, Data curation, Methodology, Supervision, Writing – review & editing

Alessandra Gamberucci: Conceptualization, Data curation, Methodology, Writing – review & editing

Massimo Valoti: Data curation, Investigation, Methodology, Supervision, Writing – review & editing

Daniela Giustarini: Investigation, Methodology, Writing – review & editing

Paolo Fiorenzani: Methodology

Beatrice Gorelli: Methodology

Valeria Francardi: Conceptualization, Data curation, Investigation, Methodology, Writing – review & editing

Maurizio Botta: Resources

Elena Dreassi: Conceptualization, Data curation, Formal analysis, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing – review & editing

Luis Eduardo M Quintas: Editor

PMCID: PMC7259609 PMID: 32470081

Abstract

In pre-hypertension, moderate control of blood pressure (BP) can be obtained by a nutritional approach. The effects of a diet enriched with defatted larvae of the mealworm Tenebrio molitor (Coleoptera: Tenebrionidae) (TM) endowed with ACE inhibitory activity was studied in both spontaneously hypertensive rats (SHR) and in the age-matched normotensive Wistar Kyoto strain. These were fed for 4 weeks with standard laboratory rodent chow supplemented with or without TM or captopril. In SHR, the TM diet caused a significant reduction in BP, heart rate and coronary perfusion pressure, as well as an increase in red blood cell glutathione/glutathione disulphide ratio. Rat brain slices of SHR were more resistant to oxidative stress and contained lower levels of inflammatory cytokines, while vascular and liver enzyme-activities were not affected. These results suggest that TM can be considered a new functional food that can lower BP in vivo and thus control cardiovascular-associated risk factors such as hypertension.

Introduction

Hypertension is the main risk factor for diseases such as coronary artery disease, stroke, heart failure, atrial fibrillation and dementia. The incidence of cardiovascular diseases and related deaths is expected to rise worldwide. The Seventh Joint National Committee (JNC7) report recently proposed the concept of prehypertension (systolic blood pressure 120–139 mmHg and/or diastolic blood pressure 80–89 mmHg), which can progress to hypertension, impairing cognitive function, increasing left ventricular mass and causing end-stage renal disease and arteriosclerosis [1]. Instead of drug therapy, dietary approaches to stop hypertension (DASH) and the Mediterranean diet associated with appropriate lifestyle could be ideal measures to implement in cases of prehypertension [2], particularly in patients with concomitant cardiovascular risk factors. Since diet plays a crucial role in maintaining healthy blood pressure, health-conscious consumers seek functional foods to reduce cardiovascular risk factors and improve vascular health.

Edible insects were recently proposed as novel functional food by virtue of their antitumor, antiobesity, antimicrobial, antioxidant and antihypertensive properties [3]. For example, the protein fraction from gastrointestinal [4] or alcalase enzyme hydrolysis [5] of larvae of Tenebrio molitor (Coleoptera Tenebrionidae) (TM) shows ACE inhibitory activity. Considering the ease of breeding TM larvae, they may be used as a functional ingredient in fortified food and/or as a dietary supplement to lower blood pressure for cardiovascular prevention in pre-hypertensive patients. The aim of the present study was to investigate the effects of the protein obtained from the larval stage of TM, in vivo and ex-vivo, in spontaneously hypertensive rats (SHRs) and in age-matched rats of the normotensive Wistar Kyoto strain (WKY). The ACE inhibitor captopril was used in parallel as a positive control. The diet supplemented with TM was found to limit blood pressure increase and to protect the brain against oxidative stress, thus indicating TM as a possible functional food.

Materials and methods

T. molitor larva meal

Larvae of TM were obtained by mass rearing at CREA-DC (Italy), as previously described [4]. Specimens were kept in plastic boxes in a climatic room (24°C ±2 and 70%±5 relative humidity) in the dark. The breeding diet was composed of yeast (5%), wheat flour (47.5%) and oat flour (47.5%) plus carrot pieces as source of water. The larvae were not starved before being killed. They were dried at 50°C under vacuum and ground using a food processor before fat extraction with ethanol (99.5%) [6]. The solvent was then removed under vacuum at 25°C for 24 h and the larva meal used for feed preparation. The nutritional value and other details of defatted TM are reported in S1 File. Mucedola Srl (Italy) prepared the standard diet (4RF25, for composition see www.mucedola.it) supplemented with larva protein or with captopril (see S1 Table).

Animals and ethical statement

The procedures used complied with European legislation on the use and care of laboratory animals (EU Directive 2010/63) and National Institutes of Health guidelines, and were approved by the Italian Ministry for Health (185/2015PR).

Nine-week old male spontaneously hypertensive rats (SHRs, n = 24) and age-matched WKY rats as controls (n = 18) were purchased from Charles River (Italy, www.criver.com). Two to four rats per cage were housed in a room maintained at constant temperature (23–24°C) and humidity (50–60%), on a 12 h light/dark cycle, with free access to food and water.

Experimental design

At ten weeks of age, when hypertension develops [7], hypertensive and WKY rats were randomly divided into three groups, and fed different diets for four weeks as indicated in Fig 1. At the end of this period, micturition frequency was assessed: all rats were then anaesthetized (for details, see S2 File), blood samples were collected and heart, aorta, liver, kidney and brain were quickly explanted.

Fig 1 — After a one-week housing period, both Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHRs) were randomly assigned to standard diet (SD), SD supplemented with *Tenebrio molitor* or captopril. Both strains were fed for 4 weeks, when body weight (BW), blood pressure (BP) and heart rate (HR) were measured on a weekly basis. After four weeks, the rats were sacrificed and their blood, heart, aorta, liver and brain harvested.

Doses

Since the diets were supplemented with 4.5% larva protein or 100 mg/kg captopril, and on the basis of rat body weight (see S2 Table) and on average daily food intake (18.7 g, see S3 Table), a daily dose of ~2.9 g/kg of larva protein and ~8 mg/kg body weight captopril was estimated. Captopril reduces blood pressure [8, 9] and inhibits plasma ACE activity [10] in SHRs. It is used to treat mild to moderate hypertension in humans [11].

Blood pressure and heart rate measurement

Systolic blood pressure was measured weekly by the non-invasive “tail-cuff” method [12]. After warming the body of the rat to 37°C for 5 min, the average of three consecutive blood pressure readings, taken between 9am and 12am, was considered. In parallel, heart rate, i.e. heart beats per minute, was also recorded.

Plasma and brain ACE and inhibitory activity

ACE was detected in plasma and brain homogenates by measuring the amount of hyppuric acid cleaved by ACE from the substrate N-hyppuryl-L-histidyl-L-leucine. ACE inhibitory activity was assessed according to a previous protocol [13], based on quantification of the hyppuric acid released in samples by addition of a known amount of ACE. Finally, hyppuric acid was determined by RP-HPLC according to Wang et al. [13]. Details are reported in S3 File.

RBCs and plasma thiols

Blood (~0.5 ml) was collected in tubes containing 50 μl N-ethylmaleimide solution, 310 mM. After 1 min, blood was centrifuged at 10,000xg for 20s and plasma was collected. The pellet was washed twice with saline and the purified RBCs were deproteinized by addition of 1 volume 15% (w/v) trichloroacetic acid. Glutathione (GSH) and glutathione disulphide (GSSG) were measured in the supernatant by UV-Vis HPLC and the GSH recycling method, respectively, while hemoglobin concentration was measured spectrophotometrically (Jasco V-530 instrument, Jasco Europe Srl, Italy) [14].

