Ameliorative effects of Penthorum chinense Pursh on insulin resistance and oxidative stress in diabetic obesity db/db mice

Jilei Hu; Leyu Zheng; Xi Fan; Hongmei Lang; Huibo Xie; Ning Lin

doi:10.1371/journal.pone.0311502

. 2024 Oct 7;19(10):e0311502. doi: 10.1371/journal.pone.0311502

Ameliorative effects of Penthorum chinense Pursh on insulin resistance and oxidative stress in diabetic obesity db/db mice

Jilei Hu ^1,², Leyu Zheng ^2,³, Xi Fan ², Hongmei Lang ⁴, Huibo Xie ², Ning Lin ^1,^*

Editor: Mehran Rahimlou⁵

PMCID: PMC11458015 PMID: 39374222

Abstract

Background

Penthorum chinense Pursh (PCP), a medicinal and edible plant, has been reported to protect against liver damage by suppressing oxidative stress. Type 2 diabetes mellitus (T2DM) is associated with liver dysfunction and oxidative stress. In the present study, we aim to investigate the hypoglycemic effect of PCP on db/db mice and further explore the underlying mechanisms.

Methods

Thirty-two db/db mice were randomized into four groups, including a diabetic model control group (MC) and three diabetic groups treated with low (LPCP, 300 mg/kg/d), medium (MPLP, 600 mg/kg/d), and high doses of PCP (HPCP, 1200 mg/kg/d), and the normal control group (NC) of eight db/m mice were included. Mice in the NC and MC groups received the ultrapure water. After four weeks of intervention, parameters of fasting blood glucose (FBG), insulin resistance (IR), blood lipid levels, hepatic oxidative stress, and enzymes related to hepatic glucose metabolism were compared in the groups.

Results

PCP administration significantly reduced FBG and IR in diabetic db/db mice, and improved hepatic glucose metabolism by increasing glucose transporter 2 (GLUT2) and glucokinase (GCK) protein expression. Meanwhile, PCP supplementation ameliorated hepatic oxidative stress by decreasing malonaldehyde content and increasing the activities of superoxide dismutase and glutathione peroxidase in db/db mice. Furthermore, PCP treatment reduced obesity and food intake in db/db mice, and improved dyslipidemia demonstrated by increasing high-density lipoprotein cholesterol (HDL-C) while decreasing total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (HDL-C). All doses of PCP treatment decreased the values of LDL-C/HDL-C in a dose-response relationship.

Conclusion

PCP significantly alleviated hyperglycemia, hyperinsulinemia, hyperlipidemia, and obesity, inhibited hepatic oxidative stress, and enhanced hepatic glucose transport in T2DM mice. Based on the above findings, the hypoglycemic effect of PCP may be attributed to the activation of the GLUT2/GCK expression in the liver and the reduction of hepatic oxidative stress.

Introduction

Diabetes mellitus (DM) is a chronic metabolic disease featured by hyperglycemia, which ranks in the top 10 causes of death worldwide. According to the report from the International Diabetes Federation, diabetes may increase from 463 million to 700 million by 2045 [1, 2]. Diabetes has become a severe health problem worldwide due to its high prevalence, disability, and mortality [3]. The characteristic symptoms of diabetes are polyuria, polydipsia, polyphagia, hyperhidrosis, and unexpected weight loss, in addition to hyperglycemia and abnormalities in serum lipids [4]. If not strictly controlled, chronic hyperglycemia can cause organ damage and serious complications such as angiocardiography, neuropathy, and nephropathy [5]. Type 2 diabetes mellitus (T2DM) is the most common form of diabetes, accounting for 90–95% of the diabetic population [6]. The etiology and pathogenesis of T2DM are not clear, but the distinctive pathophysiological feature is the reduced ability of insulin to regulate glucose metabolism manifested as insulin resistance (IR), and/or relatively reduced insulin secretion due to defective islet β-cell function.

Obesity/overweight, genetics, environment, and sedentary lifestyle may be factors associated with T2DM. A cross-sectional survey from China showed that the prevalence of diabetes among normal-weight, overweight, and obese people was 8.8%, 13.8%, and 20.1% respectively [7]. Moderate weight loss in overweight or obese people with T2DM can improve glycemic control and IR, as well as reduce the need for glucose-lowering medication [8]. Additionally, there is compelling evidence that oxidative stress contributes to more severe T2DM by reducing insulin secretion and function. Chronic hyperglycemia leads to the production of excess reactive oxygen species (ROS) in the body causing oxidative stress, which can result in disruption of insulin signaling in target tissues, as well as exacerbate IR and T2DM [9]. Several studies have shown that diabetic patients tend to exhibit higher malonaldehyde (MDA) content and lower superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity in serum and tissues. Furthermore, the levels of oxidative stress markers were reduced after treatment in patients with T2DM [10]. Therefore, the attenuation of oxidative stress is considered an effective strategy to improve IR and T2DM.

