Anti-inflammatory response of Metformin Nanoparticles and/or low doses γ-Irradiation on Acute Pancreatitis in Rats via regulation of inflammatory cytokine

Anti-inflammatory response of Metformin Nanoparticles and/or low doses γ-Irradiation on Acute Pancreatitis in Rats via regulation of inflammatory cytokine | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Anti-inflammatory response of Metformin Nanoparticles and/or low doses γ-Irradiation on Acute Pancreatitis in Rats via regulation of inflammatory cytokine Neamat Ahmed Osman, Mohamed Mosleh, Faten Zahran This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5805377/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Repeated pancreatic insult inflammation leads to the development of pancreatitis. In these studies the evaluation of nano-formulated metformin drug with chitosan (Cs-Met NPs) and/or low-doses of γ-irradiation (IR) against acute pancreatitis (AP) induced by L-arginine in an animal model. Thirty adult of Albino rats were divided randomly into 5 groups. Normal control, L-Arginine treated (AP); AP + IR; AP + Cs-Met NPs and AP + IR + Cs-Met NPs. Histopathological studies of pancreatic and biochemical parameters (serum amylase and lipase levels, Random blood glucose (RBS), plasma insulin level, Insulin growth factor 1, tumor necrosis factor-alpha (TNF-α), IL-6, IL-8, IL-10, and CRP were measured in the pancreatitis model. AP induced by L-Arginine- treatment elevates the serum pancreatic amylase & lipase levels, significantly increases IL-6, IL-8, TNF-α, RBS, and CRP, and significantly decreases IL-10, insulin levels, and Insulin growth factor 1. Microscopic examination revealed loss of the pancreatic lobular architecture, marked fibrosis, acinar degeneration, inflammation, and marked oedema. All the serological parameters and the histopathological observations were markedly improved by Cs-Met NPs administration and/or low doses of γ-irradiation treatment. In conclusion, Cs-Met NPs and/or low doses of γ- irradiation have a therapeutic effect on acute pancreatitis model induced in rats. Applied Biochemistry Biochemical Research Methods General Biochemistry Drug Delivery Acute pancreatitis Metformin nanoparticles Gamma Irradiation low-dose radiation inflammatory cytokine Figures Figure 1 Figure 2 1. Introduction Acute pancreatitis (AP), is defined as an inflammatory disorder of the pancreas. It is one of the most common and severe gastrointestinal diseases. It leads to a systemic inflammatory response to multiple organ dysfunction syndrome ( 1 , 2 ). In the body an amino acid called L-arginine (L-arg) is necessary to make proteins, improves wound healing, and decreases recovery time after surgery ( 3 ). many authors reported that L-arg induces AP in experimental animals ( 4 , 5 ). Metformin is an antidiabetic, reduces hepatic glucose production and insulin resistance, and is an oral therapy for type 2 diabetes ( 6 ). Also, it has been found to possess some interesting properties such as antioxidant, anticancer, anti-inflammation, and anti-fibrosis, which are interesting for its use as an adjuvant in oncology( 7 ). Metformin has been used in clinical settings for good safety and limited toxicity. Many reports showed that it has no effect on glucose levels in nondiabetic individuals and the use of as adjuvant therapy ( 8 ). Metformin lower cancer incidence and cancer-specific deaths have been reported among diabetics compared to diabetics on other anti-diabetic medications ( 9 ). Biomedical nanotechnology has made technological fabrication in drugs delivery. Formulating of drugs into nanoparticulate drug delivery has demonstrated distinct advantages over traditional dosage forms ( 10 ). For oral drug delivery, nanoparticulate of drugs could significantly improve dissolvability, stability and oral absorption efficiency than free drugs in conventional formulations ( 11 ). Chitosan is a modified biopolymer, originates from the crustacean shells of creatures like prawns and crabs. The USA FDA has approved that ita highly basic polymer for tissue engineering and drug delivery ( 13 ) used in the majority of pharmaceutical industries and research. Chitosan as a nanocarriers are effective at low dosage with a controlled release profile while reducing the side effects accompanied by high dosage administrations. ( 14 – 15 ). Pre-treatment with non-lethal low doses of gamma radiation have a protective effect against oxidative injury in animal tissues. Low doses of irradiation are generally regarded as safe and its effect is considered to be negligible. Low-dose of gamma irradiation has been extensively indicated by induction of hormesis and adaptive response ( 16 ). Low-dose of gamma irradiation was reported to stimulate some biological activities in vitro as well as in vivo, activation of immune functions in addition, it has been shown to enhance the efficacy of chemotherapy and immunotherapy ( 17 ). The current study prepared to investigate the possible Anti-inflammatory response of Cs-Met NPs and/or low doses of γ- irradiation against acute pancreatitis in rat's model. 2. Materials and Methods 2.1 Materials: 2.1.1 Drugs and chemicals: Metformin; L-Arginine; and Chitosan were purchased from (Sigma Chemicals Co., U.S.A). 2.1.2. Irradiation Process: Gamma irradiations were carried out in the National Center for Radiation Research and Technology (NCRRT), Egyptian Atomic Energy Authority. For preparation of nanoparticles, Chitosan was gamma-irradiated with 200 kGy, using Canadian Co 60 γ-cell-220 sources, at a dose rate of 1.14 kGy/h. On the other hand, experimental rats were exposed to 0.25 Gy twice/week for 2 weeks’ whole-body gamma radiation using Canadian Cs-137 γ-cell-40, at a dose rate of 0.423 Gy/min which was calculated according to the dosimetry department guidelines at the NCRRT at the time of the experiment. 2.1.3 Preparation of Cs-Met NPs: Metformin-loaded chitosan Nanoparticles were successfully formulated by physical degradation methods, using γ-irradiation (200 KGy) as follow; 1. Colloidal water-soluble chitosan nanoparticles were synthesized via γ-radiolysis technique in which chitosan (1.5 g) was dissolved in 100 mL of 1% (v/v) aqueous acetic acid at 60 °C using a magnetic stirrer for 2 h until the polymer becomes completely soluble. The solubilized chitosan was then irradiated (200 kGy γ-irradiation) and neutralized by using 1 M NaOH solution. By filtration and removed of water insoluble part a clear solution of neutralized water-soluble chitosan was obtained. The degradation yield and formation of water-soluble chitosan was calculated by the following equation; Yield % = (W1- W2)/W1) x100, Where W1 and W2 represent the weights of the original chitosan and the water-insoluble chitosan after neutralization, respectively. The yield was found 96% and this result is consistent with Choi et al. (18). 2. After that, for therapeutic applications, 1.5 g metformin was added to 100 mL of neutralized water-soluble chitosan nanoparticles at room temperature using a magnetic stirrer for 3 hours until the metformin became completely soluble. 2.1.4 Characterization of Cs-Met NPs: UV–Vis spectrophotometry as an analytical tool was used to investigate the formation of water-soluble chitosan nanoparticles. UV–Vis measurements were taken by a Unicam double beam UV–Vis spectrophotometer. The structural analysis was performed using a Fourier Transform Infra-Red (FTIR) spectrophotometer (Bruker Vertex 70). On the other hand, the shape and size of the synthesized nanoparticles were obtained via a high-resolution transmission electron microscopy, HR-TEM (JEOL-JEM-100 CX). 2.1.5 Experimental animals: Male Swiss albino rats weighing (100 ± 20) g were used in this study. They were purchased from the Egyptian National Authority for Drug Research and Control, Ministry of Health, Cairo, Egypt. The animals were housed under regular conditions (12-h light/12-h dark cycle, normal temperature, good ventilation, and humidity level) in specially built plastic cages, ten per each. Throughout the study, drinking water and food were supplied ad libitum . All experimental procedures were performed in compliance with the standards and guidelines of the National Research Centre Ethics Committee, issued by the U.S. National Institutes of Health, “Guide for the treatment and use of laboratory animals” for the use and protection of experimental animals (NIH publication No. 85–23, 1996). 2.1.6 Animal classification (Induction of acute pancreatitis and drug treatment): The rats were equally classified into five groups of six rats each, as follows: 1. Control group [G1]: Rats in this group were kept as controls. 2. Acute pancreatitis (AP) group [G2]: Rats were injected intraperitoneally (i.p.) with L-arginine (250 mg/100g b.wt., twice at 1-hour intervals), day over day for 14 days to induce acute pancreatitis (19). 3. AP + IR group [G3]: Rats were injected with an L-arginine-like group [2] and after that, they were exposed to whole body γ-radiation at a dose level of 0.25 Gy twice/week for 2 weeks (0.5 Gy/ week, X2). 4. AP + Cs-Met NPs group [G4]: Rats were injected with an L-arginine-like group [2] and after that, they were treated with Cs-Met NPs at a dose of 46.8 mg/kg body weight, daily for 14 days(15). 5. AP + IR + Cs-Met NPs group [G5]: Rats were injected with an L-arginine-like group [2] and after that, they were exposed to whole body γ-radiation like group [3] and were treated with Cs-Met NPs like group [4]. At the end of the experiment, all rats were anesthetized by using urethane before being sacrificed and then the blood was collected via heart piercing by using disposable plastic syringes. The coagulated blood samples were centrifuged at 3000 rpm for 15 min and the serum was collected for different estimates of biochemical parameters. For further biochemical research, pancreatic tissues were dissected, rinsed in ice-cold isotonic saline, blotted dry with a filter paper, and stored at −20 °C. Portions of pancreatic tissue were rinsed and set for histopathological examination in 10% neutralized formalin. 