Protective effect of rivastigmine against lung injury in acute pancreatitis model in rats via Hsp 70/IL6/ NF-κB signaling cascade

Abstract

Acute lung injury (ALI) that develops as a result of AP can progress to acute respiratory distress syndrome. Some hypotheses are proposed to explain the pathophysiology of AP and its related pulmonary hazards. This experiment aimed to evaluate the mitigating action of rivastigmine (Riva) in lung injury that occurs on the top of acute pancreatitis (AP) induced in rats. Thirty-two male Wister rats were randomized to one of four groups: control, Riva-treated, acute pancreatitis (AP), and acute pancreatitis treated by Riva. Serum amylase and lipase levels were assessed. Pulmonary oxidative stress and inflammatory indicators were estimated. A pancreatic and pulmonary histopathological examination, as well as an immunohistochemical study of HSP70, was carried out. Riva significantly attenuated the L-arginine-related lung injury that was characterized by increased pulmonary inflammatory biomarkers (interleukin-6 [IL‐6]), nuclear factor kappa B (NF‐κB), tumor necrosis factor‐α (TNF‐α), increased pulmonary oxidative markers (total nitrite/nitrate [NOx]), MDA, decreased total antioxidant capacity (TAC), and reduced glutathione level (GSH)) with increased caspase-3 expression. Therefore, Riva retains potent ameliorative effects against lung injury that occur on the top of AP by relieving oxidative stress, inflammation, and apoptosis via HSP70/IL6/NF-κB signaling.

Introduction

The most critical factor influencing death in people with severe acute pancreatitis (SAP) is organ failure. Acute respiratory distress syndrome (ARDS) may develop from acute lung injury (ALI) brought on by SAP. ¹ Respiratory failure, which accounts for roughly 37% of fatalities throughout the early and late stages of AP, is the most frequent type of organ failure. The lack of early organ failure prediction and management may be the primary factor behind the high mortality rate. Additionally, ARDS is mostly avoidable, and clinical outcomes may improve following effective therapies in the initial stages of ARDS. ² Furthermore, ARDS advancement in AP is considerably provoked by the increased production of many cytokines, and ALI is a result of an intense systemic inflammatory response. By boosting the transfer of bacterial endotoxins, the gut, which is made more permeable by a number of toxins, inflammatory mediators, and pancreatic enzymes, promotes lung damage through the gut–lymph–lung axis. ³

Tumor necrosis factor (TNF-α) and interleukins (IL-1 β and IL-6) are actually produced by activated neutrophils and macrophages. These cytokines, when bind to their receptors, start the AKT, ATAT3, and nuclear factor-kappa beta (NF-κβ) signaling cascades and exacerbate the pathogenic SAP-ALI process.^4,5 Besides, one of the most significant controllers of pro-inflammatory genes expression is NF-κβ. It controls the synthesis of cytokines like TNF-α and IL-6. ⁶

Acinar cells can be harmed, and multi-organ dysfunction can be exacerbated by severe inflammation and activated inflammatory cells, which can also increase reactive oxygen species (ROS) production and potentiate oxidative stress. ⁷ Accordingly, cell apoptosis, oxidative stress, and inflammation are the main contributors to the pathogenesis of SAP-ALI. ⁸ Several diseases are assessed using an arterial blood gas (ABG); one of them is ARDS, so ABGs are frequently ordered by emergency medicine, anesthesiology, and pulmonology clinicians. ⁹

Acetyl and butyrylcholinesterase inhibitors, such as rivastigmine (Riva), alter the inflammatory cascade and increase ACh to make up for the deficient Ach in neurodegenerative and inflammatory illnesses. Additionally, Riva showed antioxidant, anti-inflammatory, and anti-apoptotic properties that were effective in rheumatoid arthritis-infected rats.^10,11 Furthermore, Riva relieved bone marrow toxicity ¹² and ulcerative colitis in mice. ¹³ Shifrin and co-workers demonstrated that Riva can retain Ach, including the ACh generated by vagal nerve terminals, stimulating the nicotinic receptors in circulating macrophages, and suppressing the cytokine discharge. ¹³

Currently, there is no curative remedy for reducing SAP-ALI mortality. Consequently, the present experiment was conducted to examine the outcome of Riva, as one of the new therapeutic strategies, against induced AP and reveal the potential mechanisms of this alleviating function.