Most patients with T2DM require long-term oral medications or insulin injections to control blood glucose, however, their administration may be accompanied by adverse effects including gastrointestinal symptoms, hepatorenal functions, and hypoglycemia [11]. Thus, it is vital to search for natural, safe, and effective substitutes for these medicines.

Chinese edible herbal has the advantage of being more effective and having fewer side effects [12]. Penthorum chinense Pursh (PCP) is a traditional food and medicine widely distributed in East Asian countries. It has been demonstrated to possess potent antioxidant capabilities protecting against hepatic oxidative stress induced by alcohol and carbon tetrachloride [13, 14], but its effect on oxidative stress associated with T2DM has not been reported. In vitro studies found that ethyl acetate extract from PCP activates the Nrf2 antioxidant pathway by binding to Keap1 protein, and significantly reduces the vascular inflammation induced by high glucose levels [15]. Meanwhile, a previous study had shown that PCP possessed a modest hypoglycemic effect by increasing insulin secretion in diabetic rats induced by streptozotocin (STZ) [16]. STZ is a broad-spectrum antibiotic with selective destruction of islet β-cells and is used to induce T1DM or T2DM models by varying doses of it. However, there is a large proportion of diabetic patients characterized by IR, whose insulin levels are equal to or even higher than normal. Consequently, the treatment strategy for this population is to increase insulin sensitivity rather than promote its secretion.

Genetic models, especially db/db mice, are regarded as the closest animal models to human T2DM with IR, which absorb excessive food chronically and spontaneously develop symptoms of obesity, IR, and hyperglycemia [17]. They are more like clinical patients in terms of diabetes production due to their lack of interference from artificially induced factors. To our knowledge, no study has investigated the hypoglycemic effects of PCP on db/db mice. This study aimed to observe the effects of PCP on hyperglycemia, IR, obesity, and oxidative stress in spontaneously T2DM mice, as well as explore the underlying mechanisms. The results of our study will provide more evidence for its application in the treatment of diabetes.

Materials and methods

Herbal materials and preparation

Dried PCP was obtained from Gulin Hongan Pharmaceutical Co., Ltd. (Luzhou, China). PCP samples were ground into powder and filtered through a 65-mesh sieve. Five hundred grams of the powder was immersed in 5000 mL ultra-pure water for 1h and exhaustively extracted at 100°C for 3 rounds. The three filtrates were mixed, concentrated to 1 g/mL, and refrigerated at 4°C.

Animal experiments

All animal experiments were approved by the Ethics Committee of Southwest Medical University (No.201910-235). The 8-week-old male db/db and db/m mice were provided by Changzhou Cavens Co., Ltd. (Changzhou, China). All animals were fed in the SPF Animal Laboratory Center of Southwest Medical University at 12 h light/dark cycle, (23±2) °C, and (60±5) % relative humidity. All mice accessed food and water freely, whose padding was changed daily to keep them dry.

After seven days of acclimatization, the fasting blood glucose (FBG) was monitored by glucometer and glucose test strips (Roche Co., Germany). The db/db mice detected with FBG over 11.1 mmol/L at three executive mornings were defined as diabetic mice. Eight db/m mice were involved in the normal control (NC) group and forty diabetic db/db mice were randomly divided into four groups: model control (MC) group, low dose of PCP (LPCP) group, medium dose of PCP (MPCP) group and high dose of PCP (HPCP) group. The LPCP, MPCP, and HPCP groups were administered with a dose of 300, 600, and 1200 mg/kg/d, respectively. Allometric scaling to a person with a body weight of 60 kg, these doses correspond to 2-, 4-, and 8-gram PCP daily. 8 grams of PCP daily is the recommended upper intake level for humans. Only ultrapure water was given to the animals in the NC and MC. All treatments were administered by gavage once per day for four weeks.

Food consumption and body weight were measured weekly. All animals were given glucose (2g/kg) when an oral glucose tolerance test (OGTT) was conducted, and tail venous blood was collected at 0, 30th, 60th, and 120th minutes after oral gavage to measure blood glucose concentration.

Biochemical analysis

After 4 weeks of intervention, mice were fasted the night before blood and tissue collection, then anesthetized with 1% sodium pentobarbital solution (1.0 mL/100g) according to Animal Veterinary Medical Association Guidelines for the Euthanasia of Animals, and executed. Glycated hemoglobin A1c (HbA1c) and insulin were detected by ELISA kits (Milian Co., Ltd., Shanghai, China). IR is indicated by the homeostasis model assessment of insulin resistance (HOMA-IR) with the calculation formula of FBG (mmol/L) × serum insulin (μU/mL)/22.5. The content of serum lipids and the hepatic antioxidant enzyme was tested by commercial kits (Jiancheng Co., Ltd., Nanjing, China).

Hematoxylin and eosin staining in liver tissues

Biochemical Analyses. Liver tissue was fixed in a 4% paraformaldehyde solution and embedded in paraffin wax. The slices were sliced, dewaxed, hematoxylin, and eosin stained (H&E), and sealed in sequence. The vacuolar degeneration and steatosis of the hepatic tissue were observed under a light microscope.

