Paeonol alleviates acute pancreatitis in mice by inhibiting inflammatory response and oxidative stress and activating Nrf2/HO-1 signaling pathwa

To study the immunologic mechanism in pathogenesis of the acute pancreatitis (AP) and the intervention effects of sandostatin and magnolol. Ninety BALB/c mice were divided into negative control group, caerulein-induced model group, sandostatin-treatment group, magnolol-treatment group, combined sandostatin- and magnolol-treatment group by means of random number table, with 18 mice in each group. AP model was reproduced by seven intraperitoneal injections of caerulein at an interval of 1 hour. Every 30 minutes before the caerulein challenge, sandostatin was injected sub- cutaneously. Magnolol was injected intravenously immediately after the AP model was reproduced. Then at 9, 12, 24 hours after modelling, blood was drawn from orbital vein and serum was separated. Serum amylase (SA), interleukin-10 (IL-10) and gamma-interferon (IFN-gamma) were determined after the mice were sacrificed, and pancreas and spleen were harvested . The pathological changes of pancreas were observed, and the number and the ratio of myeloid- dendritic cells (MDCs) to lymphoid dendritic cells (LDCs) were measured with flow cytometry. Compared with control group [SA (1.12 + or - 0.05) kU/L, pancreatic score (PS) 0.09 + or - 0.10], both indexes increased progressively in the model group [SA (26.11 + or - 1.96) kU/L, PS 5.32 + or - 0.19, both P<0.01]. The ratio of MDCs/LDCs showed downward tendency at every time-point especially at 9th hour (0.421 + or - 0.049 vs. 1.712 + or - 0.372, P<0.05), while the ratio of IL-10 to IFN-gamma did not show significant differences. Compared with model group, both SA and PS significantly decreased in all the three drug intervention groups [SA (kU/L): 18.25 + or - 1.09, 17.32 + or - 1.26, 17.62 + or - 0.56, PS: 4.55 + or - 0.15, 4.16 + or - 0.18, 4.10 + or - 0.13, all P<0.01]. There was no significant difference in the two ratios of MDCs/LDCs and IL-10/IFN-gamma between sandostatin-treatment group and model group. However, the ratio of MDCs/LDCs of the magnolol-treatment group was higher than that in sandostatin-treatment group 9, 12, 24 hours after modelling (9 hours: 4.694 + or - 0.527 vs. 0.819 + or - 0.182, 12 hours: 2.566 + or - 0.463 vs. 1.421 + or - 0.163, 24 hours : 2.343 + or - 0.359 vs. 1.421 + or - 0.113, P<0.05 or P<0.01). At every time-point, the ratio of IL-10/IFN-gamma in the magnolol-treatment group was significantly higher compared with the model group, and at the 12-hour point, it was higher than that of sandostatin-treatment group (8.000 + or - 1.738 vs. 3.558 + or - 0.362, P<0.05 ). The combined treatment group showed similar changes as the magnolol-treatment group. When AP occurs, the differentiation from T helper (Th0) to Th1 is augmented, while differentiation of Th0 to Th2 decreases, thus inducing an imbalance in the relationship of pro- and anti-inflammatory response. Magnolol can induce the differentiation from Th0 to Th2 by modulating the different subtype dendritic cells, thus improving the anti-inflammatory response, resulting in attenuating local and systemic inflammatory response in AP. However, sandostatin did not show such effect.