Total thiols were measured in plasma samples by HPLC (Agilent series 1100 instrument, Agilent Technologies, Italy) by labelling SH groups with the fluorescent probe monobromobimane (Calbiochem) after reduction of disulphide bridges with dithiothreitol [14].

Liver cytochrome P450 and b₅ content, NADPH-cytochrome P450 reductase activity

Liver microsomes were prepared according to [15] (details in S4 File). Cytochrome P450 (CYP) and cytochrome b₅ contents were measured from the CO-difference spectra of the microsomes at ΔA 450–490 nm (ε: 91 mM) and ΔA 424–490 nm (ε: 112 mM), respectively [15]. NADPH-cytochrome P450 reductase activity was measured by following cytochrome c reduction at 550 nm [15].

Brain IL-6, IL-1β and TNF-α

IL-6 (Millipore®), IL-1β (Thermo Fisher Scientific) and TNF-α (Boster PicoKine^TM ELISA) were measured according to the manufacturer’s instructions in brain homogenates prepared as detailed in S5 File. Values were normalized to the protein content of the sample.

Ex-vivo functional studies

Aorta ring mechanical activity

Rings were prepared as previously described [16]. Relaxation of phenylephrine-contracted rings to either acetylcholine or sodium nitroprusside was taken as an index of endothelial function and muscle sensitivity to NO, respectively (more details in S6 File).

Isolated heart preparation and perfusion

Spontaneously beating hearts were rapidly explanted and mounted on a Langendorff apparatus for retrograde perfusion with physiological salt solution via the aorta at a constant flow rate of 10 ml/min, as described elsewhere [17] (S7 File).

Heart contractility was measured as left ventricle developed pressure (LVDP = left ventricular systolic pressure minus left ventricular diastolic pressure) by means of a latex balloon, inserted into the left ventricle via the mitral valve and connected to a pressure transducer (BLPR, WPI, Berlin, Germany). Coronary perfusion pressure was recorded by a pressure transducer in the inflow line. Heart rate was calculated from LVDP. LVDP and coronary perfusion pressure were recorded with a digital PowerLab data acquisition system (PowerLab 8/30; ADInstruments, Australia) and analysed with Chart Pro for Windows software (PowerLab; ADInstruments, Australia).

Brain susceptibility to oxidative stress-mediated injury

Brain cortical slices were prepared according to [18] (see also S8 File). Briefly, the two brain hemispheres were divided: one was homogenised and used for cytokine assessment (S5 File); the other was cut into 400-μm slices. These were exposed (OS) or not exposed (basal) to 10 mM H₂O₂ for 1 h. Then the colorimetric MTT assay was performed to assess tissue viability [19]. Slice tissue edema, an additional index of tissue viability [20], was calculated using the formula [18]:

t i s s u e e d e m a = (wet weight - dry weight) {(dry weight)}^{- 1} .

Statistical analysis

Results are reported as mean ± SEM. Statistical significance was assessed by ANOVA and Bonferroni post-test, Student t test or one-sample t test, as appropriate (GraphPad Prism version 5.04, GraphPad Software Inc., San Diego, CA). In all comparisons, statistical significance (P) was set at 0.05.

Results

Effect of TM on body weight and food intake

No changes in skin, fur, eyes, mucous membranes or salivation were observed in any of the treated groups, nor were there tremors or death. The TM and captopril diets did not affect growth rate, which was comparable to that of rats fed the standard diet (final weight gain about 43 g, see S2 Table). Similar results were obtained for food intake, which ranged from 17.7±0.1 to 19.6±0.6 g/rat/day (see S3 Table).

Effects of TM on cardiovascular mechanics

Basal blood pressure was much higher in hypertensive than in WKY rats (Fig 2, panel a). After four weeks, only the former showed a significant increase, which was reduced by TM and captopril supplementation. No effect was observed in WKY rats. Noticeably, in SHRs, captopril maintained BP around basal values (150.0±2.8 mmHg) already from the first week of treatment, while TM took 4 weeks to regain the basal values of 146.2±6.5 mmHg. Similar results were observed for heart rate (Fig 2, panel b).

Vascular endothelial function and smooth muscle sensitivity to α₁ adrenergic agonists as well as to NO were also assessed. Aorta rings from hypertensive and WKY rats showed similar responses to phenylephrine (S1 Fig). Captopril did not modify this response. On the contrary, TM supplementation generally increased the sensitivity of the preparations to phenylephrine, reaching significance in SHR rats. Rings from untreated SHRs stimulated by phenylephrine showed lower endothelium-dependent vasodilator responses to ACh than matched WKY rats (E_max = 61.2±4.0%, vs. 86.1±2%, respectively; P<0.05) (Fig 2, panel c). This difference disappeared when rings were pre-incubated with 5 μM indomethacin. Captopril (E_max 74.7±5.0%, P>0.05 vs. WKY), but not TM (E_max 53.2±5.5%, P<0.05 vs. WKY), produced a significant increase in ACh-induced relaxation of SHR rings. The response to SNP was similar in WKY rats and untreated SHRs, as well as in SHRs treated with captopril or TM (S1 Fig) Finally, neither captopril nor TM modified the response of WKY rings to ACh or SNP (S1 Fig).

Regarding heart function, no type of diet fed to WKY or SHRs affected LVDP or heart rate as evaluated by the Langendorff perfused heart model (S2 Fig). SHRs fed the standard diet showed higher coronary perfusion pressure than did the matched normotensive strain, with both captopril- and TM -supplemented diets bringing this parameter back to control values (Fig 2, panel d).

Effects of TM on red blood cells and on plasma thiol and disulfide levels

Red blood cell glutathione (GSH) levels were similar in normotensive and hypertensive rats and were not affected by TM or captopril diets (Fig 3, panel a). TM supplementation significantly increased the GSH/GSSG ratio in SHRs (Fig 3, panel b), suggesting that in this group the diet may have been responsible for a decrease in oxidative stress. In standard diet-fed rats, plasma total homocysteine (tHcys) was significantly lower in hypertensive than in matched normotensive rats (Fig 3, panel c). This difference was partially reduced by TM and completely reversed by the captopril diet. Finally, in SHRs, TM caused a significant increase in the total level of other physiological plasma thiols (i.e. cysteine, cysteinylglycine, γ-glutamylcysteine and GSH) compared to matched normotensive rats (see S4 Table).

Effect of TM on liver CYP450, cytochrome b₅ levels and NADPH-cytochrome P450 reductase activity

Liver CYP450 and cytochrome b₅ content, as well as NADPH-cytochrome P450 reductase activity were unaffected by TM and captopril treatment (see S5 Table). Likewise, liver weight gain did not change significantly after 4 weeks on standard diet or diets supplemented with TM or captopril, averaging the value of 10.4 g (lower 95% conf. limit 9.7 g; upper 95% conf. limit 11.2 g).

Effect of TM on plasma and brain ACE content and inhibitory activity

As shown in Fig 4, panel a, plasma ACE levels were comparable in all experimental groups, while in brain its content was about 30 fold higher than in plasma (Fig 4 panel c). Furthermore, both TM—and captopril-fed SHRs showed higher brain tissue ACE levels than rats fed the standard diet, though this difference did not reach statistical significance. Brain enzyme inhibitory activity was not affected by the different diets (Fig 4, panel d); on the contrary, in plasma it was significantly higher in SHRs fed with TM and captopril than in rats fed the standard diet (Fig 4, panel b).