Materials and methods

Chemicals

Powdered L-arginine (Sigma, UT, USA) and RIVA (Novartis International AG Pharmaceutical Co.) were used. Amylase, lipase, total antioxidant capacities (TAC), reduced glutathione (GSH), and amylase (Bio-Assay, USA) were all tested colorimetrically using kits from Biodiagnostic, Egypt. We bought IL-6 and TNF ELISA kits from Elabscience Co. (Catalog numbers: EEL-R0015 96T and EEL-R0019, respectively). The ELISA Genie Co. provided NF-B ELISA kits with the catalog number RTFI00988. The Caspase-3 ELISA Kit (CSB-E08857r) was purchased from CUSBIO Co. Other chemicals were commercially obtained.

Animals

Before beginning the experiment, 32 mature male albino Wistar rats weighing 220–250 g were acquired from the National Research Center in Cairo, Egypt, and given 2 weeks for adjustment by having a usual hourly cycle of dark and light (12/12). The animals received regular rats’ diets (El-Nile Company, Egypt) and drinking water. All the experimental events were in accordance with our institutional guidelines. All of the experimental activities followed the institutionally established rules. The Laboratory Animals’ Maintenance and Usage Committee of the Faculty of Medicine, Minia University, established the protocol's ethics (Council No. 394:2022).

Sample size calculation and justification

Before the study, the number of animals was determined using the “resource equation” method. ¹⁴ According to this method, the value “E" represents the degree of freedom of the analysis of variance and should lie between 10 and 20. The formula used is as follows: E = Total number of animals−Total number of groups. The E value is set to 28 to accommodate any dropouts during this study.

Experimental design

The rats were randomized (8 rats in each) into the following groups:

• 1- Control group: each rat received the vehicle (saline).

• 2- Rivastigmine (Riva-treated group): for 2 weeks, rats received Riva (0.3 mg/kg) once per day by IP. ¹²

• 3- L-arginine-treated group: each rat was administered two IP injections of L-arginine (2.5 g/kg, 1 h apart) on the 14^th day. ¹⁴

• 4- Riva + L-arginine group: rats received both L-arginine + Riva as previously mentioned.

A final concentration of 500 mg/mL of L-arginine powder was created by dissolving it in 0.9% saline and using 5 N HCl to decrease the pH level to 7. ¹⁵

Biochemical analysis

Animals were sacrificed under urethane anesthesia (1.5 g/kg, IP) 24 h following AP induction. At room temperature, blood specimens were taken and centrifuged for 15 min at 5000 r/min. The resultant sera were kept at 80°C until further analysis. Serum amylase and lipase were quantified following the kit’s guidance.

Evaluation of arterial blood gas

Blood from the carotid artery was drawn into syringes that had been washed with heparin. The arterial blood gas analysis of the blood sample was performed to ascertain the partial pressures of oxygen and carbon dioxide (PaO2 and PaCO2, respectively) in the blood.

Analysis of lung homogenates

Lung tissues were taken from the animals after they had been sacrificed. One portion of each animal’s lung tissues was homogenized in cold potassium phosphate buffer (10 mm, pH 7.4) after being stored at 80°C. The ratio of homogenization buffer to tissue weight was 1:5. The homogenates were centrifuged for 10 min at 4°C at 5000 r/min. Malondialdehyde (MDA), TAC, GSH, total nitrate/nitrate (NOx), TNF-α, NF-kB, IL-6, and caspase-3 were all measured in the supernatant.

The key indicator of lipid peroxidation is the MDA level, which was tested using the Buege and Aust method. ¹⁶ By converting nitrate into nitrite using activated cadmium granules, followed by color development with Griess reagent in acidic media, NOx was employed as an indication of nitric oxide level. ¹⁷ TNF-α, NF-kB, IL-6, and caspase-3 were assessed according to the recommendations of the manufacturer. Finally, the remaining portion of the lung and part of the pancreas were fixed in 10% formalin for histological analysis.