Western blotting

Liver tissue was lysed with RIPA buffer to produce lysate. Proteins were divided on 10% SDS-PAGE gels and moved to the PVDF membranes (Hybond, United States). The membranes were blocked with 5% skimmed milk for 120 minutes before being incubated with the primary antibody, and secondary antibody in sequence (Abcam, United Kingdom). Finally, the protein bands were tested by chemiluminescence kits (Affinity, United States) with an ECL chemiluminescence system (Tanon-5200Multi, Shanghai, China).

Statistical analysis

All data were analyzed by SPSS 22.0 statistical software and the results are presented as the mean ± standard deviation (SD). One-way ANOVA analysis was performed to compare the mean differences among multiple groups. In pairwise comparison, the LSD test was used for those with homogeneous variances, and the Dunnett T3 test was used for those with uneven variances. A P-value of less than 0.05 was deemed statistically significant.

Results

PCP improved parameters related to hyperglycemia and IR in db/db mice

According to Fig 1A, the FBG in the NC was significantly lower than those in diabetic mice no matter before or after the intervention (P<0.01). In the second week, only the HPCP showed a significant decrease in FBG levels compared to MC (P<0.05). In the fourth week, the levels of FBG in all PCP-treatment groups significantly decreased to varying degrees compared to the MC (P<0.05). Compared with the MC, the HbA1c in diabetic mice of the LPCP, MPCP, and HPCP decreased in varying degrees, but with no significant difference (Fig 1B).

The results of OGTT were shown in Fig 1C. The levels of blood glucose among diabetic mice were always higher than that of the NC (P<0.01). After intragastric administration, the blood glucose of diabetic mice increased rapidly and reached the maximum values at the 30th minute. The blood glucose values in the HPCP were significantly lower than those in the MC at the 60th and 120th min (P<0.01, P<0.05). Additionally, high-dose PCP treatment significantly lowers the AUC of OGTT compared to that in the MC (Fig 1D, P<0.01).

The levels of insulin and HOMA-IR in each group were depicted in Fig 1E and 1F. Insulin and HOMA-IR of diabetic mice increased to different degrees when compared with the NC (P<0.01). Serum insulin was significantly alleviated after treatment with MPCP and HPCP in comparison to the MC group (P<0.05, P<0.01). Meanwhile, the HOMA-IR was significantly decreased by HPCP (P<0.05).

PCP alleviated obesity in db/db mice

After PCP treatment, the food intake of diabetic mice was decreased in various degrees when compared to the MC (Fig 2A). The food intake of the HPCP was consistently significantly lower than the MC from the 1st to 4th week (P<0.05, P<0.01), which of the MPCP was significantly decreased compared with the MC from the 2nd to 4th week after the intervention (P<0.05, P<0.01). Initial body weights of diabetic mice were of no significant differences (Fig 2B). After 2 weeks of PCP treatment, the body weights of diabetic mice in PCP-treatment groups were significantly lower than the MC (P<0.05, P<0.01).

Both medium- and high-dose PCP treatments significantly decreased serum TG, while increased HDL-C (P<0.01, P<0.05) (Fig 2C–2F). Additionally, TC and LDL-C were significantly reduced in the HPCP (P<0.05, P<0.01). TG, TC, and LDL-C in LPCP were decreased by 8.84%, 11.06%, and 11.47%, as well as HDL-C was increased by 7.23%, but the changes failed to reach statistical significance after low-dose of PCP treatment (P>0.05). The results of lipid ratio parameters were shown in Fig 2G–2I. Those in the NC, including TC/HDL-C, TG/HDL-C, and LDL-C/HDL-C were significantly higher than those in the diabetic db/db mice (P<0.01). The values of TG/HDL-C in the MPCP and HPCP were significantly lower than those in the MC (P<0.05), while those of LPCP were not significantly different from those of the MC. However, the values of TG/HDL-C in the LPCP were significantly higher than those in the HPCP (P<0.05). All doses of PCP treatment decreased the values of LDL-C/HDL-C in a dose-response relationship (P<0.05), demonstrated by significantly lower values of LDL-C/HDL-C in the HPCP than those in the LPCP and MPCP (P<0.01).

PCP improved hepatic steatosis in db/db mice

H&E stains of the liver tissue were beneficial in evaluating hepatic cell fatty degeneration and injuries (Fig 3A and 3B). In the NC group, the hepatocytes with normal morphology were arranged neatly, and the structures of the hepatic lobules and sinus were intact and clear. Conversely, the hepatocyte cords in the MC group were disorganized and massive hepatocytes with hepatic steatosis or vacuolar degeneration were observed under the light microscope. After four weeks of treatment, the results showed that all doses of PCP had extremely ameliorated fatty or vacuolar degeneration in hepatic cells when compared with the MC group. In the LPCP, there was no steatosis in hepatocytes, although lots of vacuolar degeneration was found. In the MPCP, vacuolar degeneration in hepatocytes was significantly reduced. The hepatocytes in HPCP were arranged neatly and a little microvacuolar degeneration in hepatocytes was observed.

