Effects of cycloastragenol on cardiac fibrosis induced by isoproterenol in mice

AIM: To investigate the effects of cycloastragenol (CAG) on cardiac fibrosis in mice and the mechanism involved. METHODS: The mouse model of cardiac fibrosis induced by isoproterenol (ISO) was established and treated with high- and low-dose CAG. Cardiac function was measured by echocardiography, and heart sections were stained by Masson’s trichrome for fibrosis assessment. The expression of fibrosis-related factors was assayed using RT-qPCR, Western blot and immunohistochemistry. In addition, cardiac fibroblasts isolated from neonatal factors rat ventricles were cultured and administrated with ISO followed by CAG treatment, and then the expression profile of the factors above was assayed using RT-qPCR. RESULTS: Treatment with CAG significantly alleviated ISO-induced cardiac dysfunction and fibrosis, inhibited the mRNA expression of oxidative stress-related factors NADPH oxidase 4 and inducible nitric oxide synthase, and blocked the phosphorylation of proteins associated with nuclear factor-κB signaling pathway as well as the expression of transforming growth factor-β (TGF-β). In addition, it was demonstrated that CAG also inhibited the mRNA expression of the factors above in primary cardiac fibroblasts administrated with ISO. CONCLUSION: CAG markedly rescues ISO-induced cardiac dysfunction and fibrosis via inhibition of oxidative stress, inflammation and TGF-β levels.     

Astragalus membranaceus inhibits bleomycin-induced pulmonary fibrosis in mice by antioxidation

AIM:To investigate the effect of Astragalus membranaceus on the balance of oxidation and antioxidation in bleomycin-induced pulmonary fibrosis in mice and the possible anti-fibrosis mechanism of Astragalus membranaceus.METHODS:Female KM mice (n=36) were randomly divided into 3 groups.The mice in control group were administered with saline aerosol intratracheally.The mice in fibrosis group were administered with bleomycin at dose of 3 mg/kg aerosol intratracheally.The mice in Astragalus membranaceus group were administered with bleomycin at dose of 3 mg/kg aerosol intratracheally and then intraperitoneal injected with Astragalus membranaceus parenteral solution at daily dose of 1.7 g/kg.All mice were sacrificed 14 d after the treatment,and the lungs and serum were collected for detection.Hematoxylin-eosin staining was performed in the lung tissue.The mRNA expression of superoxide dismutase 1/2/3(SOD1/2/3),catalase (CAT),NADPH oxidase 2/4(NOX2/4) and α-smooth muscle actin (α-SMA) was detected by RT-PCR,and the protein expression of α-SMA and NOX2/4 was determined by Western blot.The concentration of malondialdehyde (MDA) and total antioxidant capacity (T-AOC) in the serum were measured by a colorimetric method.RESULTS:The pathological injury was obviously observed in bleomycin group compared with control group,but was attenuated in Astragalus membranaceus group.The α-SMA mRNA and protein expression,MDA/T-AOC,NOX2,NOX4 and SOD3 mRNA expression,and NOX2 protein expression in bleomycin group were significantly higher than those in control group,while those in Astragalus membranaceus group were significantly lower than those in bleomycin group.The protein expression of NOX4 in bleomycin group was significantly lower than that in control group,while that in Astragalus membranaceus group was higher than that in bleomycin group.The mRNA expression of SOD1 and CAT in Astragalus membranaceus group and bleomycin group were decreased compared with control group.No significant difference of SOD2 mRNA expression among the 3 groups was observed.CONCLUSION:Astragalus membranaceus inhibits bleomycin-induced pulmonary fibrosis in mice by maintaining the balance of oxidation and antioxidation.     