2.2 Methods: 2.2.1 Measurement of biochemical parameters: Activities of amylase and lipase were evaluated in serum, using a diagnostic kit purchased from TRUEchemie Company, India. The results were expressed as U/I. Serum glucose was measured by the kinetic method of Kaplan (20) using a commercial kit obtained from (SPINREACT Company, Spain). IL-6 was determined by a sandwich enzyme-linked immunosorbent assay (ELISA) using rat kit given by (R&D Systems, Inc., Minneapolis, USA). While, TNF-α, IL-8, and IGF1 levels were determined by the ELISA rat kit provided by (CUSABIO, USA). IL-10, and CRP levels were determined by the ELISA rat kit provided by (MyBioSource, USA). Serum insulin was determined by the ELISA rat kit provided by (CELL BIOLABS, INC. Company, USA). According to the manufacturer’s instructions, the ELISA microplate was read using an ELISA reader with an absorbance maximum of 450 nm. The parameter levels were calculated after plotting the standard curves and expressed as pg/mL. 2.2.2 Histopathological examination: Samples of pancreas tissue were fixed in 10% formaldehyde solution and inserted in paraffin using standard methods. Sectioned tissues at 5 μm thickness were treated with hematoxylin-eosin (H&E) stain for routine examination using light microscopy according to the method of Bancroft and Stevens (21). 2.2.3 Statistical analyses: The differences in means of variables between groups were estimated using a one-way analysis of variance (ANOVA) with the Least Significant Difference (LSD). The obtained results were expressed as the mean ± SE and were examined by Statistical Package for Social Science (SPSS) version 20 for Windows (SPSS® Chicago, IL, USA) software program. At P <0.05 or P<0.01, the probability was considered significant and was considered highly significant at P<0.001. 3. Results 3.1 Characterization of Cs-Met NPs: 3.1.1 FTIR analysis The FTIR spectra of Cs, water-soluble chitosan nanoparticles (Cs NPs), Met. and CS-Met. NPs are presented in Fig. 1 . (C) The spectrum of CS reveals a broad band at 3388 cm-1 which is related to –NH2 and –OH stretching vibration on the CS chains. The two bands at 2980 − 2880 cm-1 are assigned to the CH stretching vibrations. The peak at 1645 cm-1 is attributed to C = O stretching in the amide group due to the partially acetylated amino groups. The peak of 1575 cm-1 is attributed to N–H bending in the amine group that is present in protonated form (NH3+). The band at 1385 cm-1 is due to the C-N stretching vibration. In addition, the peak at 1463 cm-1 may be assigned to the O–H/C–H deformations. The two bands at 1152 cm-1 and 1077 cm-1 correspond to the asymmetric stretching vibration of the C–O–C of glycosidic linkage and C–O–C stretching in the saccharide structure (22). The FT-IR spectrum of irradiated chitosan (Cs NPs) revealed all characteristic peaks as presented in pure CS. This result indicates that no functional groups were added or deleted but some were modified due to oxidation, as no other chemical agents or moieties were added during the irradiation process. The increase in intensity of peaks at 1714 cm − 1 can be ascribed to increased C = O bonds formed by chain scission reaction and oxidation of chitosan due to irradiation ( 23 , 24 ). The intensity of peak at 3400 cm − 1 increased after irradiation which indicates a possibility of increment in a number of -OH groups resulting from the scission of glycosidic bonds leading to the formation of hydroxyl group ( 25 ). The MET drug contains three types of N-H groups which show three bands at 3366, 3300, and 3153 cm-1. The CH stretching vibrations are assigned at 2980 − 2880 cm-1. The bands at 1629 and 1552 cm-1 are due to C = N stretching vibrations. The CH deformations are assigned at 1461 and 1384 cm-1. The C-N stretching vibration occurs at 1220 − 1020 cm-1. The peaks at 938 and 809 cm-1 are due to N-H wagging( 26 ). Compared with the FTIR spectra of CS and CS-MET, there was no significant change in the vibration of the backbone of CS before and after MET addition, which indicated that the adsorbed MET did not alter the formed structure. Furthermore, the reduced intensity of the metformin N-H group at 3379 cm-1 indicates the possibility of hydrogen bonding between the metformin and CS nanoparticles ( 27 ). 3.1.2 Morphological Characterization The shape, dispersion, and average particle size of as prepared materials were determined by Transmission Electron Microscopy (TEM) analysis. As shown in Fig. 1 (A, B), the TEM analysis of chitosan nanoparticles (Cs NPs) and chitosan-metformin nanoparticles (Cs-Met NPs) confirms that the particles were in the nano-dimensional range with spherical shape with the inter-particular connection among them, smooth surface and size range of approximately 8 nm (Fig. 1 A) and 12.5 nm (Fig. 1 B) respectively. The spherical appearance of Cs NPs can be attributed to the shortening CS chains densely packed with each other into particles, while Cs-Met NPs have fewer regular surfaces and edges due to the attachment of metformin on the chitosan nanoparticles surface. 3.2 Effect of Cs-Met NPs and low doses γ radiation on the grade of AP. 3.2.1 Histological findings In the normal control section of the pancreas stained with H&E stain, the normal histological structure was observed. The pale staining of the spreading islets of Langerhans containing a spherical cluster of polygonal cells with fine secretory granules (endocrine gland) among the dark staining well-organized densely packed pyramidal-shaped cells of the acini (exocrine gland) linked by connective tissues and blood vessels (Figs. 2 , 1 & 2 ). Meanwhile, histopathological changes appeared in the pancreas in rats treated with Arg. After 24 h. of all treatments end showing a cluster of immune cells, lymphoid aggregates, hemorrhage, and necrotic acini (Fig. 2 ( 3 , 4 )). Furthermore, sections in the pancreas of albino rats suffering from pancreatitis exposed to low doses of γ- radiation showed the normal histological appearance of tissue structure, the normal architecture of islets of Langerhans and dark staining acini with slight detachment of secretory cells after 24 h post treatments end (Fig. 2 ( 6 )). Treatment of albino rats suffering from pancreatitis by Cs-Met NPs recording normal appearance islets of Langerhans, normal acini (arrowhead), intralobular duct, and interlobular duct after 24 h of treatments end (Fig. 2 ( 5 )). Whenever albino rats suffering from pancreatitis treated by Cs-Met NPs and exposed to low doses of γ- radiation recording normal appearance islets of Langerhans, normal acini, intralobular duct, and interlobular duct after 24 h post treatments end (Fig. 2 ( 7 , 8 )). 3.2.2 Serological findings: 3.2.2.1 Pancreatic enzymes activities The clinical key criteria for the diagnosis of acute pancreatitis were mylasemia and lipasemia (high amylase and lipase activity) are among. In this study, the activities of serum amylase and lipase in the L-arginine-treated group were elevated when compared to the control at the end of the experiment suggesting a successful induction of AP. However, treating the animals suffering from acute pancreatitis with Cs-Met NPs and/or exposed to γ- radiation produced substantial suppression of amylase and lipase activity (Table 1). 3.2.2.2 Glycaemic parameters One of the complications of AP is the endocrine dysfunction, specifically impaired glucose metabolism. Moreover, the glucose level is closely correlated with the inflammatory responses in AP, which can affect the progression of the disease. The data of the current study represented in table (2) revealed that after the induction of AP in rats was associated with a slight elevation in the glucose levels accompanied with low levels of insulin, when compared with the control group. On the other hand, treatment with fractionated low doses of γ-IR and Cs-Met NPs either alone or in combinations reversed these results and resulted in maintaining levels of both glucose and insulin. Therefore, a high level of glucose can be used as an indicator for evaluating the severity of AP. 3.2.2.3 Inflammatory markers Pancreatitis is identified by the destruction of acinar cells besides the activation of inflammatory cells (macrophages and neutrophils) thus, a significant change in the levels of many inflammatory mediators was observed. Systemic manifestations of the AP are mediated by a variety of pro- and anti-inflammatory mediators released from the injured pancreas. Local recruitment and activation of inflammatory cells in AP lead to the production of inflammatory markers, such as IL-6 & IL-8, TNF-α, and CRP which are important markers in predicting the severity of AP. As shown in Table (3) The obtained data showed a dramatic increase in the levels of pro-inflammatory cytokine (IL-6, IL-8, TNF-α, and CRP) levels in the pancreatic tissues of rats injected with L-arginine (AP) relative to control levels. • IL-6 : The obtained data showed a dramatic increase in the levels of IL-6 in the pancreatic tissues of rats with AP relative to control levels. In contrast, exposing rats with AP to low doses of gamma radiation, or treatment with Cs-Met NPs either alone or in combinations for two weeks, significantly reduced the IL-6 when compared to the animals with acute pancreatitis. • IL-8 : As shown in table (3) a significant increase in the levels of IL-8 was detected in pancreatic tissues of the animals injected with L-arginine as compared to the control group. However, treating AP-bearing rats with low doses of IR, and Cs-Met NPs either alone or in combinations for two weeks, resulted in a notable inhibition of the IL-8 levels when compared to the AP group. • TNF-α : The statistical comparison of the levels of TNF-α in the pancreatic tissues between the different studied groups revealed that it was significantly increased in the L-arginine group (AP) as compared to the control group. Conversely, there was a marked decrease in the levels of TNF-α upon treatments with low doses of IR and Cs-Met NPs either alone or in combinations for two weeks as shown in table (3). • CRP : CRP is a well-known marker of the severity of AP. The obtained data in table (3) showed a significant increase in the levels of CRP in the pancreatic tissues of the AP group compared to the control group. In contrast, exposing rats with AP to low doses of gamma radiation, or treatment with Cs-Met NPs either alone or in combinations for two weeks, significantly lowered the levels of CRP compared to the pancreatitis group. • IL-10 As shown in Table (4), the levels of pancreatic IL-10 markedly reduced in the pancreatitis group compared with the control group (P < 0.001), while using different treatments significantly elevated the levels of IL-10 compared to the AP group (P < 0.001). • Insulin-like growth factor 1 Insulin-like growth factor 1 (IGF-1) is a protein that belongs to the IGF axis. IGF-1 plays a role in the regulation of β-cell mass and the regulation of insulin secretion and sensitivity. Lots of studies show close links between the IGF axis and glucose metabolism, including pancreatic diseases. Many pro-inflammatory cytokines (IL-6, TNF-α) impair the activity of the IGF-1 axis. The obtained data in table (4) pointed out a remarkable decline in the concentration of IGF1 in the pancreatic tissues of the AP group compared to the control group. While, exposing rats suffering from AP to γ-IR, or treatment with Cs-Met NPs either alone or in combinations for two weeks, significantly enhanced and increased the concentration of IGF1 compared to the AP group. 