Objective To investigate the effect and mechanism of Bafiomycin A1 on acute pancreatitis in mice. Methods 60 BALB/c mice were randomly divided into control group, model group, low dose group and high dose group (15 mice in each group). Model group, low dose group and high dose group of mice by intraperitoneal injection of caerulein 50 g/kg, 1/h, a total of 7 times to establish the model of acute pancreatitis. Mice in low dose group and high dose group were intraperitoneally injected with 25 and 75 μg/kg Bafiomycin A1. Mice in the control group and model group were injected with equal volume of normal saline intraperitoneally. Mice in four groups were sacrificed in 24 h after treatment. Serum amylase in mice (AMY), inflammatory factor tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were analyzed by enzyme linked immunosorbent assay (ELISA). The mouse pancreatic lesions and score (Schmidt score) were analyzed by hematoxylin and eosin (HE) staining. autophagy substrate p62 and microtubule-associated protein 1 light chain 3 (LC3) were detected by Western blotting. The pancreatic tissue p62 were analyzed by immunofluorescence. Results Compared with the model group [(4 910.98±349.43) U/L, (109.43±10.43) pg/L, (329.16±20.91) pg/L, 9.43±3.98], the AMY, TNF-α, IL-6 and Schmidt scores of the low dose group [(3 109.13±301.44) U/L, (80.18±9.34) pg/L, (215.32±18.43) pg/L, 6.48±3.18] and the high dose group [(1 432.33±187.43) U/L, (45.19±8.14) pg/L, (153.22±12.63) pg/L, 3.14±1.02] significantly decreased (P=0.000). Compared with the low dose group, the AMY, TNF-α, IL-6 and Schmidt scores of the high dose group more significantly decreased. Compared with model group (0.15±0.06, 0.56±0.06), p62 and LC3 in pancreatic tissue of mice in low dose group (0.39±0.09, 0.75±0.09) and high dose group (0.61±0.07, 0.99±0.08) obviously enhanced (P=0.000). Compared with the low dose group, the accumulation of p62 and LC3 in the pancreatic tissue of the high dose group more obviously accumulated (P=0.000). Conclusion Bafiomycin A1 can significantly inhibit the level of autophagy in pancreatic tissue of mice with acute pancreatitis and play a protective role in pancreatic tissue. Key words: Acute pancreatitis; Autophagy; Bafiomycin A1; Inflammation

Objective To observe the effects of massage on inflammation, oxidative stress and autophagy during the repair of acute contusion of skeletal muscles so as to explore its biological mechanisms. Methods Forty-two adult Sprague-Dawley rats were randomly divided into a control group (n=6), a model group (n=18), and a treatment group (n=18). Acute contusion of the gastrocnemius muscles of the rats in the model and treatment groups was inflicted using a home-made impactor. Beginning forty-eight hours later, 15 minutes of massage was administered daily for two weeks. After one, 7 and 14 days of the massage treatment, the injured gastrocnemius was resected from 6 rats of both the model and treatment groups. Morphological changes were observed using haematoxylin and eosin (HE) staining. The serum content of tumor necrosis factor alpha (TNF-α), interleukin1β (IL-1β), C reactive protein (CRP) and prostaglandin E2 (PGE2) were detected using enzyme-linked immunosorbent assay (ELISA). The serum content of superoxide (SOD) and malondialdehyde (MDA) were detected using spectrophotometry. The expression of microtubule-associated protein 1 light chain 3(LC3), Bcl-2 homeodomain protein Beclin1 and ubiquitin binding protein P62 were detected using Western blotting. Results The HE staining showed more significant collapse and swelling of cells in the model group than in the control group at each time point. New muscle cells were observed at days 7 and 14 in the model group. At each time point, significantly better recovery was observed in the treatment group compared to the model group, with more new muscle cells and better cell morphology. According to the ELISA results, a significant increase in serum pro-inflammatory factors occurred in the model group compared to the control group and compared to the treatment group after one day and 7 days of treatment. The average serum content of SOD and MDA in the model group was significantly higher than in the control group, while the average serum content of SOD in the treatment group was significantly higher than in the model group and that of MDA was significantly lower. Western blotting showed a significant decrease in LC3 (II/I) and Beclin1, as well as a significant increase in P62 in the model group at each time point compared with the treatment group and the controls. Conclusion Inflammation and oxidative stress increase significantly in a skeletal muscle after injury, but autophagy decreases significantly. Massage can effectively reduce the inflammatory response and oxidative stress and promote autophagy, which leads to quicker repair of skeletal muscles. Key words: Massage; Skeletal muscles; Blunt injury; Inflammation; Oxidative stress; Autophagy