Establishment of pancreatic β cell iron overload model and β cell injury induced by iron overload

AIM: To establish rat insulinoma INS-1 cell iron overload model, and to study the effect of iron overload on the viability, insulin secretion, mitochondrial defect and oxidative stress change in the INS-1 cells. METHODS:INS-1 cells were cultured with ferric ammonium citrate (FAC) at different concentrations (0, 5, 10, 20, 40, 80, 160 and 320 μmol/L). Labile iron pool (LIP) were calculated by detecting calcein-AM fluorescence in 24 h, 48 h and 72 h. The cell viability was measured by CCK8 assay. Iron overload model was established by screening for the best combination to ensure both high LIP level and cell viability. Reactive oxygen species (ROS) level was further analyzed by flow cytometry after fluorescent probe staining. The function of insulin secretion was measured by ELISA. The mitochondrial membrane potential was detected by JC-1 kit, and the mitochondrial changes were observed by transmission electron microscopy. RESULTS: Intracellular LIP levels were significantly increased in FAC groups in a concentration-dependent manner (P<0.05). The viability of INS-1 cells was suppressed with increasing FAC concentration or culture time (P<0.05). The highest LIP level and cell viability over 50% were observed with the condition of exposure to FAC for 48 h, indicating that INS-1 cell iron overload model was established. With the increase in the FAC concentrations, the insulin secretion was also increased and then decreased, and that in 160 and 320 mol/L groups showed statistical difference compared with control group (P<0.05). The ROS level was significantly increased by FAC exposure as compared with control group (P<0.05). Mitochondrial membrane potential was decreased with the increase in the iron concentration (P<0.05). After iron overload, the mitochondria of INS-1 cells were swollen, the internal cristae were expanded, and the normal structure was lost. With the increase in the FAC concentration, the mitochondrial structure was destroyed more obviously. CONCLUSION: Co-culture of INS-1 cells with FAC for 48 h successfully establish the iron overload model. Iron overload significantly damages mitochondrial structure and increases intracellular ROS level. The viability of pancreatic β cells is sensitive to iron, even lower doses of iron can damage β cells. The insulin secretion is reduced when the number of β cells is decreased to a certain extent.     

Effect of atorvastatin on myocardial fibrosis induced by aldosterone

AIM: To investigate the effects and mechanisms of atorvastatin on myocardial fibrosis induced by aldosterone in SD rats. METHODS: Forty male uninephrectomized SD rats were limited to drink 1% NaCl water for 4 weeks and assigned to the follow groups: vehicle control rats (CON group); aldosterone treated rats (ALD group); spironolactone + aldosterone treated rats (SPI+ALD group); atorvastatin + aldosterone treated rats (ATO+ALD group). Blood pressure was measured by catheterization. Expression of platelet-derived growth factor (PDGF-A, PDGF-B), platelet-derived growth factor receptor (PDGFR-α, PDGFR-β) and ectodermal dysplasia-1 (ED-1) were analyzed by immunohistochemistry. Collagen volume fraction (CVF) and perivascular collagen area (PVCA) were analyzed by Sirius-Red staining. Myocardium osteopontin protein was detected by Western blotting analysis. RESULTS: Mean arterial blood pressure in ALD group, SPI+ALD group and ATO+ALD group was elevated, and significant difference was observed between the three groups and vehicle control group (P<0.01 or P<0.05). Myocardial fibrosis was observed in ALD group. Compared to other three groups, the index of CVF and PVCA was increased significantly (P<0.01 or P<0.05). No significant difference of the index of CVF and PVCA between ATO+ALD group and SPI+ALD group was observed (P>0.05). Compared to other groups, the levels of PDGF-A, PDGF-B, PDGFR-α, ED-1 and OPN in ALD group were significantly increased (P<0.01 or P<0.05). The levels of PDGF-A, PDGF-B, PDGFR-α and OPN were no significant difference between ATO+ALD group and SPI+ALD group (P<0.01 or P<0.05). However, the level of ED-1 in ATO+ALD group was significantly decreased compared to SPI+ALD group (P<0.05). No significant difference of PDGFR-β level among four groups was found (P>0.05). CONCLUSION: These results suggest that atorvastatin may attenuate myocardial fibrosis induced by aldosterone. The mechanisms concern with reduction of macrophage infiltration, expression of inflammatory cytokines OPN, partially inhibition of platelet-derived growth factor and its receptor expression.     