Brain IL-1, IL-6 and TNF-α content

Diet supplementation had no effect on brain cytokine levels in WKY rats, whereas SHRs fed the standard diet showed significantly higher brain IL-1ß and TNF-α, but not IL-6 content, than the matched normotensive strain (Fig 5 panel a and b and S3 Fig). This effect was reversed by TM supplementation, while captopril only reduced IL-1ß. Finally, IL-6 was not affected by diet in SHRs (S3 Fig).

Effects of TM on brain cortical slice susceptibility to oxidative stress-mediated injury

Hydrogen peroxide challenge caused a significant loss in brain tissue viability in both groups of rats fed the standard diet (Fig 5, panel c). Interestingly, TM treatment protected the viability of slices from this injury in both rat strains. Captopril was effective in SHRs, but not in WKY rats. Tissue edema, taken as an additional index of viability, mostly mirrored the results of the MTT assay (Fig 5, panel d). Indeed, oxidative stress induced a significant increase in tissue water gain in both groups of rats. Brain cortical slices of both groups of rats fed with TM had a significantly lower water content than the same strain fed the standard diet. In addition, captopril reduced tissue edema, although it was only effective in WKY rats.

Discussion

The present results highlight interesting in vivo antihypertensive, cardio- and neuro-protective effects of protein hydrolysates derived from the larval stage of TM that could be useful for treating borderline blood pressure values in humans. Dietary supplementation with TM appeared to be safe, as no significant effects on food intake, weight gain or physical attributes of the animals were recorded, as already reported by [21]. Similar effects were obtained with captopril, although this drug did not counteract oxidative stress in brain slices. More importantly, TM did not affect liver microsomal CYP content or the activity of the enzymes responsible for their kinetics, suggesting that TM intake is without drug-interaction effects. Modulation of CYP-dependent enzyme activities by nutraceuticals could in fact alter the pharmacokinetic properties and/or toxicity of a concomitantly administered drug [22]. The fact that TM did not inhibit and/or induce CYPs linked to drug metabolism sustains the possibility of co-therapies without risk of an increase in drug-related adverse reactions.

A feature of particular relevance shown by TM was its ability to prevent the marked decrease in blood pressure and heart rate observed in SHRs, known to be associated with vascular and heart protection [23]. The negative chronotropic effect was not, however, observed in ex vivo Langendorff isolated hearts. Absence of the physiological humoral background could explain the discrepancy existing between in vivo and ex vivo chronotropic effects of the TM-enriched diet. Hypertensive rats fed the standard diet showed higher coronary perfusion pressure than the normotensive strain, as previously described [24]. Interestingly, the TM-enriched diet reduced coronary perfusion pressure to the same extent as captopril in WKY rats, bringing its value back to control level without changing heart function. Finally, the ACE inhibitory activity recorded in plasma of SHRs (standard diet, TM and captopril) was significantly correlated with the decrease in blood pressure recorded at the end of the 4 weeks (r² = 0.9975). This suggests that the mechanism of action underlying the antihypertensive effect of TM in lowering blood pressure can be ascribed to TM-induced inhibition of ACE.

As weight gain in the TM diet group of SHRs seemed less than that of the SHR control group, it can be postulated that a slight reduction in fat mass could contribute to the hypotensive effect exerted by TM. Indeed, Seo and colleagues reported an antiobesity effect of TM supplementation in mice fed a high fat diet [25]. Although the increase in blood pressure in the SHR group is mostly genetically driven, we cannot exclude that a slight reduction in body fat might be involved. If so, TM supplements for treating pre-hypertension are even more interesting and warrant further investigation.

In hypertension, oxidative stress plays a critical role in the molecular mechanisms associated with cardiovascular and renal injury. Hypertension itself can contribute to oxidative stress [26]. The GSH/GSSG ratio in red blood cells is a biomarker of oxidative stress. Neither GSH nor GSH/GSSG values changed in SHRs, in line with a previous report [27], indicating a good blood antioxidant barrier against the burst of radicals associated with hypertension. Although the captopril diet lowered blood pressure, it did not modify GSH-GSSG balance, as already reported in humans [28]. In contrast, the TM diet increased the GSH/GSSG ratio, suggesting that its antihypertensive effect could be linked to the reduction in GSSG in red blood cells or vice versa.

A close relation exists between intracellular and extracellular thiol pools [29]. The low molecular mass Hcys in plasma is also considered an independent risk factor in cardiovascular disease as it causes endothelial dysfunction [30]. However according to other reports, SHRs have lower Hcys levels than WKY rats [31], suggesting that Hcys per se is not linked to the development of hypertension in this strain. Paradoxically, the TM diet partially, and the captopril diet completely restored Hcys levels to control values, once more demonstrating their potential to restore physiological balance in hypertensive animals.

Regression of structural and/or functional alterations of the vascular endothelium occurring in hypertension is an important target of antihypertensive therapy. In this study, impaired relaxation of aorta rings of SHRs was observed in response to ACh, in line with reports in the literature [32]. Among other things, this may result from impaired NO synthesis [33] and/or an increase in superoxide anions, as well as various contracting factors released by a dysfunctional endothelium. Partial recovery of response to ACh was recorded in SHRs treated with captopril, but not in those treated with TM. This can be ascribed to a protective effect of the drug towards endothelial function, rather than to improved sensitivity of vascular smooth muscle to NO. Indeed, no differences in the vasodilator capacity of the NO donor SNP were detected in endothelium-denuded rings of the different groups, indicating that TM did not improve vascular function, at least in ex vitro assays. Furthermore, while captopril did not affect the sensitivity of SHRs vascular smooth muscle to phenylephrine-induced stimulation, TM increased it, thus ruling out a possible involvement of the sympathetic system in its systemic, beneficial activity. In fact, TM significantly lowered blood pressure after 3–4 weeks of treatment, as did captopril after only one week: therefore, it would be worth investigating whether vascular function might also be improved by extending TM treatment.

A hyperactive brain renin-angiotensin system, oxidative stress and neuroinflammation in brainstem cardiovascular centres and other brain regions increase sympathetic activity in hypertension [34]. The pro-inflammatory cytokines TNF-α, IL-1β and IL-6 act as neuromodulators in the paraventricular nucleus (PVN) of the hypothalamus by stimulating production of cytotoxic reactive oxygen species that further propagate the sympatho-excitatory effect [35]. Standard-diet-fed SHRs showed significantly higher IL-1ß and TNF-α brain content than the matched normotensive group, as previously described [36]. IL-6, on the contrary, was similar in hypertensive and WKY rats, in contrast with what was previously observed at PVN level [37]. When considering the present results, however, it cannot be ruled out that IL-6 is indeed increased in discrete areas such as the PNV, but this could not be detected measuring whole-brain cytokine content. Interestingly, the TM-enriched diet restored cytokine content to basal values, unlike captopril which was effective only towards IL-1ß. This observation indicates that the antihypertensive activity of TM can at least partly be ascribed to its anti-inflammatory effect.