Cannabinoids Ameliorate Pain and Reduce Disease Pathology in Cerulein-Induced Acute Pancreatitis

Cordycepin protects against acute pancreatitis by modulating NF-κB and NLRP3 inflammasome activation via AMPK

The objective of this study was to investigate the potential protective effects of fucoidan, an L- and P-selectin modulator, in 2 murine models of acute pancreatitis. Acute pancreatitis was induced in mice either by the retrograde infusion of taurolithocholic acid sulfate into the pancreatic duct or by intraperitoneal injections of cerulein (50 μg/kg per hour). The experimental groups received fucoidan (25 mg/kg, intravenously) before pancreatitis induction, whereas control groups received only saline. After 24 hours, serum amylase, lipase, interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), and nitrite were measured. In addition, myeloperoxidase (MPO) activity (lung and pancreas) and histological assessment (pancreas) were determined. Serum amylase, lipase, nitrite, TNF-α, and IL-1β, and pancreatic and lung MPO were increased in both taurolithocholic acid sulfate and cerulein acute pancreatitis compared with the respective control groups. Fucoidan significantly decreased the augmented levels of amylase, lipase, pancreatic and lung MPO, TNF-α, IL-1β, and nitrite in both models. Pancreas histological changes observed in both acute pancreatitis models were significantly attenuated by fucoidan. Fucoidan reduced the severity of acute pancreatitis in mice by decreasing neutrophil infiltration and systemic inflammation, suggesting that modulation of selectins may constitute a promising therapeutic approach.

Adipose Triglyceride Lipase–Mediated Adipocyte Lipolysis Exacerbates Acute Pancreatitis Severity in Mouse Models and Patients

Background Danshen (Salvia miltiorrhiza Bunge) and its main active component Tanshinone IIA (TSA) are clinically used in China. However, the effects of TSA on acute pancreatitis (AP) and its potential mechanism have not been investigated. In this study, our objective was to investigate the protective effects of TSA against AP via three classic mouse models. Methods Mouse models of AP were established by caerulein, sodium taurocholate, and L-arginine, separately. Pancreatic and pulmonary histopathological characteristics and serum amylase and lipase levels were evaluated, and changes in oxidative stress injury and the ultrastructure of acinar cells were observed. The reactive oxygen species (ROS) inhibitor N-Acetylcysteine (NAC) and nuclear factor erythroid 2-related factor 2 (Nrf2) knockout mice were applied to clarify the protective mechanism of the drug. Results In the caerulein-induced AP model, TSA administration reduced serum amylase and lipase levels and ameliorated the histopathological manifestations of AP in pancreatic tissue. Additionally, TSA appreciably decreased ROS release, protected the structures of mitochondria and the endoplasmic reticulum, and increased the protein expression of Nrf2 and heme oxygenase 1 of pancreatic tissue. In addition, the protective effects of TSA against AP were counteracted by blocking the oxidative stress (NAC administration and Nrf2 knockout in mice). Furthermore, we found that TSA protects pancreatic tissue from damage and pancreatitis-associated lung injury in two additional mouse models induced by sodium taurocholate and by L-arginine. Conclusion Our data confirmed the protective effects of TSA against AP in mice by inhibiting oxidative stress via the Nrf2/ROS pathway.