Histological and immunohistochemical analysis

For histological examination, the tissues were cut into small portions and fixed in 10% formalin for 24 h. Samples were dehydrated, and paraffin blocks were made from which 5 um-thick sections were cut and stained with:

• • Hematoxylin and eosin (H&E).

• • Immunohistochemical staining to detect heat shock protein (HSP70) activity in the pancreas and lung cells; it is a mouse monoclonal antibody (Lab Vision Corporation Laboratories, USA).

For immunostaining, the specimens were deparaffinized in xylene, rehydrated, boiled for 10 min in citrate buffer (pH 6) for antigen retrieval, and cooled for 30 min. The sections were incubated with the primary antibody against HSP70 (1:100 dilution) for 1 h. Immunostaining was completed using the ultra-vision detection system. Then, the sections were counterstained with Mayer’s hematoxylin. The negative control was obtained by repeating the steps but substituting phosphate buffered saline for the primary antibody. ¹⁸

Morphometric analysis

For the study of HSP 70 immunoreactivity, five different fields were assessed randomly from five different animals at a magnification of 400, and the area% of the immunoreaction was measured using the image analyzer (image-j) in the pancreas and lung sections.

Statistical analysis

All data were displayed as means ± standard error of the mean (SEM) and analyzed using one-way analysis of variance (ANOVA) and the Tukey–Kramar post-analysis test. p-Values for significance were established at less than .05. For statistical computations, GraphPad Prism (version 5.01 for Windows; GraphPad Software, San Diego, California, USA; https://www.graphpad.com/) was implemented.

Results

Influence of Riva treatment on serum amylase and lipase

Being pancreatic damage indicators, serum amylase and lipase in the L-arginine group were significantly higher than those in the Riva and control groups. In contrast to the L-arginine group, Riva pre-administration significantly reduced serum levels of lipase and amylase (Figure 1).

Figure 1.

Effect of Riva treatment on the serum levels of lipase (a) and amylase (b) in L-arginine-induced acute pancreatitis: Results represent the mean ± S.E. (n = 8). ^aSignificant (p < .05) difference from the control group; ^bsignificant (p < .05) difference from the Riva group (Riva: rivastigmine); ^csignificant (p < .05) difference from the L-arginine group.

Influence of Riva treatment on pulmonary oxidative stress indicators

Table 1 showed that pulmonary MDA and NOx were significantly elevated and pulmonary TAC and GSH were reduced with L-arginine administration in comparison to the control and Riva groups. When compared to the L-arginine group, Riva pre-administration in the mixed group significantly mitigated oxidative stress parameters.

Table 1.

Influence of Riva on pulmonary oxidative stress indicators.

Groups	GSH (nmol/g tissue)	MDA (nmol/g tissue)	NOx (nmol/g tissue)	Caspase-3 (nmol/g tissue)	TAC (nmol/g tissue)
Control	72.50 ± 4.90	73.13 ± 4.6	58.13 ± 2.4	4.63 ± 0.49	66.25 ± 3.86
Riva	68.75 ± 4.43	70.75 ± 3.26	66.75 ± 3.68	4.75 ± 0.53	72.88 ± 3.68
L-arginine	26.25 ± 2.11a, b	300.4 ± 6.72a, b	94.13 ± 2.88a, b	16.50 ± 0.98a, b	32.88 ± 2.67a, b
L-arginine+Riva	62.01 ± 3.31 c	75.38 ± 3.95 c	65.63 ± 2.76 c	7.0 ± 0.46 c	62.50 ± 3.78 c

Results show the mean ± S.E. (n = 8).

GSH = reduced glutathione; MDA = malondialdehyde; NOx = total nitrite/nitrate; TAC = total antioxidant capacity.

^aSignificant (p < .05) difference from the control group.

^bSignificant (p < .05) difference from the Riva group. (Riva: rivastigmine).

^cSignificant (p < .05) difference from the L-arginine group.

Influence of Riva on pulmonary inflammatory indicators

Table 2 showed a significant elevation in pulmonary IL-6, TNF-α, and NF-kβ with L-arginine treatment when compared to the control and Riva groups. Riva pre-administration in the mixed group displayed a significant reduction in these markers when compared to the L-arginine group (Table 2).