Both peripheral and brain renin-angiotensin systems play a fundamental role in hypertension, hence ACE inhibitors are currently used to treat it. Brain ACE content was unchanged in WKY rats, while it seemed elevated in SHRs on the TM and captopril diets, although the increase did not reach statistical significance. However, chronic administration of captopril at high doses increases ACE in some brain area of SHRs, owing to suppression of inhibitory feedback of angiotensin II on ACE synthesis [38]. The apparent lack of activity of TM and captopril on brain ACE content may be explained by the low dose of captopril, which may only partly affect ACE levels. Moreover, since the enzyme was measured in whole brain, the possibility that ACE might reach higher values in specific areas cannot be ruled out.

Chronic hypertension is accompanied by brain damage caused by a hypoxic/ischemic mechanisms, in which oxidative stress plays a pivotal role [39]. High blood pressure is also related to milder, subtle and chronic forms of brain damage, especially those concerning cognitive function. This is why we assessed the susceptibility of the brains of rats fed with TM or captopril to oxidative-stress-mediated injury. Tissue viability was sharply improved in SHRs fed with TM and captopril diets. Among possible mechanisms of neuroprotection, ACE inhibition leading to an increase in brain bradykinin, which in turn reduces formation of reactive oxygen species [40], can be hypothesized. Neuroprotection against oxidative stress can also be ascribed to an increased GSH/GSSG ratio and a decrease in pro-inflammatory cytokines, which were more pronounced in TM- than in captopril-treated rats, the former being more resistant than the latter, even in the WKY strain. The observation that acute injection of captopril up to 50 mg/kg does not reduce brain infarct area in rats subjected to middle cerebral artery occlusion [41] supports this hypothesis.

In conclusion, a diet enriched with protein derived from the larval stage of the mealworm Tenebrio molitor exerts antihypertensive, cardio- and neuro-protective effects. Since TM inhibits intrinsic blood coagulation pathways [42] and has antiobesity properties [25], it can be considered a multi-target functional food. TM larvae are already produced on a commercial scale, are inexpensive, nutritious [43] and devoid of toxicity [21], as well as being easy to raise and having less environmental impact than other solutions [44]. Taken together, all these characteristics make TM an optimal candidate for the development of a multi-target functional food for non-pharmacological treatment of prehypertension or mild hypertension in the framework of life-style advice. Since some of its effects are not shared by captopril, TM may also have added value in preventing hypertension and mitigating other severe cardiovascular risk factors. These are good prerequisites for a pharmacological characterization of the bioactive peptide components of TM with a view to treating pre-hypertension, a disease with high social impact.

Supporting information

S1 File. Defatting of T. molitor larvae.

Defatted larvae used in the paper were obtained extracting twice with ethanol (99.5%) for 1 h as reported by Zhao et al. [45]. The extraction was accomplished using a solvent to material ratio of 5 mL/g, at 40°C for 1 h. For the dry matter determination 3 g exactly weighed ground larvae were dried overnight at 104°C [46]. Fat content was determined by Soxhlet extraction method using petroleum spirit for six hours starting from 5 g of sample. The solvent was removed at 40° under rotary evaporator and extracted fat weighted after the flask reaching constant weigh after heating at 104°C. Crude protein were determined with the Kjeldahl method using a protein-to-nitrogen conversion factor of 6.25 [2]. Ash were determined by weighting after incineration of 2 g of sample at 550°C for 3 h and cooling in a desiccator [2]. Each assay was run at least in triplicate. The analysis gave in the starting material dry matter (95.8±0.08%), fat content (32.7±0.84%), crude proteins (50.8±0.72%) and ash (4.8±0.09%), extracted fat (33.8±0.92%) and crude protein content in the final product (77.5±0.59%). These values were superimposable to those of Zhao et al. [45]. Nutritional value of TM protein as well as the in vitro digestibility was already accomplished [47]. Proteins extracted as water-soluble fraction (supernatant) or as water-insoluble fraction (pellet) were characterized by SD-PAGE and LC-MS/MS [48]. The most abundant in the supernatant were hemolymph protein (~12 kDa), alpha-amylase (~50 kDa, a putative allergen), and muscle proteins (e.g. actin 30–50 kDa) in the pellet fraction. Also the amino acid pattern and the essential amino acid index were already described [49] and [doi 10.3844/ajabssp.2009.319.331], showing that TM contain all the essential amino acid in the amount necessary to human [50]. The sum of total amount of amino acids (TAA) per g crude protein of TM was 910 mg/g, while the calculated essential amino acid index (EAAI) was slightly higher than that of soybean, but lower than that of casein. Moreover, the protein digestibility-corrected amino acid score (PDCAAS) of defatted TM was 0.80, further confirming that it has the ability to meet human dietary requirements for specific amino acids (0.79–0.82 for PDCAAS) [doi.org/10.3920/JIFF2018.0048]. Toxicological aspects such as subchronic toxicity of TM diet, including potential hypersensitivity, after orally administration at dose up to 3000 mg/kg/day for 90 days, was already assessed in both sexes of Sprague-Dawley rats [21]. TM was also proven to be non-genotoxic ([51], erratum in [52]).

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S2 File. Details of animal anaesthesia.

SHR and WKY rats were fully anaesthetized by intraperitoneal injection of 15 mg/kg Zoletil 100® (Virbac Srl, Milano, Italy) and 4 mg/kg Xylor® (Bio 98, San Lazzaro, Italy). Zoletil 100® is a 1:1 mixture of tiletamine (dissociative anaesthetic acting as NMDA receptor antagonist) and zolazepam (benzodiazepine). Xylor® is composed by the α₂ adrenergic receptor agonist xylazine endowed with sedation and muscle relaxation activities.

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S3 File. RP-HPLC determination of plasma and brain ACE and its inhibitory activity.

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S4 File. Liver cytochrome P450- and b5-content, NADPH-cytochrome P450 reductase activity: preparation of liver microsomes.

After the sacrifice the livers were perfused in situ through the vena cava with ice-cold physiological saline solution, excised and weighed, chopped, suspended in 4 volumes of 0.25 M sucrose and homogenized in a Potter-Elvejhem homogeniser fitted with a teflon pestle. Homogenates were then centrifuged (10,000 g for 20 min) and the resulting supernatants were further centrifuged at 105,000 g for 1 h. The resulting microsomal pellets were suspended in 0.01 M Tris-HCl buffer, pH 7.6, containing 151 mM KCl, 1 mM EDTA and 20% glycerol, and stored in liquid nitrogen until use.

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S5 File. Brain homogenate preparation for cytokine assay.

Half brain hemispheres were quickly frozen in liquid nitrogen and stored at -80°C until analysis. The day of the assay, brains were thawed, homogenated in cold phosphate buffer 0.1 M pH 7.4 (tissue weigh: buffer volume = 1:10) and centrifuged at 10,000 g x 10 min at 4°C. Afterward the supernatant was collected and used for the ELISA assay, whose sensitivity and assay range were as follows: TNF-α sensitivity < 1 pg/ml, assay range: 7.8–500 pg/ml; IL-6: sensitivity 5.3 pg/ml, assay range 18.8–1200 pg/ml; IL-1ß sensitivity < 12 pg/ml, assay range: 25.6–2500 pg/ml.

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S6 File. Vascular functionality of aorta rings.