AIMTo explore the pharmacokinetics and pharmacodynamics of Shengjiang decoction (SJD) in rats with acute pancreatitis (AP) for protecting against multiple organ injury.METHODSAn AP model was established by retrograde perfusion of 3.5% sodium taurocholate into the biliopancreatic duct, and a control group (CG) received 0.9% sodium chloride instead. Twelve male Sprague-Dawley rats were randomly divided into a CG treated with SJD (CG + SJD) and a model group treated with SJD (MG + SJD), both of which were orally administered with SJD (5 g/kg) 2 h after surgery. Blood samples were collected via the tail vein at 10, 20, and 40 min and 1, 2, 3, 4, 6, 8, and 12 h after a single dose of SJD to detect its main components using high-performance liquid chromatography-tandem mass spectrometry. The pharmacokinetic parameters were compared. In the pharmacodynamic experiment, 18 male Sprague-Dawley rats were randomly divided into a CG, an AP model group (MG), and an SJD treated AP group (SJDG). Serum amylase, lipase, and inflammatory cytokines were measured, and heart, lung, liver, spleen, pancreas, kidney, and intestine tissues were collected for pathological examination.RESULTSThe MG + SJD displayed significantly shorter mean residence time (MRT) and higher clearance (CL) for emodin and aloe-emodin; significantly shorter time of maximum concentration and T1/2 and a lower area under curve (AUC) for aloe-emodin; a significantly higher AUC and lower CL for rhein; and longer MRT and lower CL for chrysophanol than the CG + SJD. In the pharmacodynamic experiment, the amylase, interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-α levels in the MG were higher than those in the CG (P < 0.05). After the herbal decoction treatment, the SJDG had higher IL-10 and lower TNF-α levels than the MG (P < 0.05). The MG had the highest pathological scores, and the pathological scores of the lung, pancreas, kidney, and intestine in the SJDG were significantly lower than those in the MG (P < 0.05).CONCLUSIONAP may have varying effects on the pharmacokinetics of the major SJD components in rats. SJD might alleviate pathological injuries of the lung, pancreas, kidney, and intestine in rats with AP via regulating pro- and anti- inflammatory responses, which might guide the clinical application of SJD for AP treatment.

Oxidative stress plays a crucial role in the pathogenesis of acute pancreatitis (AP). Isoliquiritigenin (ISL) is a flavonoid monomer with confirmed antioxidant activity. However, the specific effects of ISL on AP have not been determined. In this study, we aimed to investigate the protective effect of ISL on AP using two mouse models. In the caerulein-induced mild acute pancreatitis (MAP) model, dynamic changes in oxidative stress injury of the pancreatic tissue were observed after AP onset. We found that ISL administration reduced serum amylase and lipase levels and alleviated the histopathological manifestations of pancreatic tissue in a dose-dependent manner. Meanwhile, ISL decreased the oxidative stress injury and increased the protein expression of the Nrf2/HO-1 pathway. In addition, after administering a Nrf2 inhibitor (ML385) or HO-1 inhibitor (zinc protoporphyrin) to block the Nrf2/HO-1 pathway, we failed to observe the protective effects of ISL on AP in mice. Furthermore, we found that ISL mitigated the severity of pancreatic tissue injury and pancreatitis-associated lung injury in a severe acute pancreatitis model induced by L-arginine. Taken together, our data for the first time confirmed the protective effects of ISL on AP in mice via inhibition of oxidative stress and modulation of the Nrf2/HO-1 pathway.

Objective To investigate the intervention effect of Qingxin-Ⅱ Recipe on myocardial cell apoptosis and its fibrosis in viral myocarditis mice and its mechanism. Methods A total of 120 Balb/c mice were randomly divided into the model group (n=50), the treatment group (n=50) and the control group (n=20). Mice in the model and treatment groups were given intraperitoneal injection of CVB3 to establish the experimental model of viral myocarditis. The control and model groups were treated with normal saline and water by intragastric administration respectively and the treatment group was given Qingxin-Ⅱ Recipe. The expression level of nuclear factor-κB (NF-κB), tumor necrosis factor (TNF-α), interleukin-1β(IL-1β), collagen type I-A1 (COL1-A1), collagen type Ⅲ-A1 (COL3-A1), matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metallproteinase-1 (TIMP-1), myocardial interstitial collagen volume fraction (CVF) and apoptosis rate (Rapo) were assessed after treatment. Results The levels of NF-κB, TNF-α, IL-1 in cardiomyocytes, serum levels of COL1-A1, MMP-9, TIMP-1, the mRNA expression levels of COL1-A1, COL3-A1, MMP-9, TIMP-1 in cardiomyocytes, CVF and Rapo were lower in the treatment and control groups than in the model group (F=21.541, 12.146, 32.583, 4.212, 6.863, 4.868, 9.089, 8.662, 7.307, 6.646, 4.324, 41.237, respectively, all P 0.05) were found, but serum MMP-9 level was higher in the treatment group than in the control group (P=0.033). Conclusions Qingxin-Ⅱ Recipe can effectively reduce inflammatory cytokines levels, protect myocardial cells from inflammatory damages and inhibit myocardial fibrosis in viral myocarditis. Key words: Myocarditis; Apoptosis; Fibrosis