4. Discussion In medicine, natural products like chitosan are used as drug carriers due to the encapsulation of a broad range of therapeutic agents that deliver to the target site efficiently. Chitosan-based nanoparticles have good biodegradation and biodistribution in the biological milieu, which have made it as one of the most attractive nanocarriers for delivering different therapeutic agents to the tumor cells ( 28 ). Nowadays, chitosan nanoparticles have become of great interest in nanomedicine, biomedical engineering, and the development of new therapeutic drug release systems with improved bioavailability increased specificity and sensitivity, and reduced pharmacological toxicity ( 29 ). On the other hand, Metformin is the most commonly used oral anti-diabetic drug in the world. It has been in clinical use for more than 50 years and has a good safety record with limited toxicity. Lower cancer incidence and cancer-specific deaths have been reported among diabetics on metformin compared to diabetics on other anti-diabetic medications ( 30 ). Therefore, our study was designed to evaluate the anti-inflammatory and protective effect of the Chitosan-Metformin nanocomposites (Cs-Met NPS) on L-arginine-induced acute pancreatitis and its complications on body tissues in Wistar albino rats. Acute pancreatitis (AP) is a common severe critical illness with a high mortality rate ( 31 ). Death due to AP may result from systemic inflammatory response syndrome or multiple organ dysfunction syndromes. Sometimes repeated attacks of AP lead chronically to loss of pancreatic function and fibrosis( 32 ). AP is characterized by inflammation, apoptosis of pancreatic acinar cells, and the release of pancreatic enzymes, including amylase and lipase ( 33 ). In the present study, arginine injection effectively induced acute pancreatitis in rats, markedly through histological changes, higher pathological scores in the pancreas. Pancreatitis was further evidenced by hyperamylasemia and hyperlipasemia, which is consistent with the earlier results of ( 34 ). Our results revealed that injection of L-arginine induced acute pancreatitis represented by a remarkable elevation of the pancreatic amylase and lipase levels. These results are in accordance with that of Salem et al. ( 35 ) who reported that the elevated levels of the pancreatic enzymes, mainly amylase and lipase, may be caused by the production of hydrolytic enzymes in AP which hydrolyse phospholipids to liberate arachidonic acid and lysophospholipids and the latter has a cytotoxic function, causing acinar cells necrosis. Furthermore, Wang et al. ( 36 ) reported that L-arginine selectively destroys pancreatic acinar cells by inducing amino acid imbalance, decreasing the synthesis of polyamine, nucleic acid and proteinase and resulting in excessive activation of the zymogen. On the other hand, Yang et al.( 37 ) indicated that the interstitial leakage of pancreatic lipase triggered adipose lipolysis and increased levels of unsaturated fatty acids. These toxic fatty acids stimulate the excessive release of inflammatory markers and an inflammatory storm that can drive disease progression with eventual multi-organ failure. AP is an inflammatory disease of the pancreas with the involvement of both local tissues and distant organs ( 38 ). The obtained results revealed a remarkable increase in the levels of IL-6, IL-8, TNF-α and CRP accompanied with a significant reduction in the levels of IL-10 in the group of acute pancreatitis induced by L-arginine. Our results were similar to that of Al-Hashem ( 39 ) who found that toxic doses of L-arginine induced pancreatic tissue injury and increased the pro-inflammatory mediators such as TNF-α coupled with a reduction in the anti-inflammatory cytokine IL-10. The prevalence of AP may lead to a systemic illness that may progress to multiple organ dysfunction and even death. Inflammatory cytokines like TNF-α, IL-6 and IL-8 generated during the pathogenesis of AP are considered responsible for the development of multiple organ failure ( 40 ). It was reported that high-level glucose can be used as one of the reference indicators for evaluating the severity of AP in clinical practice ( 41 ). The obtained results revealed a remarkable increase in the levels of glucose coupled with a significant decrease in the insulin levels in the group of acute pancreatitis induced by L-arginine compared to the control group. this is in accordance with Shoman and Nafeh ( 42 ) who reported that AP affects not only exocrine pancreatic function, manifested by significantly higher serum amylase and lipase levels, but also affects pancreatic endocrine function as manifested by decreased fasting plasma insulin (FPI) levels in association with hyperglycemia. Herein, treatment with metformin-loaded chitosan nanoparticles alone or with low-dose gamma radiation ameliorated the pancreatic injury induced by L-arginine. This was attributed to the antioxidant, anti-inflammatory, antibacterial, and immunomodulatory properties of chitosan ( 43 ). Moreover, Borai et al.( 44 ) reported that ChNPs alone and together with low doses of γ-radiation markedly reduced the pancreatic enzymes, lipase and amylase, and diminished the excessive release of the proinflammatory cytokines, TNF-α due to their anti-inflammatory antioxidative activity. Metformin acts not only as a glucose-lowering drug but exhibits additional benefits, including moderate anti-inflammatory and anti-oxidative effects ( 45 ). Sena et al.( 46 ) reported that the anti-inflammatory actions of metformin were by suppressing the main components of inflammation (endothelial cells and smooth muscle cells, monocytes, macrophages and other cell types) and restoring cell functions. Moreover, metformin not only reduced common inflammatory cytokines such as TNF-α, IL-1β and IL-6 but also counteracted macrophage infiltration and M1 polarisation into anti-inflammatory macrophages (M2) in a mouse model of olanzapine-induced insulin resistance ( 47 – 48 ). It has been suggested that metformin improves metabolic parameters such as hyperglycemia, and insulin resistance thereby reducing chronic inflammatory responses ( 49 ). Metformin reduces blood glucose levels primarily by decreasing hepatic glucose production through suppression of gluconeogenesis, ameliorating insulin signaling leading to reduction the intestinal glucose absorption, and improving glucose uptake by peripheral tissues, such as skeletal muscle and adipose tissue ( 50 – 51 ). Interestingly, it was reported that metformin could have direct protective effects on β-cells under metabolic stress, including non-diabetic and T2D human islet cells via alleviating the oxidative stress and endoplasmatic reticulum stress which are responsible for pancreatic β-cells destruction ( 52 ). Moreover, loading metformin with Chitosan NPs enhanced the beneficial effect of metformin because Chitosan NPs can influence the cells of the immune system too. They can enhance the response of the immune system by increasing the maturation of some antigen-presenting cells named dendritic cells. Furthermore, by the means of apoptotic pathway activation, these NPs are also able to start the function of innate immunity( 53 ). It was reported that a low dose of IR is essential to life, acknowledging that the natural production of ROS that is adequate to stimulate the protective systems and provoke a beneficial health effect which is known as radiation hormesis ( 54 ). Our results show that LDR was associated with a decrease in the circulating levels of some markers of inflammation, such as CRP concentrations, and an increase in the levels of the anti-inflammatory cytokines IL-10. Paradoxically LDR (0.5–1.5 Gy) acts on cells (endothelial cells, polymorphonuclear leukocytes, and macrophages) involved in the inflammatory response, producing anti-inflammatory effects, and increased production of cytokines (IL-10) by endothelial cell and immune cells. Presumably, it is in this phase where LD-RT to both lungs could be effective by acting as a powerful anti-inflammatory agent against the cascade of proinflammatory cytokines ( 55 ). From the previous discussion we conclude that Cs-Met NPs exhibits strong therapeutic effects in the course of AP. Hence, the use of Cs-Met NPs as adjuvant treatment in AP is recommended. However, further studies must be carried out to determine the proper dose and route of administration to achieve the best outcome for treatment. Declarations Declaration of competing interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper. The authors declare that they did not receive any financial support from any organization for the research, authorship, and/or publication of this article. Acknowledgments: We thank Dr. Mohamed Bekhet (Radiation Research of Polymer Chemistry Department, National Center for Radiation Research and Technology, Egyptian Atomic Energy Authority) for preparation of Cs-Met NPs. Ethics approval: The experimental animals have been handled under the standards and guidelines of the National Research Center Ethics Committee published by the U.S. National Health Institutes (NIH publication No. 85-23, 1996). Additionally, the present study was approved by the Institutional Animal Care and Use Committee Research Ethic Board, Faculty of Medicine, Zagazig University, Egypt (Approval No. ZU-IACUC/1/F/28/2019). Availability of data and materials: All data obtained from this study are included in the current manuscript. 