Vascular reactivity was evaluated on aortic rings by acetylcholine and sodium nitroprusside relaxations. Briefly, the thoracic aorta was cleaned of adherent fat and rings 2.5 mm long were cut and placed between stainless-steel hooks for isometric tension recording in organ chambers. Contractile tension was recorded with a digital PowerLab data acquisition system (PowerLab 8/30; ADInstruments, Castle Hill, Australia) and analysed by using LabChart 7.3.7 Pro (Power Lab; ADInstruments). Rings were stretched to 1 g of tension and equilibrated for 1 h. After pre-contraction with phenylephrine (0.3 μM) the presence of endothelium was verified by the ability of acetylcholine (ACh, 10 μM) to induce relaxation. Concentration-response curves of aortic rings with endothelium to ACh (100 pM–10 μM) and sodium nitroprusside (SNP, 100 pM–100 μM) were performed with and without indomethacin (5 μM; 30 min). In these experiments, the vasodilator responses to ACh and SNP were expressed as percentages of phenylephrine contraction.

(DOCX)

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S7 File. Isolated heart preparation and perfusion.

Spontaneously beating hearts were rapidly explanted and mounted on a Langendorff apparatus for retrograde perfusion via the aorta at a constant flow rate of 10 ml/min with a physiological salt solution (PSS) of the following composition (mM): NaCl 118, KCl 4.7, CaCl₂ 2.5, MgSO₄ 1.2, NaHCO₃ 25, KH₂PO₄ 1.2, glucose 11.5, Na pyruvate 2, and EDTA 0.5, continuously bubbled with a 95% O₂−5% CO₂ gas mixture (pH 7.4), and kept at 37°C [53–55]. The presence of the Ca²⁺ buffer EDTA (0.5 mM) in PSS assures a constant and controlled free Ca²⁺ concentration of 2 mM (EQCAL for Windows software, Biosoft, Cambridge, UK) throughout the experimental session.

(DOCX)

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S8 File. Preparation of brain cortical slices.

The preparation of cortical slices were performed according to standard experimental protocols already reported [18–20]. In particular, the brain was quickly washed in artificial cerebrospinal fluid (ACSF) (composition in mM: 120 NaCl; 2 KCl; 1 CaCl₂; 1 MgSO₄; 25 HEPES; 1 KH₂PO₄; 10 glucose, pH 7.4 and previously bubbled with a 95% O₂−5% CO₂ gas mixture for 20–30 min) and divided into the two hemispheres. One was used for cytokine assessment, while the other was dissected and cut into 400 μm-thickness cortex slices by using a manual chopper (Stoelting Co., Wood Dale, IL). Slices were transferred into sterile 24-well culture plates containing 0.5 ml ACSF/well previously filtered by passage through a 0.2 μm sterile filter and left at room temperature (25°C) for 60 min to recover from slicing trauma (equilibration phase). During this period, the medium was removed and replaced with fresh, oxygenated filtered ACSF every 15 min (equilibration phase). After the equilibration phase, oxidative stress was induced by treating the tissue with 10 mM H₂O₂ for 1 h [56]. At the end of the treatment, the colorimetric MTT method was used to assess tissue viability as already reported [19]. Slice viability was expressed as a percentage of untreated slices (Basal). Tissue water gain, taken as an index of tissue edema was also assessed and it was calculated by using the formula TE = (wet weight–dry weight)(dry weight)⁻¹ and given as g H₂O (g dw)⁻¹ [20].

(DOCX)

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S1 Table. Diets composition and ingredients used in the present study.

(DOCX)

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S2 Table. Effects of the feeding with standard laboratory rodent chow (SD), or SD supplemented with either TM or captopril (C) for 4 weeks on body weight.

(DOCX)

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S3 Table. Effects of the feeding with standard laboratory rodent chow (SD), or SD supplemented with either TM or captopril (C) for 4 weeks on food intake.

(DOCX)

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S4 Table. Effects of the feeding for 4 weeks with standard laboratory rodent chow (SD) or SD supplemented with either Tenebrio molitor (TM) or captopril (C) on plasma total low molecular mass thiols (sum of thiol and disulphide forms) in WKY e SHR rats.

(DOCX)

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S5 Table. Effects of the feeding for 4 weeks with standard laboratory rodent chow (SD) or SD supplemented with either Tenebrio molitor(TM) or captopril (C) on liver cytochrome P450- and b5-content and NADPH-cytochrome P450 reductase activity in WKY e SHR rats.

(DOCX)

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S1 Fig. Effects of the feeding for 4 weeks with standard laboratory rodent chow (SD) or SD supplemented with either Tenebrio molitor (TM) or captopril (C) on vascular function.

Panel a: 0.3 μM phenylephrine-induced contraction in rat aorta rings. Panel b-d: smooth muscle sensitivity to (b,c) exogenous and (d) endogenous NO: concentration-response curve to (b,c) sodium nitroprusside (SNP) of endothelium-denuded, rat aorta rings or to (d) acetylcholine (ACh) of endothelium-intact rat aorta rings pre-contracted with 0.3 μM phenylephrine. On the ordinate scale, response is reported as mg (panel a) or as percentage of the contraction induced by phenylephrine (phe), taken as 100% (panel b-d). Values are reported as mean ± SEM. *P<0.05 vs SHR SD (ANOVA followed by Dunnett post test).

(TIF)

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S2 Fig. Effects of the feeding for 4 weeks with standard laboratory rodent chow (SD) or SD supplemented with either Tenebrio molitor (TM) or captopril (C) on left ventricular pressure (LVDP, panel a) and heart rate (panel b) on isolated heart.

Data are reported as mean±SEM.

(TIF)

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S3 Fig. Effects of the feeding for 4 weeks with standard laboratory rodent chow (SD) or SD supplemented with either Tenebrio molitor (TM) or captopril (C) IL-6 contents.

Values are reported as mean ± SEM.

(TIF)

Click here for additional data file.^{(1.3MB, tif)}

Acknowledgments

The authors wish to thank Jessica Pinassi, Dr. Ettore Vanni and Dr. Ilenia Gaudiosi for the assistance with some of the experiments.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This research was carried out under the project “INSECT CARD” funded by the Tuscan Regional Administration's Call for Research Projects in the Nutraceutical Industry. Executive Decrees No. 4741 of 21 October 2014 and No. 5418 of 17 November 2014, PI Valeria Francardi. Lead Discovery Siena Srl provided support in the form of research materials. The funders did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0233788.r001

Decision Letter 0

Luis Eduardo M Quintas

13 Jan 2020

PONE-D-19-31063

Antihypertensive, cardio- and neuro-protective effects of Tenebrio molitor (Coleoptera: Tenebrionidae) defatted larvae in spontaneously hypertensive rats

PLOS ONE

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Reviewer #1: Antihypertensive, cardio- and neuro-protective effects of Tenebrio molitor 2 (Coleoptera: Tenebrionidae) defatted larvae in spontaneously hypertensive rats

Reviewer

The present manuscript studied the effects of a diet enriched with defatted larvae of the mealworm Tenebrio molitor (TM) endowed with ACE inhibitory activity in spontaneously hypertensive rats (SHR) and in the normotensive Wistar Kyoto. This is an important study considering it suggests a dietary approaches to stop hypertension and using the foods to reduce cardiovascular risk factors and improve vascular health.

Comments:

1. Abstract:

Line 38-39: ……Hypertensive rat brain slices were more resistant to oxidative stress and contained lower levels of inflammatory cytokines, while vascular and liver enzyme-activities were not affected…

Authors should inform if this response is from TM group or only for the SHR.

Besides it was demonstrated the TM induced a more resistance to oxidative stress in the brain, this reviewer would like to see its effects at cardiac cells also.