Pancreatic kininogenase protects against renal fibrosis in rat model of unilateral ureteral obstruction

AIM To assess the beneficial effects of pancreatic kininogenase （PK） on renal fibrosis in rat model of unilateral ureteral obstruction （UUO）. METHODS Male Sprague-Dawley rats were randomly assigned to 5 groups and treated daily with PK for 7 d and 14 d. Masson trichrome and HE staining were used to assess the degree of tubulointerstitial fibrosis, and immunohistochemistry and Western blot were employed to evaluate the expression of profibrotic and proinflammatory cytokines, and apoptosis- and autophagy-related proteins. RESULTS PK treatment significantly decreased expression of profibrotic and proinflammatory cytokines, and these were paralleled with attenuation of tubulointerstitial inflammation ［monocyte chemoattractant protein-1 （MCP-1） and Toll-like receptor 2 （TLR-2）］ and fibrosis ［transforming growth factor β1 （TGF-β1） and connective tissue growth factor （CTGF）］ in a time-dependent manner. Oxidative stress induced by UUO manifested by augment of oxidant enzymes ［NADPH oxidase-2 （NOX-2） and NOX-4］ and decrease in antioxidant enzymes ［superoxide dismutase 1 （SOD1） and SOD2］, which was closely associated with dysregulation of apoptosis- and autophagy-related proteins subsequent excessive apoptotic （Bcl-2/Bax and cleaved caspase-3） and autophagy （LC3B, beclin-1 and P62）, and all of these were eliminated by administration of PK. CONCLUSION PK treatment protects against the progression of renal fibrosis in obstructed kidneys probably by interfering oxidative stress and programmed cell death.     

Effects of FGF-21 and Nrf2 on BLM-induced pulmonary fibrosis in mice

AIM:To investigate the effect of fibroblast growth factor-21 (FGF-21) on bleomycin (BLM)-induced inflammatory response and oxidative stress in the lung, and to further explore the molecular mechanism of FGF-21 against pulmonary fibrosis. METHODS:The lung fibrosis model was induced by BLM intratracheal instillation. A total of 40 mice were randomly divided into control group, BLM group, FGF-21 (1, 2 and 5 mg/kg)+BLM groups. Western blot was used to detected the protein expression of collagen I, fibronectin and nuclear factor E2-related factor 2 (Nrf2). The reactive oxygen species (ROS) production was measured by DCFH-DA staining. The levels of inflammatory cytokines were measured by ELISA. The content of malondialdehyde (MDA), the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx), and the content of hydroxyproline (HYP) were detected by commercially available assay kits. RESULTS:Treatment with FGF-21 notably attenuated BLM-induced the expression levels of inflammatory mediators tumor necrosis factor-α, interleukin-1β and interleukin-6 in the lung tissue. In addition, FGF-21 treatment remarkably reduced the generation of ROS and the content of MDA trigged by BLM, accompanied with the enhanced activity of anti-oxidative enzymes SOD and GPx (P<0.05). Furthermore, treatment with FGF-21 obviously reduced the extracellular matrix (ECM) accumulation by suppressing the expression of collagen I and fibronectin induced by BLM, accompanied with the decreases in the levels of TGF-β1 and HYP. Silencing of Nrf2 expression abolished the protective effect of FGF-21. CONCLUSION:FGF-21 relieves BLM-induced pulmonary fibrosis by reducing the inflammatory response, mitigating oxidative damage and decreasing the ECM deposition via Nrf2 activation, thus providing the basis for the therapeutic effect of FGF-21 on the lung fibrosis.     