PCP improved hepatic oxidative stress in db/db mice

Significant increase of activities of SOD in diabetic mice after medium- and high-dose of PCP treatment (P<0.01), together with a reduction of MDA in all PCP-treatment groups were presented by comparison with the MC (P<0.05, P<0.01) (Fig 4A–4C). Additionally, the LPCP, MPCP, and HPCP were found to increase GSH-Px activities of db/db mice by 2.31%, 3.31%, and 5.68%, respectively, when compared with the MC, but no significant differences were found among MC and PCP-treatment groups.

Fig 4 — Effects of PCP on enzymatic antioxidant activities of (A) SOD and (B) GSH-Px, and the lipid peroxidation levels of (C) MDA in the liver of *db/db* mice. Data are expressed as mean ± SD; n = 8 for each experimental group. *P<0.05, **P<0.01 vs. normal control group. ^#P<0.05, ^##P<0.01 vs. model control group.

PCP upregulated the protein expression of GLUT2 and GCK in db/db mice

As shown in Fig 5A and 5B, the protein expression of GLUT2 and GCK was significantly lower in diabetic db/db mice by comparison to the NC (P < 0.01, P < 0.05). However, treatment with PCP significantly increased the expression of GLUT2 and GCK compared to the MC (P < 0.01, P < 0.05). All doses of PCP increased GLUT2 protein expression in the liver, with HPCP being significantly more effective in upregulating GLUT2 expression than MPLP and LPCP (P < 0.01). Compared with the MC group, the relative expression of GCK protein increased by 22.55%, 28.51%, and 33.69% in the LPCP, MPCP, and HPCP groups, respectively (P < 0.05, P < 0.01), but it was not significant in PCP-treatment groups.

Discussion

T2DM is characterized by abnormalities in glucose, lipid, and protein metabolism, as well as impairment in insulin secretion and/or IR [18]. In the present study, we demonstrated that PCP significantly reduced FBG, fasting insulin levels, and body weight in db/db mice, and ultimately ameliorated oral glucose tolerance, IR, dyslipidemia, and obesity. Meanwhile, we found that PCP inhibited hepatic oxidative stress and steatosis by increasing SOD and GSH-Px activities and reducing MDA content, with the high dose of PCP being significantly more effective. Mechanistically, the improvement of FBG and IR in db/db mice by PCP may be attributed to the activation of the GLUT2/GCK expression and reduction of hepatic oxidative stress.

Genetic models and a high-fat diet combined with low-dose STZ-induced models are probably the most used models of T2DM at present. Db/db mice, due to gene mutation of the leptin receptor, are insensitive to leptin and absorb excessive food chronically, spontaneously developing symptoms of obesity, IR, and hyperglycemia within the 8th week [19]. In comparison tests between the two models, db/db mice kept hyperglycemia in the whole experiment, whereas the blood glucose of STZ-induced models spontaneously declined after three weeks [20]. It was possible because a part of islet β-cells could repair themselves after STZ failure, which may affect the outcome of the experiment. Therefore, db/db mice were chosen for this study to remove the interference of human intervention on the results.

Insulin, secreted by islet β-cells located in the central part of the pancreas, is the only hormone with a hypoglycemic effect in the body [21]. The fasting serum insulin of db/db mice significantly declined after the intervention of PCP, and HOMA-IR was negatively correlated with the dose of PCP, which suggested that PCP significantly reduced IR in diabetic mice. Our findings were different from Huang’s [16], which considered that PCP could enhance insulin secretion instead of reducing IR in diabetic rats. We further analyzed the reasons, which may be the different animal models chosen for the two studies. Their models were established by STZ, which is selectively toxic to the β-cells, to build diabetic models by disrupting the β-cells function and reducing insulin secretion [22]. In contrast, db/db mice are chronically overfed resulting in obesity, IR, and hyperglycemia, which occurs due to reduced insulin sensitivity rather than decreased insulin secretion [23].