3. Line 40: …..TM can be considered a new functional food that can improve BP in vivo……

Also, when authors inform: line 42: ….and control certain hypertension-associated risk factors…. What authors want to say with…. Certain hypertension-associated risk… it should be more specific.

Introduction

4. Line 44: ……Hypertension is the main risk factor for pathologies such as coronary artery disease……

Pathology is the study of the way diseases and illnesses develop. Authors should avoid use this term as a synonym of disease.

Methods

5. Line 158 : …..Heart contractility was measured as left ventricle pressure (LVP= left ventricular systolic pressure 159 minus left ventricular diastolic pressure)…. I suggest that authors name it as …developed left ventricular systolic pressure, as it is not the absolute systolic pressure, but the systolic minus diastolic pressure.

Also, authors should inform the value of diastolic pressure in the left ventricle, as diastolic pressure is an important determinant of systolic pressure. This is described by Starling Law. That states the systolic pressure increases in response to an increase in the diastolic pressure or diastolic volume, before contraction (the end diastolic volume), when all other factors remain constant. It is important considering the ability of the heart to change its force of contraction in response to changes in diastolic pressure or volume.

Please, inform the diastolic pressure or volume in the hearts.

6. Line 709: Authors informed that nutrition solution for heart perfused at Langendorff preparations had CaCl2 2.5. My question is, usually, rats need a smaller extracellular calcium concentration, around 1.2 to 1.8 mM. Calcium concentration higher them this, usually induces calcium overload and the preparations does not survive longer. I would like to know why authors used this calcium concentration and how long did the hearts survived without demonstrate signal of calcium overload. It is specially complicated when temperature is kept 37 0C.

7. Line 710 to 711: Authors informed that NaHCO3 25 mM, bubbled with a 95% O2–5% CO2 gas mixture (pH 7.4). I would like to know if, at this NaHCO3 concentration, pH was maintained constant during all the experimental time. Did the authors measure the pH in the beginning and in the end of the experiments, or was it estimated? This reviewer would like to see the values. What is the propose of EDTA 0.5 mM?

8. Line 116: why authors measured brain ACE activity and not cardiac? Also, why measure brain IL-6, IL1B and TNP and not vascular iNOS expression to correlate it with those pro-inflammatory mediators?

9. Line 148 to 152: Ex-vivo functional studies and Aorta ring mechanical activity.

Authors should conduct more experiments with the vasoconstriction to phenylephrine using dose-response curves, to analyze the vascular vasoreactivity.

Also, authors should measure de vascular ROS and NO. The reduced NO bioavailability can be attributed to a decreased NO production and/or an increased NO degradation.

I suggested to use some regular technics (for ex DHE and DAF) and also Western blott for protein expression of iNOS, eNOS and nNOS. Uncoupling of eNOS has been reported in essential hypertensive patients with endothelial dysfunction. So, authors should measure uncoupling eNOS after TM treatment.

Other important enzyme, NADPH oxidase, plays a crucial role in eNOS uncoupling, producing ROS, which leads to oxidation of co-factor BH4 inducing oxidative stress. It should be measured.

Results and Discussion

10. Isolated heart preparation and perfusion was described at methods section, but no results were presented at Results section. Please include it.

11. Lines 295 to 301: …..Pre-hypertension is a warning sign of increased risk of cardiovascular disease and end-organ 296 damage. When not detected early, it can progress to more harmful hypertension. However, blood pressure values of pre-hypertensive patients are not high enough to warrant prescription of antihypertensive drugs. This gap has prompted research into functional foods as a potential source 299 of alternative therapies. In this scenario, food containing peptides that are inactive in the parent protein, but become ACE inhibitors when released during enzymatic digestion or food processing, can play an important role in reducing blood pressure ….

This sentence is not necessary at discussion section as it seems a replay of introduction. I would suggest take it out.

12. Authors inform in the manuscript that: …A feature of particular relevance shown by TM was the marked decrease in blood pressure and hear rate observed in SHRs….

It seems that TM avoided the increment on blood pressure better than to reduce it. In this study. It would be the case if the authors had permitted the BP to increase, them, treated with TM to make it reduce.

Reviewer #2: The manuscript "Antihypertensive, cardio- and neuro-protective effects of Tenebrio molitor (Coleoptera: Tenebrionidae) defatted larvae in spontaneously hypertensive rats” is very well written, and easy to understand. The topic is interesting and important because it deals with potential functional food as a source of alternative therapies for pre-hypertensive patients. Methods section provide enough details for the general reader to repeat the experiments. Аuthors report that diet enriched with defatted larvae of the mealworm in SHR caused a significant reduction of systolic blood pressure, heart rate and coronary perfusion pressure, also increased ACE inhibitory activity in plasma, and decreased inflammatory cytokines IL-1β and TNFα levels in brain homogenates. Further, hypertensive rat brain slices were more resistant to oxidative stress. I suggest that in the future studies focus is on the mechanistic investigations and explaining in more detail the cellular and molecular mechanisms of action of proteins from defatted larvae of the mealworm in hypertensive rats.

**********

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PLoS One. 2020 May 29;15(5):e0233788. doi: 10.1371/journal.pone.0233788.r002

Author response to Decision Letter 0

30 Mar 2020

Comments:

1. Abstract: Line 38-39: ……Hypertensive rat brain slices were more resistant to oxidative stress and contained lower levels of inflammatory cytokines, while vascular and liver enzyme-activities were not affected…Authors should inform if this response is from TM group or only for the SHR.

As the sentence reported “hypertensive rat brain slices”, we though that it was clear that data refers to SHR rats. We agree however that the sentence could be misleading, and thus it has been changed into “Rat brain slices of SHR …”.

2. Line 38-39:……Hypertensive rat brain slices were more resistant to oxidative stress and contained lower levels of inflammatory cytokines, while vascular and liver enzyme-activities were not affected. Besides it was demonstrated the TM induced a more resistance to oxidative stress in the brain, this reviewer would like to see its effects at cardiac cells also.

In the present study, hearts of normotensive and hypertensive rats were used to perform the experiments with Langendorff-apparatus. For this reason, it was not possible to assess oxidative stress on cardiac cells immediately after the end of the treatments. The present results, however, encouraged us to deepen the effects of TM in a following in vivo and ex-vivo study, which will be focused on the changes induced at cardiovascular level by the proteins from defatted larvae of the mealworm in hypertensive rats, as also suggested by Referee #2.

3. Line 40: …..TM can be considered a new functional food that can improve BP in vivo……

Authors should clarify what they want does it means when they say, TM improved blood pressure. It is a general word that does not inform the mechanism or the physiological effect of TM on blood pressure. Also, when authors inform: line 42: ….and control certain hypertension-associated risk factors…. What authors want to say with…. Certain hypertension-associated risk… it should be more specific.

We agree with the Referee that the sentence in line 40 is not sufficiently clear. It has been changed as follows:

These results suggest that TM can be considered a new functional food that can lower BP in vivo and thus control cardiovascular-associated risk factors such as hypertension.

Introduction

4. Line 44: ……Hypertension is the main risk factor for pathologies such as coronary artery disease……Pathology is the study of the way diseases and illnesses develop. Authors should avoid use this term as a synonym of disease.

We agree with the Referee and the text has been amended accordingly.