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Polym Degrad Stab 78(3):533–538 Dawra R, Saluja AK (2012) L-arginine-induced experimental acute pancreatitis. The Exocrine Pancreas Knowledge Base, Pancreapedia Kaplan A (1984) Glucose. Clin Chem The CV Mosby Co st Louis Toronto Princeton. :1032-6 Bancroft J, Stevens A (1996) Theory and practice of histological techniques. P. 1996;185:282 Ali ZI, Mosallam FM, Sokary R, Afify TA, Bekhit M (2021) Radiation synthesis of ZnS/chitosan nanocomposites and its anti-bacterial activity. Int J Environ Anal Chem 101(3):379–390 Alqahtani MS, Al-Yousef HM, Alqahtani AS, Rehman MT, AlAjmi MF, Almarfidi O et al (2021) Preparation, characterization, and in vitro-in silico biological activities of Jatropha pelargoniifolia extract loaded chitosan nanoparticles. Int J Pharm 606:120867 Muley AB, Shingote PR, Patil AP, Dalvi SG, Suprasanna P (2019) Gamma radiation degradation of chitosan for application in growth promotion and induction of stress tolerance in potato (Solanum tuberosum L). Carbohydr Polym 210:289–301 El-Sawy NM, Abd El-Rehim HA, Hegazy E, Elbarbary AM (2013) Preparation of low molecular weight natural polymers by γ-radiation and their growth promoting effect on zea maize plants. Chem Mater Res 3(13):66–78 Gunasekaran S, Natarajan R, Renganayaki V, Natarajan S (2006) Vibrational spectra and thermodynamic analysis of metformin. Indian J Pure Appl Phys 44(7):495–500 Chinnaiyan SK, Deivasigamani K, Gadela VR (2019) Combined synergetic potential of metformin loaded pectin-chitosan biohybrids nanoparticle for NIDDM. Int J Biol Macromol 125:278–289 Alizadeh L, Zarebkohan A, Salehi R, Ajjoolabady A, Rahmati-Yamchi M (2019) Chitosan-based nanotherapeutics for ovarian cancer treatment. J Drug Target 27(8):839–852 Sharifi-Rad J, Quispe C, Butnariu M, Rotariu LS, Sytar O, Sestito S et al (2021) Chitosan nanoparticles as a promising tool in nanomedicine with particular emphasis on oncological treatment. Cancer Cell Int 21(1):1–21 Whitburn J, Edwards CM, Sooriakumaran P (2017) Metformin and prostate cancer: a new role for an old drug. Curr Urol Rep 18(6):1–7 Xia S, Wang J, Kalionis B, Zhang W, Zhao Y (2019) Genistein protects against acute pancreatitis via activation of an apoptotic pathway mediated through endoplasmic reticulum stress in rats. Biochem Biophys Res Commun 509(2):421–428 Abdelzaher WY, Ahmed SM, Welson NN, Marraiki N, Batiha GE-S, Kamel MY (2021) Vinpocetine ameliorates L-arginine induced acute pancreatitis via Sirt1/Nrf2/TNF pathway and inhibition of oxidative stress, inflammation, and apoptosis. Biomed Pharmacother 133:110976 Najenson AC, Courreges AP, Perazzo J, Rubio MF, Vatta MS, Bianciotti LG (2018) Atrial natriuretic peptide reduces inflammation and enhances apoptosis in rat acute pancreatitis. Acta Physiol 222(3):e12992 Kononczuk T, Lukaszuk B, Miklosz A, Chabowski A, Zendzian-Piotrowska M, Kurek K (2018) Cerulein-induced acute pancreatitis affects sphingomyelin signaling pathway in rats. Pancreas 47(7):898–903 Salem F, Lokman M, Kassab R (2014) Ameliorative eff ects of watery extracts of boswella serrata and syzygium aromaticum on L-arginine induced acute pancreatitis in rats. World J Pharm Res 3(10):71–87 Wang N, Zhang F, Yang L, Zou J, Wang H, Liu K et al (2017) Resveratrol protects against L-arginine-induced acute necrotizing pancreatitis in mice by enhancing SIRT1-mediated deacetylation of p53 and heat shock factor 1. Int J Mol Med 40(2):427–437 Yang J, Tang X, Wu Q, Ren P, Yan Y (2022) A Severe Acute Pancreatitis Mouse Model Transited from Mild Symptoms Induced by a Two-Hit Strategy with L-Arginine. Life 12(1):126 Mallick B, Tomer S, Arora SK, Lal A, Dhaka N, Samanta J et al (2019) Change in serum levels of inflammatory markers reflects response of percutaneous catheter drainage in symptomatic fluid collections in patients with acute pancreatitis. JGH Open 3(4):295–301 Al-Hashem F (2021) Suppression of L-Arginine-Induced Acute Necrotizing Pancreatitis in Rats by Metformin Associated with the Inhibition of Myeloperoxidase and Activation of Interleukin-10. Int J Morphology 39(1):102–108 Rehman K, Rashid U, Jabeen K, Akash MSH (2021) Morin attenuates L-arginine induced acute pancreatitis in rats by downregulating myeloperoxidase and lipid peroxidation. Asian Pac J Trop Biomed 11(4):148 Sun Y-f, Song Y, Liu C-s, Geng J-l (2019) Correlation between the glucose level and the development of acute pancreatitis. Saudi J Biol Sci 26(2):427–430 Shoman AA, Nafeh NY (2014) Serum Ghrelin and Plasma Insulin Levels were altered during Disease Course of L-Arginine induced Acute Pancreatitis. Int J 2(12):611–625 Mei Q, Deng G, Huang Z, Yin Y, Li C, Hu J et al (2020) Porous COS@ SiO2 nanocomposites ameliorate severe acute pancreatitis and associated lung injury by regulating the Nrf2 signaling pathway in mice. Front Chem. :720 Borai IH, Hanafi N, Elgawish MA, Kandil E, Arafa MIK (2017) Role of Chitosan Nanoparticles in Reducing Pancreatitis in Rats Treated with L-arginine. Int J Adv Sci Tech Res 3(7):44–58 Kuryłowicz A, Koźniewski K (2020) Anti-inflammatory strategies targeting metaflammation in type 2 diabetes. Molecules 25(9):2224 Sena CM, Matafome P, Louro T, Nunes E, Fernandes R, Seiça RM (2011) Metformin restores endothelial function in aorta of diabetic rats. Br J Pharmacol 163(2):424–437 Hattori Y, Hattori K, Hayashi T (2015) Pleiotropic benefits of metformin: macrophage targeting its anti-inflammatory mechanisms. Diabetes 64(6):1907–1909 Guo C, Liu J, Li H (2021) Metformin ameliorates olanzapine-induced insulin resistance via suppressing macrophage infiltration and inflammatory responses in rats. Biomed Pharmacother 133:110912 Saisho Y (2015) Metformin and inflammation: its potential beyond glucose-lowering effect. Endocrine, Metabolic & Immune Disorders-Drug Targets (Formerly Current Drug Targets-Immune. Endocr Metabolic Disorders) 15(3):196–205 Sliwinska A, Drzewoski J (2015) Molecular action of metformin in hepatocytes: an updated insight. Curr Diabetes Rev 11(3):175–181 Adeva-Andany MM, Rañal-Muíño E, Fernández-Fernández C, Pazos-García C, Vila-Altesor M (2019) Metabolic effects of metformin in humans. Curr Diabetes Rev 15(4):328–339 Moon JS, Karunakaran U, Elumalai S, Lee I-K, Lee HW, Kim Y-W et al (2017) Metformin prevents glucotoxicity by alleviating oxidative and ER stress–induced CD36 expression in pancreatic beta cells. J Diabetes Complicat 31(1):21–30 Jia J, Zhang Y, Xin Y, Jiang C, Yan B, Zhai S (2018) Interactions between nanoparticles and dendritic cells: from the perspective of cancer immunotherapy. Front Oncol 8:404 Lau YS, Chew MT, Alqahtani A, Jones B, Hill MA, Nisbet A et al (2021) Low Dose Ionising Radiation-Induced Hormesis: Therapeutic Implications to Human Health. Appl Sci 11(19):8909 Conti P, Ronconi G, Caraffa A, Gallenga C, Ross R, Frydas I et al (2020) Induction of pro-inflammatory cytokines (IL-1 and IL-6) and lung inflammation by Coronavirus-19 (COVI-19 or SARS-CoV-2): anti-inflammatory strategies. J Biol Regul Homeost Agents 34(2):327–331 Tables Tables 1 to 4 are available in the Supplementary Files section. Additional Declarations The authors declare no competing interests. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5805377","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":400598447,"identity":"77e4909c-8b67-49e2-a82d-a84f4e2b1fb4","order_by":0,"name":"Neamat Ahmed Osman","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYPCCA0DM+ODAByDFxk6UjgSQFmaDgzNAWphJ0cLMA+IQ0mLO3vx0w88fd+TN2Q8zHrZt2ybPx8zA+OFjDm4tlj3HzG72JDwz3NmTzHA4t+22YRszA7PkzG24tRjcSDC7wZNwmHHDgfwDIC2MQC1szLz4tNx//u3mn4TD9hvOP2Y4bNl2256wlhs8ZreBtiRuuAF0GGPb7UTCWs7klN2WSTucvOHGY4aDPeduJ7cxMzbj98vx49tuvrE5bLvhfDLzhx9lt23ntzcf/PARjxZUwMgGJhuIVQ8Cf0hRPApGwSgYBSMFAACMqVt+R0q/uwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-9528-9780","institution":"National Center for Radiation Research and Technology, Egyptian Atomic Energy Authority","correspondingAuthor":true,"prefix":"","firstName":"Neamat","middleName":"Ahmed","lastName":"Osman","suffix":""},{"id":400602953,"identity":"a5317eab-446b-4b55-8537-f69b6e28a687","order_by":1,"name":"Mohamed Mosleh","email":"","orcid":"","institution":"Faculty of Science, Zagazig University","correspondingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"","lastName":"Mosleh","suffix":""},{"id":400603920,"identity":"3f5c0c49-2568-4e64-9b45-d8c429c72001","order_by":2,"name":"Faten Zahran","email":"","orcid":"","institution":"Faculty of Science, Zagazig University","correspondingAuthor":false,"prefix":"","firstName":"Faten","middleName":"","lastName":"Zahran","suffix":""}],"badges":[],"createdAt":"2025-01-10 16:55:16","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5805377/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5805377/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73622793,"identity":"38994fa0-b1ae-4ed1-8b40-478f684cdf64","added_by":"auto","created_at":"2025-01-13 04:40:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":413138,"visible":true,"origin":"","legend":"\u003cp\u003eTEM image of the particle size distribution of water-soluble chitosan nanoparticles \u003cstrong\u003e(A), \u003c/strong\u003eand metformin/ water-soluble chitosan nanoparticles \u003cstrong\u003e(B). \u003c/strong\u003eFTIR spectra of chitosan, water soluble chitosan nanoparticles, Metformin, and Metformin/water soluble chitosan nanoparticles (C).