Effect of aldosterone on rat peritoneal fibrosis induced by peritoneal dia-lysis

AIM: To investigate the pathologic role of aldosterone and protective effect of aldosterone receptor antagonist on peritoneal fibrosis in peritoneal dialysis rats. METHODS: A peritoneal fibrosis rat model was established by intraperitoneal injection of lipopolysaccharide(at 1 d, 3 d, 5 d and 7 d, 0.6 mg/kg) and dialysate(daily intraperitoneal injection of 4.25% dialysate, 100 mL/kg). At the same time, spironolactone(an aldosterone receptor antagonist, 100 mg·kg^-1·d^-1) was given to the model rats. After 4 weeks, the expression of aldosterone synthase CYP11B2, 11β-hydroxysteroid dehydrogenase type 2(11β-HSD2), mineralocorticoid receptor(MCR), and inflammatory factors were detected by immunohistochemistry, real-time PCR and Western blotting. RESULTS: The rat model of peritoneal fibrosis was successfully established. At the same time, the injury of mesothelial cells, deposition of collagen fibers and thickness of peritoneal were increased. Moreover, the infiltration of macrophages in the peritoneum/dialysate was increased. The level of aldosterone and the expression of MCR, 11β-HSD2 and CYP11B2 in fibrotic peritoneum were obviously up-regulated as compared with normal rats. The expression of NF-κB/MCP-1 was also increased. However, treatment with spironolactone alleviated peritoneal fibrosis and reduced the expression of NF-κB/MCP-1. CONCLUSION: Local aldosterone is involved in the process of peritoneal fibrosis via NF-κB/MCP-1 pathway. Spironolactone alleviates peritoneal fibrosis of peritoneal dialysis.     

Experimental research on cardiac fibrosis induced by recombinant mouse interleukin-11 in mice

AIM To observe the effect of recombinant mouse interleukin-11 （rmIL-11）injected subcutaneously into mice on heart structure and function and to determine its pro-fibrotic effect. METHODS C57BL/6 mice were randomly divided into experimental group and control group.The mice in experimental group were injected subcutaneously with recombinant mouse IL-11 at the dose of 100 μg·kg^-1·d^-1 for 3 consecutive weeks, while the control group were given equal volume of normal saline in the same way. After the experiment was finished, the parameters of heart function were measured by echocardiography.The heart weight was weighed and the cardiac weight index (CWI) was calculated. HE staining and Masson's trichrome staining were performed to observe the pathological changes and the extent of myocardial fibrosis in mouse myocardia respectively, and the cardiac collagen volume fraction (CVF) was calculated. The expression levels of extracellular matrix proteins in the myocardial tissues of mice, including type Ⅰ collagen, type Ⅲ collagen and fibronectin, were determined by Western blot. RESULTS Left ventricular ejection fraction and left ventricular fraction shortening in experimental group were obviously lower than those in control group (P<0.01), however left ventricular end-diastolic diamension and left ventricular end systolic dimension were significantly higher than those in control group (P<0.05).Compared with control group, the CWI was increased (P<0.01), the myocardial arrangement was disorder, the necrosis of cardiac myocytes was increased, and excessive deposition of collagen was observed in the myocardial tissues in experimental group. Correspondingly, the CVF and protein levels of type Ⅰ collagen, type Ⅲ collagen and fibronectin in the left ventricle in experimental group were increased significantly (P<0.05). CONCLUSION Injection of rmIL-11 into the mice subcutaneously induces fibrogenesis in the heart, which implies that IL-11 is likely a novel pro-fibrotic factor.     