Objective To investigate the effects of resolvin D1 on autophagy in the prevention of acute pancreatitis (AP) in mice. Methods Thirty C57BL/6 mice were divided into control group, AP group and resolvin D1 group. AP model was established by intraperitoneal injection of cerulein at 50 μg·kg-1·h-1. Resolvin D1 was intraperitoneally given at 50 μg/kg one hour before and four hours after modeling. The mice of control group were intraperitoneally injected the same volume of 0.9% sodium chloride solution. The serum levels of amylase and lipase were measured by colorimetric method. The pathological injury of the lung and pancreatitis were observed under optical microscope. Autophagic vacuoles in acinar cells of pancreas of mice were evaluated by transmission electron microscope. And the expressions of autophagy related markers Beclin-1, p62 and LC3-Ⅱ at the mRNA and protein levels in pancreas of mice were detected by real time quantitative polymerase chain reaction (RT-qPCR) and Western blotting method. One-way analysis of variance and SNK-q were performed for statistical analysis. Results There were statistically significant differences in serum amylase and lipase levels between control group, AP group and resolvin D1 group (F=62.99 and 149.69, both P<0.01). The serum amylase and lipase levels of mice in resolvin D1 group were lower than those of AP group ((525.08±41.12) U/L vs. (752.62±42.03) U/L, (758.24±134.77) U/L vs. (1 201.06±112.53) U/L), and the differences were both statistically significant (both SNK-q test and P<0.01). In addition, there were statistically significant differences in the ratio of the pancreas and lung wet mass to body mass between control group, AP group and resolvin D1 group (F=11.36 and 18.51, both P<0.05). Pathological injury scores of pancreas and lung of resolvin D1 group were both lower than those of AP group (3.3±0.6 vs. 5.6±0.6, 5.4±0.5 vs. 8.8±0.4), and the differences were statistically significant (both SNK-q test and P<0.05). The results of transmission electron microscopy observation revealed that the number of autophagic vacuole of resolvin D1 group was less than that of AP group, and the size was smaller. Moreover, there were statistically significant differences in Beclin-1, p62 and LC3-Ⅱ mRNA between control group, AP group and resolvin D1 group (F=270.95, 151.83 and 124.77, all P<0.05). The relative expression mRNA levels of Beclin-1, p62 and LC3-Ⅱ of resolvin D1 group were 1.59±0.12, 2.75±0.27 and 1.34±0.14, respectively, which were lower than those of AP group (2.68±0.13, 3.32±0.30 and 3.37±0.26, respectively), and the differences were statistically significant (all SNK-q test and P<0.05). There were statistically significant differences in the expressions of Beclin-1, p62 and LC3-Ⅱat the protein level between control group, AP group and resolvin D1 group (F=116.63, 384.40 and 192.45, all P<0.05). The expressions of Beclin-1, p62 and LC3-Ⅱ at protein level of resolvin D1 group were 0.98±0.03, 0.57±0.04 and 0.31±0.03, respectively, which were lower than those of AP group (1.34±0.07, 1.02±0.03 and 0.48±0.04, respectively), and the differences were statistically significant (all SNK-q test and P<0.05). Conclusion Resolvin D1 ameliorates the severity of AP by attenuating the impaired autophagy and restoring autophagic flux in AP mice. Key words: Autophagy; Acute pancreatitis; Resolvin D1; Mice