IR is manifested by a decrease in insulin sensitivity that the normal amount of insulin secreted is insufficient to move glucose into target tissues including the liver, adipose, and muscle, which are commonly involved in the regulation of whole-body fuel metabolism [24, 25]. Previous studies have consistently concluded that PCP has a unique protective effect on the liver, which may be its target tissue [26]. The liver, a primary organ of glucose metabolism, is responsible for maintaining blood glucose homeostasis in the body. In hepatocytes, IR is reflected by a decrease in glucose uptake and utilization, a reduction in hepatic glycogen synthesis, and an increase in hepatic glucose production and output, which subsequently leads to fasting hyperglycemia [27]. Non-alcoholic fatty liver disease (NAFLD) is the most common liver injury in diabetes and is characterized by excessive lipid deposition in hepatocytes. According to statistical data, the incidence of NAFLD in the general adult is 20–30%, but it occurs in obese and diabetic patients with an incidence of up to 70–80%. NAFLD is believed to promote insulin resistance and type 2 diabetes to form a vicious circle [28]. In this study, we observed that PCP significantly reduced liver steatosis or vacuolar degeneration in db/db mice. Similarly, Guo et al. [29] found that PCP exerted protective effects against hepatic steatosis and oxidative stress by enhancing the expression of AMP-activated protein kinase (AMPK). It has been demonstrated that the activation of AMPK in the liver can promote glucose metabolism and IR [30]. AMPK is a serine/threonine protein kinase, and the activated AMPK can increase glucose transporter protein expression and glucose uptake, and reduce IR to a greater degree [31]. Therefore, we further examined liver enzymes associated with glucose metabolism. GLUT2 and GCK are mainly secreted exclusively by hepatocytes and work together to maintain hepatic glucose metabolism homeostasis. GLUT-2 transports glucose in hepatic parenchymal cells, but the rate of glucose uptake is limited by the rate of glucose phosphorylation catalyzed by GCK [32]. Increasing the activity of GLUT2 and GCK inhibits hepatic glucose output and promotes glucose phosphorylation, hepatic glycogen synthesis, and aerobic metabolism of glucose in hepatocytes [33]. In the present study, PCP treatment significantly increased the expression of GLUT2 and GCK in db/db mice, with a high dose of PCP showing the best therapeutic effect and a dose-response relationship between the three groups. Based on the above findings, we proposed the hypothesis that PCP may decrease FBG and improve IR by upregulating the expression of GLUT2 and GCK to promote hepatic glucose uptake and utilization.

Hyperglycemia and IR are intimately linked to the overproduction of ROS and oxidative stress [34]. Sun et al. [15] found polyphenols from PCP show strong protective effects against high glucose-induced vascular inflammation by scavenging ROS and enhancing antioxidant activity, suggesting it to be a potential resource for anti-diabetes, which is consistent with our findings. Previous studies have shown that patients with T2DM produce large amounts of ROS during chronic hyperglycemia and that the liver is a major organ of ROS attack, disrupting its ability to scavenge free radicals and creating oxidative stress, thereby causing abnormalities in the insulin signaling pathway [35]. Therefore, the reduction of hyperglycemia-associated oxidative stress is considered to be an effective strategy to improve IR and T2DM [36]. Oxidative stress is usually assessed by measuring parameters including MDA, SOD, and GSH-Px. MDA, a product of lipid peroxidation, indirectly reflects the severity of free radical attack. SOD is responsible for catalyzing the breakdown of free radicals and reducing superoxide levels, which reflects the ability to scavenge free radicals [37]. The main role of GSH-Px is to protect the structural and functional integrity of cell membranes and to reduce the damage and attack of superoxide on the body [38]. In this study, the activities of SOD and GSH-Px were augmented and the levels of MDA were decremented in T2DM mice after PCP treatment, which is consistent with previous findings [39, 40]. These findings demonstrated that PCP can improve free radical scavenging ability by increasing SOD and GSH-Px activity and decreasing MDA content, thereby preventing liver damage under hyperglycemic conditions and regulating blood glucose and IR levels.

In addition to severe hyperglycemia, db/db mice exhibit severe symptoms including overeating and obesity [41]. There is strong evidence that obesity is among the most prominent hazard factors for IR and T2DM [42, 43]. Administration of PCP could potently reduce food consumption and alleviate body weight. No study discussed the effect of PCP on overeating and obesity in db/db mice before our research. However, Li et al. found daily oral administration of PCP for 8 weeks decreased body weight, dyslipidemia, and insulin resistance in high-fat diet-induced non-alcoholic fatty liver disease (NAFLD) mice by regulating the gut microbiota and bile acid metabolism [44]. Furthermore, quercetin considered one of the key ingredients for PCP has been reported to restrain body weight gain and food intake in obese mice [45, 46]. We supposed that the anti-obesity effect of PCP might be moderately attributable to quercetin, but the hypothesis needs to be validated by further studies.

However, there are some limitations to our study. Firstly, PCP was only approved to be consumed in the form of tea bags in China. Therefore, db/db mice were selected to be intervened with an aqueous extract of PCP to simulate the way of human consumption. Secondly, in consideration of the fact that the markers of PCP are still unclear, we have not purified and identified its components. We will explore more deeply to determine the active components of PCP in alleviating hyperglycemia and IR. Finally, HbA1c in PCP treatment groups was lower than the MC, but no significant difference was observed. HbA1c is formed by continuous and irreversible glycosylation reaction and reflects the blood glucose levels in the previous two or three months [47]. We considered that the intervention time was too short to change HbA1c levels. Whether PCP treatment with a prolonged experimental period can improve the HbA1c of db/db mice needs further study.