Effects of sex on chronic pancreatitis induced by L-arginine in Kunming mice

AIM: To explore the effect of sex on the formation of chronic pancreatitis (CP) by comparing the differences in L-arginine-induced CP model between male and female mice.METHODS: Male (n=42) and female (n=42) Kunming mice were randomly divided into 4 groups:female control group, female CP group, male control group and male CP group (n=18 in each control group, n=6 at each time point; n=24 in each CP group, n=8 at each time point). The mice in CP groups were intraperitoneally injected with 20% L-arginine (3 g/kg, twice/d, 1 d/week). After modeling for 2 weeks, 4 weeks and 6 weeks, the mice were anesthetized and sacrificed. The morphological changes and the degree of fibrosis in the pancreas were observed by HE and Masson staining. The positive expression rate of F4/80 in the pancreatic tissues was observed by the method of immunohistochemistry. The mRNA expression of interleukin-6 (IL-6), α-smooth muscle actin (α-SMA) and fibronectin (FN) in the pancreas were detected by real-time PCR. The protein of pancreas was extracted to detect the expression of α-SMA and FN by Western blot.RESULTS: At 2 weeks, 4 weeks and 6 weeks after intraperitoneal injection of L-arginine, the pancreatic tissues were obviously injured and exhibited different degrees of fibrosis in female and male CP groups. At the same time, there were significant differences in the degree of pancreatic injury between male and female mice, and the degree of pancreatic lesion in the male mice was significantly more severe. The positive rate of F4/80 in the pancreas of male CP mice was significantly higher than that in female CP group at the same time point after modeling. At every time point, the mRNA expression of IL-6 in the pancreas was increased in both female and male CP groups, but that in male CP group was higher (P<0.05). The fibrosis indexes, α-SMA and FN, were both highly expressed at mRNA and protein levels after modeling, but compared with the female group, the time of positive expression in male mice was earlier and the expression level was higher (P<0.05).CONCLUSION: The CP model is successfully established by intraperitoneal injection of 20% L-arginine, and a difference in the degree of pathologic alteration in pancreas between male and female mice exists. CP is more effectively induced by L-arginine in male mice, and the degree of fibrosis is more pronounced. The reason may be related to the sensitivity of male mice to L-arginine, causing more serious inflammatory response. Therefore, male mice are more suitable for establishing CP animal model.     

Effect of bitter melon on liver fibrosis induced by carbon tetrachloride

AIM: To investigate the effects of bitter melon (BM) on liver fibrosis induced by CCl₄ in Wistar rats. METHODS: Healthy male Wistar rats were randomly divided into 4 groups (with 8 each): olive oil control group (group C), olive oil CCl₄ model group (group M), CCl₄+BM at low concentration (BM 100 g/kg, group BM-L), CCl₄+ BM at high concentration (BM 200 g/kg, group BM-H). All rats except those in group C were subcutaneously injected with CCl₄ twice a week for 8 weeks to induce liver fibrosis. After injection of CCl₄ for 8 weeks, all rats were sacrificed and the samples of blood and livers were collected. The weight ratio of liver to body was measured. The serum level of MDA and the activity of SOD were tested. The contents of total protein and albumin, the activity of GSH-Px, the content of hydroxyproline and the activity of monoamine oxidase in the liver homogenate were determined. Hepatic inflammation and collagen deposition were observed under microscope with Masson staining. RESULTS: In the rats treated with BM, the weight ratio of liver to body, the serum level of MDA, the content of hydroxyproline and the activity of monoamine oxidase in the liver homogenate were lower than those in group M (P<0.01). The serum activity of SOD, the contents of total protein and albumin, and the activity of GSH-Px in the liver homogenate were enhanced (P<0.01). The livers of the model rats had remarkable inflammatory necrosis, collagen accumulation and fibrosis. The rats in BM-treated group showed slighter hepatic injury and collagen deposition, and the liver functions were much better than those in the model group. High dose of BM showed more obvious liver-protective effects. CONCLUSION: BM attenuates liver fibrosis by its antioxidant effect and the mechanisms of reducing hydroxyproline content and monoamine oxidase activity.     

Study on thrombomodulin and von Willebrand factor in microvasculature in mice lungs with pulmonary fibrosis induced by bleomycin

AIM:To explore the distribution of thrombomodulin (TM) and von Willebrand factor (vWf) on endothelial cells of lung microvasculature as well as the phenotype change of this cell in the process of pulmonary fibrosis in C57BL/6 mice. METHODS:It was carried out with dual immunofluorescent stain and quantitative analysis of fluorescent intensity of TM and vWf in normal and pulmonary fibrosis model induced by bleomycin (BLM) administration. RESULTS:① The normal lungs showed multiple continuous linear positive staining of TM and seldom positive of vWf on the surface of endothelium of alveolar capillaries. Meanwhile, blood vessels exhibited considerable positive of vWf in endothelial cell in normal C57BL/6 mice. ② The fibrotic lungs revealed a statistically significant diminution of TM expression, and at the same time, an increase of vWf expression when comparing with normal lung sections.CONCLUSION:These results suggest that TM dominant phenotype endothelial cells, rather than vWf dominant phenotype, are the major ones of alveolar capillaries in normal C57BL/6 mice lungs. TM dominant phenotype endothelial cells changed into vWf dominant ones as pulmonary fibrosis develops. Both TM and vWf antigen might be considered as markers of endothelium injury of lung microvasculature.     