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5805377/v1/ba8857f1dd4772fd9e61ad2d.png"},{"id":73622796,"identity":"e84e4b1e-d438-413e-9b43-c92d70bff1af","added_by":"auto","created_at":"2025-01-13 04:40:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":746486,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhotomicrographs of sections in the pancreas of albino rats. [1, 2]:\u003c/strong\u003e Normal tissue sections showing normal appearance of tissue structure (normal islets of Langerhans (thin arrow), normal acini (arrowhead), and normal pancreatic arteries (broken arrow). [3,4]:\u003cstrong\u003e treated with ARG \u003c/strong\u003eshows a cluster of immune cells (lymphoid aggregate) observed (↨) Hemorrhage (star) and necrotic acini (▲). [5]: \u003cstrong\u003esuffering from pancreatitis treated by Cs-Met NPs, \u003c/strong\u003erecording normal appearance islets of Langerhans (thin arrow), normal acini (arrowhead), intralobular duct (↕). [6]\u003cstrong\u003e suffering from pancreatitis exposed to low doses of γ- radiation \u003c/strong\u003eshowing normal appearance of islets of Langerhans architecture (thin arrow) and dark staining acini with a slight detachment of secretory cells (arrow head). [7,8]:\u003cstrong\u003e suffering from pancreatitis treated by Cs-Met NPs and exposed to low doses of γ- radiation, \u003c/strong\u003erecording normal appearance islets of Langerhans (thin arrow), normal acini (arrowhead), intralobular duct, and interlobular duct (●).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5805377/v1/1e191546e1e3d5ca9264867d.png"},{"id":73624408,"identity":"d06cac1f-3bd8-47da-88dc-d57b1f3138a6","added_by":"auto","created_at":"2025-01-13 05:04:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2137509,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5805377/v1/37bea9ff-b3eb-4bc8-874c-bcddabda69ff.pdf"},{"id":73622794,"identity":"c5dd5a2f-b720-48cc-a88f-92b2453ed2cc","added_by":"auto","created_at":"2025-01-13 04:40:55","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18302,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-5805377/v1/03fcbc902cf9a4ee88d902c7.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eAnti-inflammatory response of Metformin Nanoparticles and/or low doses γ-Irradiation on Acute Pancreatitis in Rats via regulation of inflammatory cytokine\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAcute pancreatitis (AP), is defined as an inflammatory disorder of the pancreas. It is one of the most common and severe gastrointestinal diseases. It leads to a systemic inflammatory response to multiple organ dysfunction syndrome (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the body an amino acid called L-arginine (L-arg) is necessary to make proteins, improves wound healing, and decreases recovery time after surgery (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). many authors reported that L-arg induces AP in experimental animals (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMetformin is an antidiabetic, reduces hepatic glucose production and insulin resistance, and is an oral therapy for type 2 diabetes (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Also, it has been found to possess some interesting properties such as antioxidant, anticancer, anti-inflammation, and anti-fibrosis, which are interesting for its use as an adjuvant in oncology(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Metformin has been used in clinical settings for good safety and limited toxicity. Many reports showed that it has no effect on glucose levels in nondiabetic individuals and the use of as adjuvant therapy (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Metformin lower cancer incidence and cancer-specific deaths have been reported among diabetics compared to diabetics on other anti-diabetic medications (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBiomedical nanotechnology has made technological fabrication in drugs delivery. Formulating of drugs into nanoparticulate drug delivery has demonstrated distinct advantages over traditional dosage forms (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). For oral drug delivery, nanoparticulate of drugs could significantly improve dissolvability, stability and oral absorption efficiency than free drugs in conventional formulations (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eChitosan is a modified biopolymer, originates from the crustacean shells of creatures like prawns and crabs. The USA FDA has approved that ita highly basic polymer for tissue engineering and drug delivery (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) used in the majority of pharmaceutical industries and research. Chitosan as a nanocarriers are effective at low dosage with a controlled release profile while reducing the side effects accompanied by high dosage administrations. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePre-treatment with non-lethal low doses of gamma radiation have a protective effect against oxidative injury in animal tissues. Low doses of irradiation are generally regarded as safe and its effect is considered to be negligible. Low-dose of gamma irradiation has been extensively indicated by induction of hormesis and adaptive response (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Low-dose of gamma irradiation was reported to stimulate some biological activities in vitro as well as in vivo, activation of immune functions in addition, it has been shown to enhance the efficacy of chemotherapy and immunotherapy (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe current study prepared to investigate the possible Anti-inflammatory response of Cs-Met NPs and/or low doses of γ- irradiation against acute pancreatitis in rat's model.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Materials:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.1 Drugs and chemicals:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMetformin; L-Arginine; and Chitosan were purchased from (Sigma Chemicals Co., U.S.A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.2. Irradiation Process:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGamma irradiations were carried out in the National Center for Radiation Research and Technology (NCRRT), Egyptian Atomic Energy Authority. For preparation of nanoparticles, Chitosan was gamma-irradiated with 200 kGy, using Canadian Co\u003csup\u003e60\u003c/sup\u003e \u0026gamma;-cell-220 sources, at a dose rate of 1.14 kGy/h. On the other hand, experimental rats were exposed to 0.25 Gy twice/week for 2 weeks\u0026rsquo; whole-body gamma radiation using Canadian Cs-137 \u0026gamma;-cell-40, at a dose rate of 0.423 Gy/min which was calculated according to the dosimetry department guidelines at the NCRRT at the time of the experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.3 Preparation of Cs-Met NPs:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMetformin-loaded chitosan Nanoparticles were successfully formulated by physical degradation methods, using \u0026gamma;-irradiation (200 KGy) as follow;\u003c/p\u003e\n\u003cp\u003e1. Colloidal water-soluble chitosan nanoparticles were synthesized via \u0026gamma;-radiolysis technique in which chitosan (1.5 g) was dissolved in 100 mL of 1% (v/v) aqueous acetic acid at 60 \u0026deg;C using a magnetic stirrer for 2 h until the polymer becomes completely soluble. The solubilized chitosan was then irradiated (200 kGy \u0026gamma;-irradiation) and neutralized by using 1 M NaOH solution. By filtration and removed of water insoluble part a clear solution of neutralized water-soluble chitosan was obtained. The degradation yield and formation of water-soluble chitosan was calculated by the following equation; Yield % = (W1- W2)/W1) x100, Where W1 and W2 represent the weights of the original chitosan and the water-insoluble chitosan after neutralization, respectively. The yield was found 96% and this result is consistent with Choi et al. (18).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2. After that, for therapeutic applications, 1.5 g metformin was added to 100 mL of neutralized water-soluble chitosan nanoparticles at room temperature using a magnetic stirrer for 3 hours until the metformin became completely soluble.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.4 Characterization of Cs-Met NPs:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUV\u0026ndash;Vis spectrophotometry as an analytical tool was used to investigate the formation of water-soluble chitosan nanoparticles. UV\u0026ndash;Vis measurements were taken by a Unicam double beam UV\u0026ndash;Vis spectrophotometer. The structural analysis was performed using a Fourier Transform Infra-Red (FTIR) spectrophotometer (Bruker Vertex 70). On the other hand, the shape and size of the synthesized nanoparticles were obtained via a high-resolution transmission electron microscopy, HR-TEM (JEOL-JEM-100 CX).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.5 Experimental animals:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale Swiss albino rats weighing (100 \u0026plusmn; 20) g were used in this study. They were purchased from the Egyptian National Authority for Drug Research and Control, Ministry of Health, Cairo, Egypt. The animals were housed under regular conditions (12-h light/12-h dark cycle, normal temperature, good ventilation, and humidity level) in specially built plastic cages, ten per each. Throughout the study, drinking water and food were supplied \u003cem\u003ead libitum\u003c/em\u003e. All experimental procedures were performed in compliance with the standards and guidelines of the National Research Centre Ethics Committee, issued by the U.S. National Institutes of Health, \u0026ldquo;Guide for the treatment and use of laboratory animals\u0026rdquo; for the use and protection of experimental animals (NIH publication No. 85\u0026ndash;23, 1996).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.6 Animal classification (Induction of acute pancreatitis and drug treatment):\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe rats were equally classified into five groups of six rats each, as follows:\u003c/p\u003e\n\u003cp\u003e1. Control group [G1]: Rats in this group were kept as controls.\u003c/p\u003e\n\u003cp\u003e2. Acute pancreatitis (AP) group [G2]: Rats were injected intraperitoneally (i.p.) with L-arginine (250 mg/100g b.wt., twice at 1-hour intervals), day over day for 14 days to induce acute pancreatitis (19).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3. AP + IR group [G3]: Rats were injected with an L-arginine-like group [2] and after that, they were exposed to whole body \u0026gamma;-radiation at a dose level of 0.25 Gy twice/week for 2 weeks (0.5 Gy/ week, X2).