Conclusion

In summary, our results showed that PCP treatment decreased FBG and IR in T2DM mice. Moreover, it inhibited oxidative stress by promoting SOD and GSH-Px activities and reducing the MDA content. In addition, our results also showed that PCP regulated glucose transport and absorption by upregulating GLUT2 and GCK protein expression. PCP dose-dependently improved the parameters of oxidative stress and upregulated GLUT2 and GCK protein expression in comparison to the model group. Therefore, a high dose of PCP is more effective in alleviating IR, inducing hypoglycemic effects, and scavenging free radicals. The present study offers a new understanding of the health-promoting role of PCP and its potential significance in the therapy of T2DM.

Supporting information

S1 Raw data

(PDF)

pone.0311502.s001.pdf^{(57KB, pdf)}

S1 Fig. Original image of western blot analysis.

(PDF)

pone.0311502.s002.pdf^{(107.8KB, pdf)}

Data Availability

All raw data and blot/gel images have been uploaded as Supporting information.

Funding Statement

This research was funded by grants from the Key Research and Development of the Science and Technology Plan Project of Luzhou City (2012-NYF-17), the Joint Research Project of the General Hospital of Western Theater Command (KY201900), and the Sichuan Cadre Healthcare Research Project (No. 2021-1303).

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PLoS One. doi: 10.1371/journal.pone.0311502.r001

Decision Letter 0

Mehran Rahimlou

28 Jun 2024

PONE-D-24-18781Ameliorative Effects of Penthorum chinense Pursh on Insulin Resistance and Oxidative Stress in diabetic obesity db/db micePLOS ONE

Dear Dr. Hu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Aug 10 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're 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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Additional Editor Comments:

Dear Dr. Jilei Hu

Based on the comments received from the reviewers regarding this manuscript, we request you to revise your manuscript based on the comments raised by the reviewers.

Many thans

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

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Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

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Reviewer #1: 1.A few grammatical and typographical errors noted. For instance, ANIMALS EXPERIMENT, instead of ANIMAL EXPERIMENTS

2. De Ritis ratio and Ferritin could have been included

3. The authors are advised to compute lipid ratios, in view of their link with both diabetes mellitus and steatosis

4. Was the ELISA kit for HbA1c ,a high sensitivity and specificity one with minimal cross reactivity?? Why did the authors not use HPLC ???

Reviewer #2: 1. Language should be improved and spelling mistakes should be corrected.

2. Please abbreviate the text at first instance.

3. Give 2-3 lines about current status of diabetes at global level.

Brauer, Michael, Roth, Gregory A., Aravkin, Aleksandr Y., Zheng, Peng, Abate, Kalkidan Hassen, Abate, Yohannes Habtegiorgis, Abbafati, Cristiana, Kavita Munjal et al., 2024. Global burden and strength of evidence for 88 risk factors in 204 countries and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021. The Lancet. 403, 2162-2203. https://doi.org/10.1016/S0140-6736 (24)00933-4.

Gauttam, V., Munjal, K., Mujwar, S., Sawale, J., Rohilla, M., Gupta, S. 2022. Comparative Study of Developed Formulation and Market Formulation for Antidiabetic Potential in Alloxan Induced Diabetic Wistar Rats. Journal of Young Pharmacists. 2022; 14(4), 387-393.

Reviewer #3: In this manuscript, the authors demonstrated that PCP significantly alleviated hyperglycemia, hyperinsulinemia, hyperlipidemia, and obesity, inhibited hepatic oxidative stress, and enhanced hepatic glucose transport in T2DM mice. Generally, the paper is well written and the results are of some scientific significance. I recommend this paper to be accepted after minor revision.

Minor points:

1. The inter-group significance shown in the picture is inconsistent with the description in the text. For example, in the result section ‘PCP improved parameters related to hyperglycemia and IR in db/db mice’ (Line 186), according to the figure, it should be ‘P<0.01’?

2. In Fig5-B, the significance may be wrong, especially HPCP Group. Please check through the manuscript to avoid these errors.”.

**********

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Reviewer #1: Yes: A.R.SRINIVASAN

Reviewer #2: Yes: Dr. Kavita Munjal

Reviewer #3: No

**********

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Attachment

Submitted filename: Accept after minor revision.docx

pone.0311502.s003.docx^{(12.4KB, docx)}

PLoS One. 2024 Oct 7;19(10):e0311502. doi: 10.1371/journal.pone.0311502.r002

Author response to Decision Letter 0

3 Aug 2024

Dear Editor and Reviewers,

We sincerely thank you for your valuable feedback which helped to improve the quality of our manuscript entitled “Ameliorative Effects of Penthorum chinense Pursh on Insulin Resistance and Oxidative Stress in diabetic obesity db/db mice” (Manuscript ID: PONE-D-24-18781). We have considered your comments word by word and have made revisions based on your suggestions question by question. We have amended the format according to the Journal Requirements, and the details can be found in the flie of Point-by-point response to reviewers and editors.The reviewers’ comments are listed in blue italics and specific concerns have been numbered. Our response is given in normal font and changes/additions to the manuscript are marked in red. The main amendments in the paper and the responses to your comments are listed as follows:

For reviewer 1#

1. A few grammatical and typographical errors were noted. For instance, ANIMALS EXPERIMENT, instead of ANIMAL EXPERIMENTS.