Table 2.

Influence of Riva on pulmonary inflammatory indicators.

Groups	IL-6 (pg/mg)	TNF-α (pg/mg)	NF-kB (ng/mg)
Control group	48 ± 4.14	54.75 ± 2.9	0.62 ± 0.02
Riva group	60.38 ± 2.6	53.63 ± 4.6	0.55 ± 0.03
L-arginine group	131.4 ± 2.65a, b	89.9 ± 3.3a, b	1.3 ± 0.12a, b
L-arginine+Riva	60.13 ± 3.56 c	60.25 ± 3.6 c	0.57 ± 0.04 c

Results show the mean ± S.E. (n = 8).

^aSignificant (p < .05) difference from the control group.

^bSignificant (p < .05) difference from the Riva group. (Riva: rivastigmine).

^cSignificant (p < .05) difference from the L-arginine group. IL-6 = interleukin-6; TNF-α = tumor necrosis factor-alpha.; NF-κB = nuclear factor kappa B.

Influence of Riva on ABG parameters

As regards PaO2, a significant decrease was noticed with L-arginine administration relative to the control and Riva groups. In contrast, Riva + L-arginine-treated rats showed a significant elevation in their values relative to the L-arginine group. In addition, PaCO2 values showed a significant elevation with L-arginine administration relative to the control and Riva groups, with a significant minimization in its value in the Riva pre-treated animals (Table 3).

Table 3.

Influence of Riva on ABG markers.

Groups	PaO2 (mmHg)	PaCO2 (mmHg)
Control group	96.63 ± 1.88	43.13 ± 2.97
Riva group	97.38 ± 2.43	45.88 ± 2.55
L-arginine group	69.63 ± 3.37a, b	65.13 ± 1.7a, b
L-arginine+Riva	92.00 ± 2.36 c	39.63 ± 1.7 c

Results show the mean ± S.E. (n = 8).

Partial pressure of oxygen in arterial blood (PaO2) and partial pressure of Co₂ in arterial blood (PaCO₂).

^aSignificant (p < .05) difference from the control group.

^bSignificant (p < .05) difference from the Riva group. (Riva: rivastigmine).

^cSignificant (p < .05) difference from the L-arginine group.

Histological examination of the pancreas

Hematoxylin and eosin

In both the control and Riva-treated rats, the pancreatic tissue lacked obvious changes and displayed closely packed pancreatic acini with acidophilic apical cytoplasm and basophilic cytoplasm. Interlobular ducts were observed, surrounded by reticular connective tissue (Figure 2). Islets of Langerhans appeared as pale-stained areas scattered among the pancreatic acini and separated by blood capillaries (Figure 2(a) and (b), respectively).

Figure 2.

Photomicrographs of sections in the rat pancreatic tissue of (a) the control rats and (b) the Riva group showing the islets of Langerhans (I) are seen surrounded by the pancreatic acini (A). The islet cells (IC) are separated by blood capillaries (arrow). Notice the interlobular duct (D). (c) The L-arginine group showing islets (I) with destroyed area (arrow) encircled by irregular acini (A) and pyknotic cells (yellow circle). Notice the congestion (*), interlobular duct with retained secretion (D) and disrupted epithelium (arrow head). (d) The L-arginine + Riva treated rats showing the islets of Langerhans (I), the packed acini (A), and the interlobular duct with cuboidal epithelium (D). Notice vacuolation (V) and congestion (*).

In the L-arginine-treated group, examination displayed variable structural alterations. Irregularities in the acini shape were detected. The acinar cells showed pyknosis. The islets appeared atrophic around the congested blood vessels. The pancreatic ducts showed retained secretions and disrupted epithelium. (Figure 2(c)).

The L-arginine + Riva group had visible intact acinar cells. The interlobular ducts were observed with cuboidal epithelium. However, vacuolation and vascular congestion were detected (Figure 2(d)).