\u003c/p\u003e\n\u003cp\u003e4. AP + Cs-Met NPs group [G4]: Rats were injected with an L-arginine-like group [2] and after that, they were treated with Cs-Met NPs at a dose of 46.8 mg/kg body weight, daily for 14 days(15).\u003c/p\u003e\n\u003cp\u003e5. AP + IR + Cs-Met NPs group [G5]: Rats were injected with an L-arginine-like group [2] and after that, they were exposed to whole body \u0026gamma;-radiation like group [3] and were treated with Cs-Met NPs like group [4].\u003c/p\u003e\n\u003cp\u003eAt the end of the experiment, all rats were anesthetized by using urethane before being sacrificed and then the blood was collected via heart piercing by using disposable plastic syringes. The coagulated blood samples were centrifuged at 3000 rpm for 15 min and the serum was collected for different estimates of biochemical parameters. For further biochemical research, pancreatic tissues were dissected, rinsed in ice-cold isotonic saline, blotted dry with a filter paper, and stored at \u0026minus;20 \u0026deg;C. Portions of pancreatic tissue were rinsed and set for histopathological examination in 10% neutralized formalin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Methods:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.1 Measurement of biochemical parameters:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eActivities of amylase and lipase were evaluated in serum, using a diagnostic kit purchased from TRUEchemie Company, India. The results were expressed as U/I. Serum glucose was measured by the kinetic method of Kaplan \u003csup\u003e(20)\u003c/sup\u003e using a commercial kit obtained from (SPINREACT Company, Spain).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIL-6 was determined by a sandwich enzyme-linked immunosorbent assay (ELISA) using rat kit given by (R\u0026amp;D Systems, Inc., Minneapolis, USA). While, TNF-\u0026alpha;, IL-8, and IGF1 levels were determined by the ELISA rat kit provided by (CUSABIO, USA). IL-10, and CRP levels were determined by the ELISA rat kit provided by (MyBioSource, USA). Serum insulin was determined by the ELISA rat kit provided by (CELL BIOLABS, INC. Company, USA). According to the manufacturer\u0026rsquo;s instructions, the ELISA microplate was read using an ELISA reader with an absorbance maximum of 450\u0026thinsp;nm. The parameter levels were calculated after plotting the standard curves and expressed as pg/mL.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.2 Histopathological examination:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSamples of pancreas tissue were fixed in 10% formaldehyde solution and inserted in paraffin using standard methods. Sectioned tissues at 5 \u0026mu;m thickness were treated with hematoxylin-eosin (H\u0026amp;E) stain for routine examination using light microscopy according to the method of\u0026nbsp;Bancroft and Stevens (21).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.3 Statistical analyses:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe differences in means of variables between groups were estimated using a one-way analysis of variance (ANOVA) with the Least Significant Difference (LSD). The obtained results were expressed as the mean \u0026plusmn; SE and were examined by Statistical Package for Social Science (SPSS) version 20 for Windows (SPSS\u0026reg; Chicago, IL, USA) software program. At P \u0026lt;0.05 or P\u0026lt;0.01, the probability was considered significant and was considered highly significant at P\u0026lt;0.001.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Characterization of Cs-Met NPs:\u003c/h2\u003e\n \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.1 FTIR analysis\u003c/h2\u003e\n \u003cp\u003eThe FTIR spectra of Cs, water-soluble chitosan nanoparticles (Cs NPs), Met. and CS-Met. NPs are presented in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. (C) The spectrum of CS reveals a broad band at 3388 cm-1 which is related to \u0026ndash;NH2 and \u0026ndash;OH stretching vibration on the CS chains. The two bands at 2980\u0026thinsp;\u0026minus;\u0026thinsp;2880 cm-1 are assigned to the CH stretching vibrations. The peak at 1645 cm-1 is attributed to C\u0026thinsp;=\u0026thinsp;O stretching in the amide group due to the partially acetylated amino groups. The peak of 1575 cm-1 is attributed to N\u0026ndash;H bending in the amine group that is present in protonated form (NH3+). The band at 1385 cm-1 is due to the C-N stretching vibration. In addition, the peak at 1463 cm-1 may be assigned to the O\u0026ndash;H/C\u0026ndash;H deformations. The two bands at 1152 cm-1 and 1077 cm-1 correspond to the asymmetric stretching vibration of the C\u0026ndash;O\u0026ndash;C of glycosidic linkage and C\u0026ndash;O\u0026ndash;C stretching in the saccharide structure (22).\u003c/p\u003e\n \u003cp\u003eThe FT-IR spectrum of irradiated chitosan (Cs NPs) revealed all characteristic peaks as presented in pure CS. This result indicates that no functional groups were added or deleted but some were modified due to oxidation, as no other chemical agents or moieties were added during the irradiation process. The increase in intensity of peaks at 1714 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e can be ascribed to increased C\u0026thinsp;=\u0026thinsp;O bonds formed by chain scission reaction and oxidation of chitosan due to irradiation (\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e). The intensity of peak at 3400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e increased after irradiation which indicates a possibility of increment in a number of -OH groups resulting from the scission of glycosidic bonds leading to the formation of hydroxyl group (\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe MET drug contains three types of N-H groups which show three bands at 3366, 3300, and 3153 cm-1. The CH stretching vibrations are assigned at 2980\u0026thinsp;\u0026minus;\u0026thinsp;2880 cm-1. The bands at 1629 and 1552 cm-1 are due to C\u0026thinsp;=\u0026thinsp;N stretching vibrations. The CH deformations are assigned at 1461 and 1384 cm-1. The C-N stretching vibration occurs at 1220\u0026thinsp;\u0026minus;\u0026thinsp;1020 cm-1. The peaks at 938 and 809 cm-1 are due to N-H wagging(\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eCompared with the FTIR spectra of CS and CS-MET, there was no significant change in the vibration of the backbone of CS before and after MET addition, which indicated that the adsorbed MET did not alter the formed structure. Furthermore, the reduced intensity of the metformin N-H group at 3379 cm-1 indicates the possibility of hydrogen bonding between the metformin and CS nanoparticles (\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.2 Morphological Characterization\u003c/h2\u003e\n \u003cp\u003eThe shape, dispersion, and average particle size of as prepared materials were determined by Transmission Electron Microscopy (TEM) analysis. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e (A, B), the TEM analysis of chitosan nanoparticles (Cs NPs) and chitosan-metformin nanoparticles (Cs-Met NPs) confirms that the particles were in the nano-dimensional range with spherical shape with the inter-particular connection among them, smooth surface and size range of approximately 8 nm (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA) and 12.5 nm (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB) respectively. The spherical appearance of Cs NPs can be attributed to the shortening CS chains densely packed with each other into particles, while Cs-Met NPs have fewer regular surfaces and edges due to the attachment of metformin on the chitosan nanoparticles surface.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Effect of Cs-Met NPs and low doses \u0026gamma; radiation on the grade of AP.\u003c/h2\u003e\n \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.1 Histological findings\u003c/h2\u003e\n \u003cp\u003eIn the normal control section of the pancreas stained with H\u0026amp;E stain, the normal histological structure was observed. The pale staining of the spreading islets of Langerhans containing a spherical cluster of polygonal cells with fine secretory granules (endocrine gland) among the dark staining well-organized densely packed pyramidal-shaped cells of the acini (exocrine gland) linked by connective tissues and blood vessels (Figs.\u0026nbsp;\u0026lt;link rid=\u0026quot;fig2\u0026quot;\u0026gt;\u003cspan class=\"InternalRef\"\u003e2\u0026lt;/link\u0026gt;\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e \u0026amp; \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eMeanwhile, histopathological changes appeared in the pancreas in rats treated with Arg. After 24 h. of all treatments end showing a cluster of immune cells, lymphoid aggregates, hemorrhage, and necrotic acini (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e (\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e)). Furthermore, sections in the pancreas of albino rats suffering from pancreatitis exposed to low doses of \u0026gamma;- radiation showed the normal histological appearance of tissue structure, the normal architecture of islets of Langerhans and dark staining acini with slight detachment of secretory cells after 24 h post treatments end (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e (\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e)).\u003c/p\u003e\n \u003cp\u003eTreatment of albino rats suffering from pancreatitis by Cs-Met NPs recording normal appearance islets of Langerhans, normal acini (arrowhead), intralobular duct, and interlobular duct after 24 h of treatments end (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e (\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e)). Whenever albino rats suffering from pancreatitis treated by Cs-Met NPs and exposed to low doses of \u0026gamma;- radiation recording normal appearance islets of Langerhans, normal acini, intralobular duct, and interlobular duct after 24 h post treatments end (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e)).