The research examined the antioxidative impact of astragalosides (AST) on experimental acute pancreatitis (AP) in mice. This study aims to assess the correlation between varying doses of astragalosides and superoxide dismutase (SOD) activity in an acute pancreatitis mouse model. By examining the interplay between astragaloside's protective effects and its antioxidant properties, we aim to deepen our understanding of its therapeutic potential in acute pancreatitis. The AP model in mice was induced by retrograde injection of sodium deoxycholate into the biliary and pancreatic ducts. Serum amylase activity was monitored at various time points following induction. Furthermore, 24 hours post-induction, levels of serum nitric oxide (NO), superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content in pancreatic tissue were assessed. The findings of this study illustrated that AST, while exhibiting a protective effect in experimental AP, could effectively lower the elevated serum NO levels, reduce MDA production, and enhance SOD activity in model mice. AST notably reduced MDA levels in the pancreatic tissue of AP mice, underscoring its ability to inhibit membrane peroxidation induced by oxygen free radicals. Furthermore, AST was observed to elevate SOD activity in scavenging oxygen free radicals in pancreatic tissue. These findings suggest that AST enhances recovery in an experimental acute pancreatitis mouse model by exerting antioxidative effects.

Inhibition of hypoxia-inducible factor-1α alleviates acinar cell necrosis in a mouse model of acute pancreatitis

Objective To investigate the protective effect of butylphenyphthalein (NBP) on RPE apoptosis induced by H2O2. Methods The human RPE cell line (human ARPE-19 cell line) were used as the experimental cells and were divided as control group, model group, NBP group. Complete medium was used in control group. The model group was stimulated with 200 μmol/L H2O2 for 2 h, and the cells were cultured in complete medium. The NBP group was cultured with 200 μmol/L H2O2 and 1 μmol/L NBP for 2 h. After changing the medium, complete medium was combined with 1 μmol/L NBP to continue the culture of the cells. Cell viability were detected by MTT assay while the morphology of RPE were observed by HE staining. Moreover, Hoechst 33258 was used to detect RPE cell apoptosis. Mitochondrial membrane potential (JC-1) staining were performed to monitor changes in cell membrane potential and the characteristic change of apoptosis in RPE cells. Furthermore, 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) staining were used to analyze the effect of NBP treatment on the expression of ROS. The effect of NBP on the expression of Heme oxygenase-1(HO-1) was analyzed by cellular immunofluorescence and western blotting. Results The results of MTT assay showed that the cells were cultured for 24 and 48 hours, cell viability of control group (t=17.710, 13.760; P<0.000 1, <0.000 1) and treatment group (t=4.857, 9.225; P=0.000 7, <0.000 1) were stronger than that of model group, and the difference was statistically significant. HE staining and Hoechst33258 staining showed that compared with the control group, the number of cells in the model group was significantly less, and the cell morphology was incomplete. Compared with the model group, the number of cells in the treatment group was significantly increased, and the cell morphology was better. The results of JC-1 assay showed that the number of apoptotic cells in the model group was significantly higher than that in the control group, and the number of apoptotic cells in the treatment group was significantly lower than that in the model group. DCFH-DA staining showed that the ROS accumulation in the model group was more than that in the control group, and the ROS accumulation in the treatment group was less than that in the model group. Immunostaining observation showed that the HO-1 fluorescence intensity of the cells in the treatment group was significantly higher than that of the control group, and the difference was statistically significant (t=10.270, P=0.000 5). Western blot analysis showed that NBP up-regulated the expression level of HO-1 in a time-dependent manner. The relative expression of HO-1 at 4, 8, and 12 h of NBP showed a clear increase trend compared with 0 h, and the difference was statistically significant (F=164.91, P<0.05). Conclusions Oxidative stress injury can down-regulate the viability of RPE cells and induce apoptosis. NBP can increase the antioxidant capacity of RPE cells, reduce cell damage and inhibit cell apoptosis by up-regulating HO-1 expression. Key words: Retinal pigment epithelium; Apoptosis; Butylphthalide; Oxidative stress