HSP70 immunohistochemical staining

(Figure 3) Pancreases from control and Riva-treated rats showed negative immunoreactivity (Figure 3(a) and (b), respectively); however, pancreases from the L-arginine group showed positive cytoplasmic immunoreaction in acinar cells (Figure 3(c)). The pancreases of the Riva + L-arginine-treated rats, on the other hand, showed dense immunoreaction in the cytoplasm of acinar cells (Figure 3(d)).

Figure 3.

Photomicrographs of sections in the rat pancreatic tissue of (a) the control rats and (b) the Riva group showing negative immunoreactivity; (c) the L-arginine-treated rats showing positive cytoplasmic immunoreactivity in the acinar cells (arrows); (d) the L-arginine + Riva-treated rats showing dense cytoplasmic immunoreactivity in the acinar cells (arrows).

Histological examination of the pulmonary tissue

Hematoxylin and eosin

(Figure 4) In the control group and Riva groups, the alveoli were separated by thin interalveolar septa (Figure 4(a) and (b)). However, the lungs of L-arginine-treated rats revealed congestion, a thick interalveolar septum, collapsed alveoli, lung emphysema, and inflammatory cellular infiltration (Figure 4(c)).

Figure 4.

Photomicrographs of sections in the rat lung of (a) the control rats and (b) the Riva group showing alveoli (A) separated by interalveolar septa (arrow); (c) the L-arginine-treated rats showing vacuolation (V), thick interalveolar septum (arrow), lung emphysema (E), and congestion (double arrows). Inset: note the inflammatory cellular infiltration (I); (d) the L-arginine + Riva-treated rats showing alveoli (A), interalveolar septa (arrow), and some congestion (double arrows).

Examination of the lungs of Riva-treated rats revealed most of the alveoli and interalveolar septa with obviously intact histology. However, some congestion was still detected (Figure 4(d)).

Immunohistochemical results of HSP70 stained sections

(Figure 5) The lungs of control and Riva-treated rats showed negative immunoreactivity (Figure 5(a) and (b), respectively), while the lungs of the L-arginine group showed positive immunoreaction in the bronchial epithelium (Figure 5(c)). In the lungs of Riva + L-arginine-treated rats, cytoplasmic immunostaining was prominent in the bronchial epithelium and alveolar septae (Figure 5(d)).

Figure 5.

Photomicrographs of sections in the rat lung of (a) the control rats and (b) the Riva group showing negative immunoreactivity; (c) the L-arginine-treated rats showing positive cytoplasmic immunoreactivity in the bronchial wall (arrows); (d) the L-arginine + Riva-treated rats showing dense cytoplasmic immunoreactivity in the bronchial wall (arrow) and interalveolar septa (double arrows).

Morphometric examination

As displayed in Table 4, the mean percent areas of HSP-70 immunoreactions were significantly different between groups (p < .05).

Table 4.

Mean area percentage ± SE of HSP-70 in the different studied groups.

Groups	Pancreas	Lung
Control	0.00 ± 0.00	0.2 ± 0.2
Riva	0.00 ± 0.00	0.2 ± 0.2
L-arginine	3.025 ± 0.39a, b	2.56 ± 0.4a, b
Riva + L-arginine	6.582 ± 0.5a, b, c	4.57 ± 0.28a, b, c

Results show the mean ± S.E. (n = 8).

^aSignificant (p < .05) difference from the control group.

^bSignificant (p < .05) difference from the Riva group. (Riva: rivastigmine).

^cSignificant (p < .05) difference from the L-arginine group.

Discussion

The most obvious symptom of extra-abdominal organ failure in pancreatitis is pulmonary inefficiency, which can range in severity from mild oxygenation malfunction to fatal ARDS. ¹⁹

Aseptic acute pancreatic inflammation develops suddenly in AP. It is regarded as one of the most well-known life-threatening illnesses in the world, with rising rates. ⁸ Multiple organ failure and systemic inflammatory response syndrome may be the leading causes of mortality. ²⁰ The purpose of the present experimental work was to evaluate Riva’s ameliorative efficiency in L-arginine-induced AP. Riva is recommended for the treatment of Alzheimer’s disease, which is usually present in old age. ¹¹

In the current experiment, L-arginine administration induced AP, which is manifested by increased amylase and lipase levels at 24 h postinduction. ²¹ According to Salem et al., ²² amylase levels increase as AP produces hydrolytic enzymes that hydrolyze phospholipids to release arachidonic acid and lysophospholipids before acting cytotoxically and causing necrosis of the acinar cell. Amylase and lipase levels increased as a result of the injured acinar cells. The current study demonstrated that blood amylase and lipase levels were dramatically restored by Riva therapy when administered prior to L-arginine-induced AP.