Response: Thank you for your meticulousness. We felt sorry about this low-grade mistake and the amendment is in red.

2. De Ritis ratio and Ferritin could have been included.

Response: Thank you for your advice. Penthorum chinense Pursh (PCP) is widely utilized in China to treat a variety of liver diseases. Previous studies have demonstrated that the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were ameliorated in a dose-dependent manner by PCP [1-4]. Therefore, we did not measure liver function-related indicators in this study and unfortunately, we were unable to calculate the De Ritis ratio either. As for ferritin, there is no published literature report on the effect of PCP on it. However, our team's ongoing but unpublished study suggests that total flavonoids of PCP were able to significantly attenuate liver injury of iron-overloaded rats by up-regulating the expression of divalent metal ion transporter protein 1 and transferrin receptor 1. Of course, if we can get new funds, we shall carry out more experiments to dig into the effect of PCP on ferritin.

3. The authors are advised to compute lipid ratios, because of their link with both diabetes mellitus and steatosis.

Response: Thank you for your advice. T2DM patients often present with characteristic plasma lipid and lipoprotein abnormalities, including low HDL-C, high LDL-C, and elevated TC and TG levels [5], which is consistent with our findings. Considering the reviewer’s suggestion, we further analyzed the lipid ratio parameters, including TC/HDL-C, TG/HDL-C, and LDL-C/HDL-C, that considered could predict the risk of T2DM complications by prior studies [6], especially for coronary heart disease. The results of lipid ratio parameters are shown in Fig 2G-I. Those in the NC, including TC/HDL-C, TG/HDL-C, and LDL-C/HDL-C were significantly higher than those in the db/db mice (P<0.01). The values of TG/HDL-C in the MPCP and HPCP were significantly lower than those in the MC (P<0.05), while those of LPCP were not significantly different from those of the MC. However, the values of TG/HDL-C in the LPCP were significantly higher than those in the HPCP (P<0.05). All doses of PCP treatment decreased the values of LDL-C/HDL-C in a dose-response relationship (P<0.05), demonstrated by significantly lower values of LDL-C/HDL-C in the HPCP than those in the LPCP and MPCP (P<0.01).

4. Was the ELISA kit for HbA1c, a high-sensitivity and specificity one with minimal cross-reactivity? Why did the authors not use HPLC?

Response: Thank you for your question. According to the literature, various methods developed for HbA1c examination include immunoassay, boronated affinity, enzymatic, capillary electrophoresis, and Ion-Exchange High-Performance Liquid Chromatography (IE-HPLC) [7], and each has its advantages and limitations. Currently, the two most used methods for HbA1c detection are IE-HPLC and immunoassay, and the results of them are in strong agreement in most cases. In addition to its advantages in detecting HbA1c, IE-HPLC methods can also detect other types of hemoglobin and the presence of hemoglobin variants, which are widely used in clinical practice. However, we did not choose IE-HPLC due to its high price. ELISA kits working on the principle of immunoassay, are one of the methods with high sensitivity, stable efficacy, and low price for the determination of HbA1c, which are widely used in animal experiments [8]. In summary, we finally chose the ELISA kits to measure the HbA1c levels of db/db mice.

For reviewer 2#:

1. Language should be improved and spelling mistakes should be corrected.

Response: Thank you for your suggestion. We have invited a native English speaker to help polish our manuscript, and we hope the revised manuscript will be acceptable to you.

2. Please abbreviate the text at the first instance.

Response: Thank you for your suggestion. We felt sorry about this low-grade mistake and now all full names with their abbreviations have been added when they first appear. The amendment is in red.

3. Give 2-3 lines about the status of diabetes at the global level.

Response: Thank you for providing valuable references. We have checked the literature carefully and added more description about the status of diabetes at the global level in the INTRODUCTION part of the revised manuscript (Lines 57-60).

For reviewer 3#:

Response: Thank you for your meticulousness. We felt sorry about this low-grade mistake and the amendment is in red.

2. In Fig5B, the significance may be wrong, especially HPCP Group. Please check through the manuscript to avoid these errors.

Response: Thank you for your meticulousness. We have checked and reanalyzed the raw data and unfortunately, the results and significance were real. As shown in Fig 5B, when compared to the MC, the relative expression of GCK protein increased in the LPCP, HPCP (P < 0.05), and MPCP (P < 0.01), respectively. However, there was no significant difference in multiple comparisons between the three PCP-treatment groups. We considered the following two possible reasons. Firstly, a smaller p-value does not mean that the difference is larger, only that more reason to think that there is a difference between the two groups. In this study, the relative expression of GCK protein in the MPCP and HPCP was significantly higher than the MC, but the p-value was smaller in the MPCP, which only means that there was more reason to think that the MPCP is different from the MC. Secondly, the above results may be due to the relatively small sample size of the experiment.