Methods

5. Line 158 : …..Heart contractility was measured as left ventricle pressure (LVP= left ventricular systolic pressure minus left ventricular diastolic pressure)…. I suggest that authors name it as …developed left ventricular systolic pressure, as it is not the absolute systolic pressure, but the systolic minus diastolic pressure.

We agree with the point raised and thus LVP has been replaced by LVDP in text, Figures and their legend.

According to referee’s suggestion we have measured the diastolic pressure (see Figure below). As already reported for LVDP (Supplementary S2 Fig.), neither Tenebrio Molitor (TM), nor captopril (C) affected the LVEDP.

Effects of the feeding for 4 weeks with standard laboratory rodent chow (SD) or SD supplemented with either Tenebrio molitor (TM) or captopril (C) on left ventricular end diastolic pressure (LVEDP) on isolated heart. Data are reported as mean±SEM.

The 2.5 mM calcium Krebs and Henseleit perfusion solution has always been used worldwide as well as in our laboratories (see for example: Saponara et al, Eur J Pharmacol 2007, 563(1-3):160-3; Gemma et al, J Med Chem 2012, 55(15):6948-67 Fusi et al, J Cardiovasc Pharmacol 2017, 70(6):405-410; Pessina et al, Biochem Pharmacol 2018;155:434-443). Moreover, it is reported in seminal papers (see Skrzypiec-Spring et al (2007) J Pharmacol Toxicol Methods;55:113-26 and chapters of books [Dhein S (2005). The Langendorff Heart in Practical Methods in Cardiovascular Research. Ed Dhein S, Mohr FW, Delmar M. Springer Nature; Doring, HJ and Dehnert H (1987). The isolated perfused heart according to Langendorff. BVM-Biomesstechnic Verlag) describing the Langendorff technique.

In addition, the presence of 0.5 mM EDTA in our solution, ensures a free calcium concentration of 2 mM (EQCAL for Windows software, Biosoft, Cambridge, UK) and we performed experiments to assess the stability of our preparations up to 3 hours from the beginning of the experiment, without any evidence of calcium overload.

The perfusion solution was continuously gassed with 5% CO2 and 95% O2 at a constant temperature of 37°C to maintain an appropriate pH of 7.4. In S7 file is now reported that bubbling was continuous, being this not clearly stated. pH was checked several times, at the beginning, during and at the end of the experimental session, and it was found to be constant.

EDTA Ca2+ buffering capacity was exploited to ensure a constant and controlled free Ca2+ concentration throughout the experimental session.

Cardiac ACE activity was not assessed, as heart were used to perform the experiments with isolated organ. As previously outlined, however, we are planning a novel in vivo and ex-vivo study, which will be focused on the changes induced at cardiovascular level by the proteins from defatted larvae of the mealworm in hypertensive rats, with particular regard to the role played by vascular NO, iNOS, eNOS and nNOS expression and inflammation (see also the answer to point 9).

9. Line 148 to 152: Ex-vivo functional studies and Aorta ring mechanical activity.

Authors used relaxation of phenylephrine-contracted rings to either acetylcholine or sodium nitroprusside as an index of endothelial function and muscle sensitivity to NO. Besides it is an universal and well established way to analyze vasodilatation and endothelial function, isolated, is has less physiological information them when it is used associated with the vasoconstriction phenylephrine dose-response curves activated, to analyze the vascular vasoreactivity. Specially because, the vascular tonus, in the physiological conditions, is regulated by sympathetic system, as the parasimpathetic system does not control aortic function. Authors should conduct more experiments with the vasoconstriction to phenylephrine using dose-response curves, to analyze the vascular vasoreactivity. Also, authors should measure de vascular ROS and NO. The reduced NO bioavailability can be attributed to a decreased NO production and/or an increased NO degradation.

A decreased NO production could be due to reduced eNOS expression/presence. The up-regulated expression of eNOS in situations of endothelial dysfunction is likely to be the consequence of an elevated production of hydrogen peroxide, which is a dismutation product of O2·−, and can increase the protein presence of eNOS. I suggested to use some regular technics (for ex DHE and DAF) and also Western blott for protein expression of iNOS, eNOS and nNOS. Uncoupling of eNOS has been reported in essential hypertensive patients with endothelial dysfunction. So, authors should measure uncoupling eNOS after TM treatment. Other important enzyme, NADPH oxidase, plays a crucial role in eNOS uncoupling, producing ROS, which leads to oxidation of co-factor BH4 inducing oxidative stress. It should be measured.

We thank the Referee for his suggestion. The vasoreactivity to phenylephrine has been analysed and discussed and now is included in the revised Supporting Figure 1, panel a. However, as TM supplementation did not improve either endothelium-dependent relaxation or smooth muscle sensitivity to phenylephrine, we believe that an in depth analysis of eNOS/NADPH oxidase expression as well as ROS production in aorta rings would not help clarifying the mechanism(s) underpinning the beneficial effect of TM treatment on systemic blood pressure and, therefore, has not been executed.

Results and Discussion 10. Isolated heart preparation and perfusion was described at methods section, but no results were presented at Results section. Please include it.

In the previous version of the manuscript Langendorff results were already reported. CPP results were reported in Fig 2d and LVDP and BPM in Suppl Fig 2 (wrongly linked to Suppl Fig 3, now properly amended).

In paragraph “Effects of TM on cardiovascular mechanics” was already stated “Regarding heart function, no type of diet fed to WKY or SHRs affected LVDP or heart rate as evaluated by the Langendorff perfused heart model. SHRs fed the standard diet showed higher coronary perfusion pressure than did the matched normotensive strain, with both captopril- and TM -supplemented diets bringing this parameter back to control values (Fig 2, panel d).

The suggestion has been taken and the text omitted.

12. Authors inform in the manuscript that: …A feature of particular relevance shown by TM was the marked decrease in blood pressure and heart rate observed in SHRs….

We agree with the Referee that the original sentence might be ambiguous, and thus it was modified accordingly as reported below:

A feature of particular relevance shown by TM was its ability to prevent the marked increase in blood pressure and heart rate observed in SHRs, known to be associated with vascular and heart protection.

We thank the Referee for the suggestion that will be followed in the next study focused on the mechanisms responsible for the effects that the proteins from defatted larvae of the mealworm exert at cardiovascular level in hypertensive rats.

Attachment

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PLoS One. doi: 10.1371/journal.pone.0233788.r003

Decision Letter 1

Luis Eduardo M Quintas

27 Apr 2020

PONE-D-19-31063R1

Antihypertensive, cardio- and neuro-protective effects of Tenebrio molitor (Coleoptera: Tenebrionidae) defatted larvae in spontaneously hypertensive rats

PLOS ONE

Dear FROSINI,

Specifically, reviewer #1 is still concerned about EDTA addition and the concentration of calcium in Langendorff experiments. Thus, I would require that you insert in the text (in supporting information S7) the explanation you presented to the reviewer (i.e., why EDTA was added, how free calcium was estimated and some literature reference about 2.5 mM CaCl2) in order to clear to the reader.

We would appreciate receiving your revised manuscript by Jun 11 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Luis Eduardo M Quintas, Ph.D.

Academic Editor

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: Authors answer:

In addition, the presence of 0.5 mM EDTA in our solution, ensures a free calcium

concentration of 2 mM (EQCAL for Windows software, Biosoft, Cambridge, UK) and we

performed experiments to assess the stability of our preparations up to 3 hours from

the beginning of the experiment, without any evidence of calcium overload.