ALI, which is defined by the accumulation of inflammatory substances in the lung, can result from SAP. Uncertainty surrounds the mechanisms underlying ALI. There aren’t many treatments that work well for it. In reality, treating ALI patients in the clinic is still difficult. Therefore, the development of new therapeutic medications and an understanding of their pharmacological effects may aid in the clinic’s management of patients with ALI.

Kupffer cells, alveolar macrophages, and peritoneal macrophages can be stimulated in AP, releasing systemic cytokines and inflammatory markers. There is growing evidence that these markers released by activated macrophages, in turn, cause the systemic inflammatory reaction and AP-related lung damage. Neutrophils are a different class of leukocyte that participate in AP-associated ALI. Localized pulmonary endothelial cell injury is induced by oxygen-radical products from the neutrophils and causes lung damage. Finally, particular effects on pancreatic enzymes, including proteases and phospholipase A2, are linked to pancreatitis-associated ALI. ²³

The current investigation demonstrated increased oxidative stress markers with significantly decreased pulmonary TAC and GSH with L-arginine administration, coinciding with the fact that oxidative stress is one of the key problems prevalent in ALI that occurs in relation to AP. ²⁴ A well-known paradigm for inducing AP that is comparable to that in humans is L-arginine-induced AP. Nitric oxide synthase converts it into nitric oxide (NO), which triggers AP and causes endoplasmic reticulum stress in the pancreas. ²⁵ Earlier studies had also demonstrated that L-arginine-produced oxidative stress was responsible for the development of AP, which is regarded as the disease’s main cause.^20,25,26

The pathophysiology of AP is extremely complex, and it has been linked to oxygen-derived free radicals. In addition to damaging pancreatic acinar cells, oxygen free radicals also harm the liver through blood circulation, which reduces the liver’s capacity to remove free radicals and intensifies the body's reaction to oxidative stress. ²⁷ As a result, preventing oxidative stress and acinar cell damage in the initial stages of AP can limit the progression of the disease.

Oxidative damage, inflammation, and elevated pancreatic enzyme levels are all aspects of the AP pathophysiology. When there is an infection, trauma, or tissue damage that results in inflammation, T-cells generate the pro-inflammatory cytokine IL-6. The progression of AP is significantly correlated with the level of IL-6, according to several studies.^28,29 TNF-α is important in the systemic course of inflammation because of its effects on the upregulation of other cytokines and inflammatory markers.

Riva pre-treatment significantly restored pulmonary inflammatory markers (IL-6, TNF-α, and NF-kβ) as well as pulmonary oxidative parameters (MDA, GSH, caspase-3, TAC, and NOx) in the current L-arginine-induced AP model.

These findings coincide with the study of Shafiey et al. (2018), ¹⁰ which states that Riva has an antirheumatoid impact in experimentally induced rheumatoid arthritis through its antioxidant effect. Additionally, Ali et al. (2016) ¹⁵ wrote that Riva has a mitigating role in osteoporosis and bone marrow toxicity by reducing oxidative stress. Numerous nAChR subunits, including α1, α7, and β4, have also been found in human polymorphonuclear neutrophils, and it has been revealed that their messenger RNA and protein production are concomitant with their regulatory activities and maturity in the area of inflammation. Furthermore, it is known that in many inflammatory disorders, a rise in the proinflammatory cytokine storm is associated with a drop in vagal nerve activity. ACh is released at the distal end of the vagal efferent upon activation, which prevents the production of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-18). ³⁰ Inflammation is reduced by cholinesterase inhibitors (ChEIs), which raise ACh levels. ACh produced from vagal nerve terminals can be preserved by them, allowing it to trigger circulating macrophages’ α7-nAChR. A ChEI called RIVA, which is used for AD, has been proven to improve experimentally produced colitis through its immune-modulatory and anti-inflammatory effects. ¹³ It prevented the generation of TNF-α from macrophages by stimulating the nicotinic receptor (7nAChR) and preventing cytokine discharge from vagal nerve endings and related nerve cells in the brain. ¹³

According to Abdelzaher et al. (2023), ³¹ Riva reduces experimentally developed acute renal damage from gentamicin by activating the cholinergic anti-inflammatory pathway via the 7nAChR, acting as an antioxidant by regulating MDA, NOx, and GSH and inhibiting caspase-3 levels.