Finally, we would like to express our thanks again for your reviews and guidance on our manuscript. We sincerely hope that with the help of your knowledge, we can complete an excellent paper and we also hope our paper can be published in your journal.

Thank you and best regards.

Yours Sincerely,

Ning Lin

Clinical Nutrition, The General Hospital of Western Theater Command, Sichuan, Chengdu 610083, China.

Email: helenmedic@yeah.net

August 3, 2024

Reference

1. Du YC, Lai L, Zhang H, Zhong FR, Cheng HL, Qian BL, et al. Kaempferol from Penthorum chinense Pursh suppresses HMGB1/TLR4/NF-κB signaling and NLRP3 inflammasome activation in acetaminophen-induced hepatotoxicity. Food & function. 2020;11(9):7925-34.

2. Jiang Y, Zhong M, Zhan H, Tao X, Zhang Y, Mao J, et al. Integrated strategy of network pharmacology, molecular docking, HPLC-DAD and mice model for exploring active ingredients and pharmacological mechanisms of Penthorum chinense Pursh against alcoholic liver injury. Journal of ethnopharmacology. 2022;298:115589.

3. Wang S, Li W, Liu W, Yu L, Peng F, Qin J, et al. Total flavonoids extracted from Penthorum chinense Pursh mitigates CCl(4)-induced hepatic fibrosis in rats via inactivation of TLR4-MyD88-mediated NF-κB pathways and regulation of liver metabolism. Frontiers in pharmacology. 2023;14:1253013.

4. Zhang H, Cui X, Liu W, Xiang Z, Ye JF. Regulation of intestinal microflora and metabolites of Penthorum chinense Pursh on alcoholic liver disease. Frontiers in pharmacology. 2023;14:1331956.

5. Bahiru E, Hsiao R, Phillipson D, Watson KE. Mechanisms and Treatment of Dyslipidemia in Diabetes. Current cardiology reports. 2021;23(4):26.

6. Gu X, Yang X, Li Y, Cao J, Li J, Liu X, et al. Usefulness of Low-Density Lipoprotein Cholesterol and Non-High-Density Lipoprotein Cholesterol as Predictors of Cardiovascular Disease in Chinese. The American journal of cardiology. 2015;116(7):1063-70.

7. Rhea JM, Molinaro R. Pathology consultation on HbA(1c) methods and interferences. American journal of clinical pathology. 2014;141(1):5-16.

8. Huang D, Jiang Y, Chen W, Yao F, Huang G, Sun L. Evaluation of hypoglycemic effects of polyphenols and extracts from Penthorum chinense. Journal of ethnopharmacology. 2015;163:256-63.

Attachment

Submitted filename: Point-by-point response to reviewers and editors.docx

pone.0311502.s004.docx^{(179KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0311502.r003

Decision Letter 1

Mehran Rahimlou

20 Sep 2024

Ameliorative Effects of Penthorum chinense Pursh on Insulin Resistance and Oxidative Stress in diabetic obesity db/db mice

PONE-D-24-18781R1

Dear Dr. Jilei Hu ,

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

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Reviewer #1: Ameliorative Effects of Penthorum chinense Pursh on Insulin Resistance and Oxidative Stress in diabetic obesity db/db mice is a sound attempt. More light can be thrown by associating the markers of oxidative stress with the available and objective indicators of insulin sensitivity/insulin resistance, both dependent and independent of lipid status

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PLoS One. doi: 10.1371/journal.pone.0311502.r004

Acceptance letter

Mehran Rahimlou

27 Sep 2024

PONE-D-24-18781R1

PLOS ONE

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Associated Data

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Supplementary Materials

S1 Raw data

(PDF)

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S1 Fig. Original image of western blot analysis.

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pone.0311502.s004.docx^{(179KB, docx)}

Data Availability Statement

All raw data and blot/gel images have been uploaded as Supporting information.

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PERMALINK

Ameliorative effects of Penthorum chinense Pursh on insulin resistance and oxidative stress in diabetic obesity db/db mice

Jilei Hu

Leyu Zheng

Xi Fan

Hongmei Lang

Huibo Xie

Ning Lin

Roles

Abstract

Background

Methods

Results

Conclusion

Introduction

Materials and methods

Herbal materials and preparation

Animal experiments

Biochemical analysis

Hematoxylin and eosin staining in liver tissues

Western blotting

Statistical analysis

Results

PCP improved parameters related to hyperglycemia and IR in db/db mice

Fig 1.

PCP alleviated obesity in db/db mice

Fig 2.

PCP improved hepatic steatosis in db/db mice

Fig 3.

PCP improved hepatic oxidative stress in db/db mice

Fig 4.

PCP upregulated the protein expression of GLUT2 and GCK in db/db mice

Fig 5. Effect of PCP on glucose metabolism-related enzyme activities in the liver of db/db mice.

Discussion

Conclusion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Mehran Rahimlou

Roles

Author response to Decision Letter 0

Decision Letter 1

Mehran Rahimlou

Roles

Acceptance letter

Mehran Rahimlou

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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