7. Line 710 to 711: Authors informed that NaHCO3 25 mM, bubbled with a 95%

O2–5% CO2 gas mixture (pH 7.4). I would like to know if, at this NaHCO3

concentration, pH was maintained constant during all the experimental time. Did the

authors measure the pH in the beginning and in the end of the experiments, or was it

estimated? This reviewer would like to see the values. What is the propose of EDTA

0.5 mM?

EDTA Ca2+ buffering capacity was exploited to ensure a constant and controlled free

Ca2+ concentration throughout the experimental session.

Reviewer question:

It still not clear why authos used EDTA 0.5 mM in order to buffer high Ca++ concentration .

If the idea was to use Calcium 2.0 mM, why did authors used a high concentration (2.5 mM), and them used EDTA to reduce it, as most of paper have been using extracecullar calcium in rat heart 1.2 to 1.8 mM.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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Reviewer #1: No

PLoS One. 2020 May 29;15(5):e0233788. doi: 10.1371/journal.pone.0233788.r004

Author response to Decision Letter 1

1 May 2020

Reviewer #1 question:

It still not clear why authors used EDTA 0.5 mM in order to buffer high Ca++ concentration .

As suggested by the Editor, in supporting information S7 the explanation about why EDTA was added, how free calcium was estimated were insert along with appropriate references (i.e. 53-55) about 2.5 mM CaCl2 in order to clear to the reader.

Attachment

Submitted filename: Rebuttal letter.docx

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PLoS One. doi: 10.1371/journal.pone.0233788.r005

Decision Letter 2

Luis Eduardo M Quintas

13 May 2020

Antihypertensive, cardio- and neuro-protective effects of Tenebrio molitor (Coleoptera: Tenebrionidae) defatted larvae in spontaneously hypertensive rats

PONE-D-19-31063R2

Dear Dr. FROSINI,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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Luis Eduardo M Quintas, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0233788.r006

Acceptance letter

Luis Eduardo M Quintas

15 May 2020

PONE-D-19-31063R2

Antihypertensive, cardio- and neuro-protective effects of Tenebrio molitor (Coleoptera: Tenebrionidae) defatted larvae in spontaneously hypertensive rats

Dear Dr. Frosini:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 File. Defatting of T. molitor larvae.

(DOCX)

Click here for additional data file.^{(19.1KB, docx)}

S2 File. Details of animal anaesthesia.

(DOCX)

Click here for additional data file.^{(12KB, docx)}

S3 File. RP-HPLC determination of plasma and brain ACE and its inhibitory activity.

(DOCX)

Click here for additional data file.^{(14.3KB, docx)}

S4 File. Liver cytochrome P450- and b5-content, NADPH-cytochrome P450 reductase activity: preparation of liver microsomes.

(DOCX)

Click here for additional data file.^{(12.2KB, docx)}

S5 File. Brain homogenate preparation for cytokine assay.

(DOCX)

Click here for additional data file.^{(12.2KB, docx)}

S6 File. Vascular functionality of aorta rings.

(DOCX)

Click here for additional data file.^{(12.3KB, docx)}

S7 File. Isolated heart preparation and perfusion.

(DOCX)

Click here for additional data file.^{(13.1KB, docx)}

S8 File. Preparation of brain cortical slices.

(DOCX)

Click here for additional data file.^{(19.7KB, docx)}

S1 Table. Diets composition and ingredients used in the present study.

(DOCX)

Click here for additional data file.^{(13.1KB, docx)}

S2 Table. Effects of the feeding with standard laboratory rodent chow (SD), or SD supplemented with either TM or captopril (C) for 4 weeks on body weight.

(DOCX)

Click here for additional data file.^{(13.5KB, docx)}

S3 Table. Effects of the feeding with standard laboratory rodent chow (SD), or SD supplemented with either TM or captopril (C) for 4 weeks on food intake.

(DOCX)

Click here for additional data file.^{(13.1KB, docx)}

(DOCX)

Click here for additional data file.^{(13.7KB, docx)}

(DOCX)

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S1 Fig. Effects of the feeding for 4 weeks with standard laboratory rodent chow (SD) or SD supplemented with either Tenebrio molitor (TM) or captopril (C) on vascular function.

(TIF)

Click here for additional data file.^{(2.2MB, tif)}

Data are reported as mean±SEM.

(TIF)

Click here for additional data file.^{(1.5MB, tif)}

S3 Fig. Effects of the feeding for 4 weeks with standard laboratory rodent chow (SD) or SD supplemented with either Tenebrio molitor (TM) or captopril (C) IL-6 contents.

Values are reported as mean ± SEM.

(TIF)

Click here for additional data file.^{(1.3MB, tif)}

Attachment

Submitted filename: Refebuttal final.pdf

Click here for additional data file.^{(239.3KB, pdf)}

Attachment

Submitted filename: Rebuttal letter.docx

Click here for additional data file.^{(12KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Antihypertensive, cardio- and neuro-protective effects of Tenebrio molitor (Coleoptera: Tenebrionidae) defatted larvae in spontaneously hypertensive rats

Federica Pessina

Maria Frosini

Paola Marcolongo

Fabio Fusi

Simona Saponara

Alessandra Gamberucci

Massimo Valoti

Daniela Giustarini

Paolo Fiorenzani

Beatrice Gorelli

Valeria Francardi

Maurizio Botta

Elena Dreassi

Roles

Abstract

Introduction

Materials and methods

T. molitor larva meal

Animals and ethical statement

Experimental design

Fig 1. Overview of experimental design.

Doses

Blood pressure and heart rate measurement

Plasma and brain ACE and inhibitory activity

RBCs and plasma thiols

Liver cytochrome P450 and b5 content, NADPH-cytochrome P450 reductase activity

Brain IL-6, IL-1β and TNF-α

Ex-vivo functional studies

Aorta ring mechanical activity

Isolated heart preparation and perfusion

Brain susceptibility to oxidative stress-mediated injury

Statistical analysis

Results

Effect of TM on body weight and food intake

Effects of TM on cardiovascular mechanics

Fig 2. Effects of feeding hypertensive and WKY rats for 4 weeks with standard laboratory rodent chow (SD) or SD supplemented with Tenebrio molitor (TM) or captopril (C) on cardiovascular systems.

Effects of TM on red blood cells and on plasma thiol and disulfide levels

Fig 3.

Effect of TM on liver CYP450, cytochrome b5 levels and NADPH-cytochrome P450 reductase activity

Effect of TM on plasma and brain ACE content and inhibitory activity

Fig 4. Effects of feeding hypertensive and WKY rats for 4 weeks with standard laboratory rodent chow (SD) or SD supplemented with Tenebrio molitor (TM) or captopril (C) on plasma and brain ACE content and inhibitory activity.

Brain IL-1, IL-6 and TNF-α content

Fig 5.

Effects of TM on brain cortical slice susceptibility to oxidative stress-mediated injury

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Luis Eduardo M Quintas

Roles

Author response to Decision Letter 0

Decision Letter 1

Luis Eduardo M Quintas

Roles

Author response to Decision Letter 1

Decision Letter 2

Luis Eduardo M Quintas

Roles

Acceptance letter

Luis Eduardo M Quintas

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Liver cytochrome P450 and b₅ content, NADPH-cytochrome P450 reductase activity

Effect of TM on liver CYP450, cytochrome b₅ levels and NADPH-cytochrome P450 reductase activity