Thus, it is believed that nicotinic activity is essential for the anti-inflammatory effects of Riva. According to Khalaf et al. (2022), ³² Riva reduces indomethacin-induced gastric mucosal damage by activating the 7nAChR and preventing oxidative stress and apoptosis.

The current findings demonstrated that Riva-administered rats had decreased caspase-3 genetic expression. Similar findings were made by Siddique et al. (2022) ³³ on the upregulation of caspase-3 in the L-arginine group and Riva's ability to decrease caspase-3 and 9 gene expression, which is a measure of apoptosis.

The PaO2 and PaCO2 values can be used to assess a patient’s degree or type of respiratory disease if they exhibit symptoms like hypoxia or CO2 retention. Lung tissue destruction and partial lung function loss can cause breathing difficulties, hypoxia, and possibly patient mortality. ³⁴ In the study conducted by Lelli et al. (2017), ³⁵ Curcumin, an antioxidant, showed several growth-promoting properties that may treat lung disorders through regulating transcription factors, cytokines, adhesion molecules, and enzymes that are crucial in inflammation and cancer.

The present experiment revealed a significant rise in PaCO2 and reduction in PaO2 in the ABG samples of the L-arginine-administered rats that Riva could restore.

On pathological examination, L-arginine revealed congestion, collapsed alveoli, a thick interalveolar septum, inflammatory cellular infiltration, and lung emphysema. The coadministration of Riva displayed most of the alveoli and interalveolar septa with restored histology except for some congestion.

L-arginine also elevated the cytoplasmic immunostaining of Hsp70 in the bronchial epithelium and alveolar septae. Hsp is a specific protein that has exhibited conservative organic evolution. HSP production can be triggered by a variety of physiological, pathological, and stress-related factors, and this is crucial for defending the body against the damaging effects of excessive stress. The HSP70 family has a close connection to pulmonary biology and is protective against lung damage through anti-inflammation, anti-oxidation, anti-apoptosis, and molecular chaperone functions.^36,37

HSP prevents type II pulmonary epithelial cells from proliferating and inhibits NF-κB, a transcription factor that promotes inflammation.^38,39 Additionally, increased apoptosis has been linked to the emergence of acute lung damage. Hsp70 has been shown to reduce apoptosis in vitro. ⁴⁰ Also, acute lung injury caused by sepsis is prevented by Hsp70 abundance. ⁴¹

Hsp70 has the ability to directly bind to caspase-3 and caspase-9, protecting specific caspase targets and promoting survival. ⁴² As a result, one of Riva’s proposed protective mechanisms appears to be mediated by the inflammatory cascade actions of Hsp70 and NF-κB on apoptosis, while the other is mediated by oxidative pathways. Additionally, that establishes the basis for the therapeutic use of Hsp70 in the management of ARDS, the prevalent, fatal condition. Future studies are in demand to investigate Riva's efficacy with various dose profiles, either alone or as an adjuvant therapy, in order to cure lung injury in AP patients.

Conclusion

Riva retains potent ameliorative effects against lung injury that occur on the top of AP by diminishing oxidative stress, inflammation, and apoptosis via the HSP70/IL6/NF-κB signaling cascade. The current results shed light on the promising role of Riva in treating patients with lung injuries like ARDs.

Ethical statement

Ethical approval

Ethical approval for this study was obtained from Laboratory Animals’ Maintenance and Usage Committee of the Faculty of Medicine, Minia University (Council No. 394:2022).

ORCID iD

Nermeen N Welson https://orcid.org/0000-0001-7854-2086

Data availability statement

Data will be made available on request.