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.2 Serological findings:\u003c/h2\u003e\n \u003cdiv id=\"Sec21\" class=\"Section4\"\u003e\n \u003ch2\u003e3.2.2.1 Pancreatic enzymes activities\u003c/h2\u003e\n \u003cp\u003eThe clinical key criteria for the diagnosis of acute pancreatitis were mylasemia and lipasemia (high amylase and lipase activity) are among. In this study, the activities of serum amylase and lipase in the L-arginine-treated group were elevated when compared to the control at the end of the experiment suggesting a successful induction of AP. However, treating the animals suffering from acute pancreatitis with Cs-Met NPs and/or exposed to \u0026gamma;- radiation produced substantial suppression of amylase and lipase activity (Table 1).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec22\" class=\"Section4\"\u003e\n \u003ch2\u003e3.2.2.2 Glycaemic parameters\u003c/h2\u003e\n \u003cp\u003eOne of the complications of AP is the endocrine dysfunction, specifically impaired glucose metabolism. Moreover, the glucose level is closely correlated with the inflammatory responses in AP, which can affect the progression of the disease. The data of the current study represented in table (2) revealed that after the induction of AP in rats was associated with a slight elevation in the glucose levels accompanied with low levels of insulin, when compared with the control group. On the other hand, treatment with fractionated low doses of \u0026gamma;-IR and Cs-Met NPs either alone or in combinations reversed these results and resulted in maintaining levels of both glucose and insulin. Therefore, a high level of glucose can be used as an indicator for evaluating the severity of AP.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec23\" class=\"Section4\"\u003e\n \u003ch2\u003e3.2.2.3 Inflammatory markers\u003c/h2\u003e\n \u003cp\u003ePancreatitis is identified by the destruction of acinar cells besides the activation of inflammatory cells (macrophages and neutrophils) thus, a significant change in the levels of many inflammatory mediators was observed. Systemic manifestations of the AP are mediated by a variety of pro- and anti-inflammatory mediators released from the injured pancreas. Local recruitment and activation of inflammatory cells in AP lead to the production of inflammatory markers, such as IL-6 \u0026amp; IL-8, TNF-\u0026alpha;, and CRP which are important markers in predicting the severity of AP.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAs shown in Table\u0026nbsp;(3)\u003c/strong\u003e The obtained data showed a dramatic increase in the levels of pro-inflammatory cytokine (IL-6, IL-8, TNF-\u0026alpha;, and CRP) levels in the pancreatic tissues of rats injected with L-arginine (AP) relative to control levels.\u003c/p\u003e\n \u003cp\u003e\u0026bull; \u003cstrong\u003eIL-6\u003c/strong\u003e:\u003c/p\u003e\n \u003cp\u003eThe obtained data showed a dramatic increase in the levels of IL-6 in the pancreatic tissues of rats with AP relative to control levels. In contrast, exposing rats with AP to low doses of gamma radiation, or treatment with Cs-Met NPs either alone or in combinations for two weeks, significantly reduced the IL-6 when compared to the animals with acute pancreatitis.\u003c/p\u003e\n \u003cp\u003e\u0026bull; \u003cstrong\u003eIL-8\u003c/strong\u003e:\u003c/p\u003e\n \u003cp\u003eAs shown in table (3) a significant increase in the levels of IL-8 was detected in pancreatic tissues of the animals injected with L-arginine as compared to the control group. However, treating AP-bearing rats with low doses of IR, and Cs-Met NPs either alone or in combinations for two weeks, resulted in a notable inhibition of the IL-8 levels when compared to the AP group.\u003c/p\u003e\n \u003cp\u003e\u0026bull; \u003cstrong\u003eTNF-\u0026alpha;\u003c/strong\u003e:\u003c/p\u003e\n \u003cp\u003eThe statistical comparison of the levels of TNF-\u0026alpha; in the pancreatic tissues between the different studied groups revealed that it was significantly increased in the L-arginine group (AP) as compared to the control group. Conversely, there was a marked decrease in the levels of TNF-\u0026alpha; upon treatments with low doses of IR and Cs-Met NPs either alone or in combinations for two weeks as shown in table (3).\u003c/p\u003e\n \u003cp\u003e\u0026bull; \u003cstrong\u003eCRP\u003c/strong\u003e:\u003c/p\u003e\n \u003cp\u003eCRP is a well-known marker of the severity of AP. The obtained data in table (3) showed a significant increase in the levels of CRP in the pancreatic tissues of the AP group compared to the control group. In contrast, exposing rats with AP to low doses of gamma radiation, or treatment with Cs-Met NPs either alone or in combinations for two weeks, significantly lowered the levels of CRP compared to the pancreatitis group.\u003c/p\u003e\n \u003cp\u003e\u0026bull; \u003cstrong\u003eIL-10\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAs shown in Table\u0026nbsp;(4), the levels of pancreatic IL-10 markedly reduced in the pancreatitis group compared with the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while using different treatments significantly elevated the levels of IL-10 compared to the AP group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\n \u003cp\u003e\u0026bull; \u003cstrong\u003eInsulin-like growth factor 1\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eInsulin-like growth factor 1 (IGF-1) is a protein that belongs to the IGF axis. IGF-1 plays a role in the regulation of \u0026beta;-cell mass and the regulation of insulin secretion and sensitivity. Lots of studies show close links between the IGF axis and glucose metabolism, including pancreatic diseases. Many pro-inflammatory cytokines (IL-6, TNF-\u0026alpha;) impair the activity of the IGF-1 axis. The obtained data in table (4) pointed out a remarkable decline in the concentration of IGF1 in the pancreatic tissues of the AP group compared to the control group. While, exposing rats suffering from AP to \u0026gamma;-IR, or treatment with Cs-Met NPs either alone or in combinations for two weeks, significantly enhanced and increased the concentration of IGF1 compared to the AP group.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn medicine, natural products like chitosan are used as drug carriers due to the encapsulation of a broad range of therapeutic agents that deliver to the target site efficiently. Chitosan-based nanoparticles have good biodegradation and biodistribution in the biological milieu, which have made it as one of the most attractive nanocarriers for delivering different therapeutic agents to the tumor cells (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Nowadays, chitosan nanoparticles have become of great interest in nanomedicine, biomedical engineering, and the development of new therapeutic drug release systems with improved bioavailability increased specificity and sensitivity, and reduced pharmacological toxicity (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOn the other hand, Metformin is the most commonly used oral anti-diabetic drug in the world. It has been in clinical use for more than 50 years and has a good safety record with limited toxicity. Lower cancer incidence and cancer-specific deaths have been reported among diabetics on metformin compared to diabetics on other anti-diabetic medications (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Therefore, our study was designed to evaluate the anti-inflammatory and protective effect of the Chitosan-Metformin nanocomposites (Cs-Met NPS) on L-arginine-induced acute pancreatitis and its complications on body tissues in Wistar albino rats.\u003c/p\u003e \u003cp\u003eAcute pancreatitis (AP) is a common severe critical illness with a high mortality rate (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Death due to AP may result from systemic inflammatory response syndrome or multiple organ dysfunction syndromes. Sometimes repeated attacks of AP lead chronically to loss of pancreatic function and fibrosis(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). AP is characterized by inflammation, apoptosis of pancreatic acinar cells, and the release of pancreatic enzymes, including amylase and lipase (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, arginine injection effectively induced acute pancreatitis in rats, markedly through histological changes, higher pathological scores in the pancreas. Pancreatitis was further evidenced by hyperamylasemia and hyperlipasemia, which is consistent with the earlier results of (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur results revealed that injection of L-arginine induced acute pancreatitis represented by a remarkable elevation of the pancreatic amylase and lipase levels. These results are in accordance with that of Salem et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) who reported that the elevated levels of the pancreatic enzymes, mainly amylase and lipase, may be caused by the production of hydrolytic enzymes in AP which hydrolyse phospholipids to liberate arachidonic acid and lysophospholipids and the latter has a cytotoxic function, causing acinar cells necrosis. Furthermore, Wang et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) reported that L-arginine selectively destroys pancreatic acinar cells by inducing amino acid imbalance, decreasing the synthesis of polyamine, nucleic acid and proteinase and resulting in excessive activation of the zymogen.\u003c/p\u003e \u003cp\u003eOn the other hand, Yang et al.(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) indicated that the interstitial leakage of pancreatic lipase triggered adipose lipolysis and increased levels of unsaturated fatty acids. These toxic fatty acids stimulate the excessive release of inflammatory markers and an inflammatory storm that can drive disease progression with eventual multi-organ failure. AP is an inflammatory disease of the pancreas with the involvement of both local tissues and distant organs (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). The obtained results revealed a remarkable increase in the levels of IL-6, IL-8, TNF-α and CRP accompanied with a significant reduction in the levels of IL-10 in the group of acute pancreatitis induced by L-arginine. Our results were similar to that of Al-Hashem (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e) who found that toxic doses of L-arginine induced pancreatic tissue injury and increased the pro-inflammatory mediators such as TNF-α coupled with a reduction in the anti-inflammatory cytokine IL-10.\u003c/p\u003e \u003cp\u003eThe prevalence of AP may lead to a systemic illness that may progress to multiple organ dysfunction and even death. Inflammatory cytokines like TNF-α, IL-6 and IL-8 generated during the pathogenesis of AP are considered responsible for the development of multiple organ failure (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt was reported that high-level glucose can be used as one of the reference indicators for evaluating the severity of AP in clinical practice (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). The obtained results revealed a remarkable increase in the levels of glucose coupled with a significant decrease in the insulin levels in the group of acute pancreatitis induced by L-arginine compared to the control group. this is in accordance with Shoman and Nafeh (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) who reported that AP affects not only exocrine pancreatic function, manifested by significantly higher serum amylase and lipase levels, but also affects pancreatic endocrine function as manifested by decreased fasting plasma insulin (FPI) levels in association with hyperglycemia.\u003c/p\u003e \u003cp\u003eHerein, treatment with metformin-loaded chitosan nanoparticles alone or with low-dose gamma radiation ameliorated the pancreatic injury induced by L-arginine. This was attributed to the antioxidant, anti-inflammatory, antibacterial, and immunomodulatory properties of chitosan (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Moreover, Borai et al.(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e) reported that ChNPs alone and together with low doses of γ-radiation markedly reduced the pancreatic enzymes, lipase and amylase, and diminished the excessive release of the proinflammatory cytokines, TNF-α due to their anti-inflammatory antioxidative activity.\u003c/p\u003e \u003cp\u003eMetformin acts not only as a glucose-lowering drug but exhibits additional benefits, including moderate anti-inflammatory and anti-oxidative effects (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Sena et al.(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e) reported that the anti-inflammatory actions of metformin were by suppressing the main components of inflammation (endothelial cells and smooth muscle cells, monocytes, macrophages and other cell types) and restoring cell functions. Moreover, metformin not only reduced common inflammatory cytokines such as TNF-α, IL-1β and IL-6 but also counteracted macrophage infiltration and M1 polarisation into anti-inflammatory macrophages (M2) in a mouse model of olanzapine-induced insulin resistance (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt has been suggested that metformin improves metabolic parameters such as hyperglycemia, and insulin resistance thereby reducing chronic inflammatory responses (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Metformin reduces blood glucose levels primarily by decreasing hepatic glucose production through suppression of gluconeogenesis, ameliorating insulin signaling leading to reduction the intestinal glucose absorption, and improving glucose uptake by peripheral tissues, such as skeletal muscle and adipose tissue (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInterestingly, it was reported that metformin could have direct protective effects on β-cells under metabolic stress, including non-diabetic and T2D human islet cells via alleviating the oxidative stress and endoplasmatic reticulum stress which are responsible for pancreatic β-cells destruction (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Moreover, loading metformin with Chitosan NPs enhanced the beneficial effect of metformin because Chitosan NPs can influence the cells of the immune system too. They can enhance the response of the immune system by increasing the maturation of some antigen-presenting cells named dendritic cells. Furthermore, by the means of apoptotic pathway activation, these NPs are also able to start the function of innate immunity(\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt was reported that a low dose of IR is essential to life, acknowledging that the natural production of ROS that is adequate to stimulate the protective systems and provoke a beneficial health effect which is known as radiation hormesis (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Our results show that LDR was associated with a decrease in the circulating levels of some markers of inflammation, such as CRP concentrations, and an increase in the levels of the anti-inflammatory cytokines IL-10. Paradoxically LDR (0.5\u0026ndash;1.5 Gy) acts on cells (endothelial cells, polymorphonuclear leukocytes, and macrophages) involved in the inflammatory response, producing anti-inflammatory effects, and increased production of cytokines (IL-10) by endothelial cell and immune cells. Presumably, it is in this phase where LD-RT to both lungs could be effective by acting as a powerful anti-inflammatory agent against the cascade of proinflammatory cytokines (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). From the previous discussion we conclude that Cs-Met NPs exhibits strong therapeutic effects in the course of AP. Hence, the use of Cs-Met NPs as adjuvant treatment in AP is recommended. However, further studies must be carried out to determine the proper dose and route of administration to achieve the best outcome for treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper. The authors declare that they did not receive any financial support from any organization for the research, authorship, and/or publication of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Mohamed Bekhet (Radiation Research of Polymer Chemistry Department, National Center for Radiation Research and Technology, Egyptian Atomic Energy Authority) for preparation of Cs-Met NPs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental animals have been handled under the standards and guidelines of the National Research Center Ethics Committee published by the U.S. National Health Institutes (NIH publication No. 85-23, 1996). Additionally, the present study was approved by the Institutional Animal Care and Use Committee Research Ethic Board, Faculty of Medicine, Zagazig University, Egypt (Approval No. ZU-IACUC/1/F/28/2019).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data obtained from this study are included in the current manuscript.\u0026emsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding of Declaration:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo Funding\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbu-Hilal M, McPhail M, Marchand L, Johnson CD (2006) Malondialdehyde and superoxide dismutase as potential markers of severity in acute pancreatitis. Jop 7(2):185\u0026ndash;192\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLankisch P, Apte M, Banks P (2015) Acute pancreatitis. 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J Biol Regul Homeost Agents 34(2):327\u0026ndash;331\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"National Center for Radiation Research and Technology, Egyptian Atomic Authority,Cairo, Egypt","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Acute pancreatitis, Metformin nanoparticles, Gamma Irradiation, low-dose radiation, inflammatory cytokine","lastPublishedDoi":"10.21203/rs.3.rs-5805377/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5805377/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRepeated pancreatic insult inflammation leads to the development of pancreatitis. In these studies the evaluation of nano-formulated metformin drug with chitosan (Cs-Met NPs) and/or low-doses of γ-irradiation (IR) against acute pancreatitis (AP) induced by L-arginine in an animal model. Thirty adult of Albino rats were divided randomly into 5 groups. Normal control, L-Arginine treated (AP); AP\u0026thinsp;+\u0026thinsp;IR; AP\u0026thinsp;+\u0026thinsp;Cs-Met NPs and AP\u0026thinsp;+\u0026thinsp;IR\u0026thinsp;+\u0026thinsp;Cs-Met NPs. Histopathological studies of pancreatic and biochemical parameters (serum amylase and lipase levels, Random blood glucose (RBS), plasma insulin level, Insulin growth factor 1, tumor necrosis factor-alpha (TNF-α), IL-6, IL-8, IL-10, and CRP were measured in the pancreatitis model. AP induced by L-Arginine- treatment elevates the serum pancreatic amylase \u0026amp; lipase levels, significantly increases IL-6, IL-8, TNF-α, RBS, and CRP, and significantly decreases IL-10, insulin levels, and Insulin growth factor 1. Microscopic examination revealed loss of the pancreatic lobular architecture, marked fibrosis, acinar degeneration, inflammation, and marked oedema. All the serological parameters and the histopathological observations were markedly improved by Cs-Met NPs administration and/or low doses of γ-irradiation treatment. In conclusion, Cs-Met NPs and/or low doses of γ- irradiation have a therapeutic effect on acute pancreatitis model induced in rats.\u003c/p\u003e","manuscriptTitle":"Anti-inflammatory response of Metformin Nanoparticles and/or low doses γ-Irradiation on Acute Pancreatitis in Rats via regulation of inflammatory cytokine","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-13 04:40:51","doi":"10.21203/rs.3.rs-5805377/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4e81bb7a-76c4-42cf-b6c6-9fc50834f549","owner":[],"postedDate":"January 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":42699211,"name":"Applied Biochemistry"},{"id":42699212,"name":"Biochemical Research Methods"},{"id":42699213,"name":"General Biochemistry"},{"id":42699214,"name":"Drug Delivery"}],"tags":[],"updatedAt":"2025-01-21T02:38:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-13 04:40:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5805377","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5805377","identity":"rs-5805377","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}