Effect of leonurine on expression of miR-1 in rats with myocardial fibrosis induced by isoproterenol

AIM: To investigate effect of leonurine on the expression of microRNA-1 (miR-1) in rats with myocardial fibrosis induced by isoproterenol (ISO). METHODS: SD rats (n=10) were used as normal control group, and 80 rats were given ISO by intraperitoneal injection daily for 2 weeks to establish the model of myocardial fibrosis. The model rats were randomly divided into 5 groups:model group, low-dose (7.5 mg·kg^-1·d^-1) leonurine group, middle-dose (15 mg·kg^-1·d^-1) leonurine group, high-dose (30 mg·kg^-1·d^-1) leonurine group and p38 mitogen-activated protein kinase (p38 MAPK) inhibitor (0.3 mg·kg^-1·d^-1) group. After the treatment for 2 weeks, the ultrastructure of left ventricular myocardial tissues was observed under electron microscope. Masson staining was used to detect collagen fibrosis, and the expression of collagen I and collagen Ⅲ was determined by the method of immunohistochemistry. The contents of endothelin-1 (ET-1) and angiotensin Ⅱ (Ang Ⅱ) were measured by ELISA. The expression of miR-1 and ET-1 mRNA was detected by real-time PCR, and the protein expression of p38 MAPK, β-myosin heavy chain (MHC) and α-MHC was determined by Western blot. RESULTS: Compared with model group, the ultrastructure of left ventricular myocardial tissues in high-dose leonurine group was attenuated, and the expression of miR-1 and the protein expression of α-MHC in left ventricular myocardial tissues of high-dose leonurine group were increased (P<0.05). Collagen volume fraction, collagen I, collagen Ⅲ, the ratio of collagen Ⅰ/collagen Ⅲ, the contents of ET-1 and Ang Ⅱ, the mRNA expression of ET-1, and the protein expression of p38 MAPK and β-MHC in high-dose leonurine group were lower than those in model group (P<0.05). CONCLUSION: Leonurine attenuates myocardial fibrosis in the rats induced by ISO, and it is potentially associated with affecting the expression of miR-1, and inhibiting ET-1/p38 MAPK signaling pathway.     

Comparison of in vivo anti-fibrotic effects of pirfenidone and nintedanib in bleomycin-induced pulmonary fibrosis model

AIM: To compare the effects of pirfenidone and nintedanib on bleomycin-induced mice pulmonary fibrosis model in different periods. METHODS: Five mice models were established according to the schedule of drug administration:a inflammatory phase model and 4 fibrotic phase models including the early prevention study group, early therapy study group, late therapy study group and full-course therapy study group. The indicators of lung inflammatory and lung fibrosis were detected respectively. RESULTS: (1) The level of anti-inflammatory and antioxidant indicators:both pirfenidone and nintedanib reduced the number of inflammatory cells and inhibited the secretion of inflammatory factors. Pirfenidone had a better effect on inhibition of interleukin (IL)-1β and IL-4 (P < 0.01), while nintedanib had a better effect on inhibition of IL-6 and IFN-γ (P < 0.05). Pirfenidone significantly increased superoxide dismutase (SOD) activity (P < 0.01), and nintedanib significantly reduced malondialdehyde (MDA) and myeloperoxidase (MPO) levels (P < 0.01). (2) Collagen content in lung tissue:the inhibitory effect of nintedanib on hydroxyproline content in mouse lung was better than that of pirfenidone in the early therapy study group, late therapy study group and full-course therapy study group of the fibrotic phase models (P < 0.05). Pirfenidone had a better inhibitory effect on hydroxyproline content than nintedanib in the early prevention study group (P < 0.01). (3) Pathological evaluation of lung tissue:both pirfenidone and nintedanib reduced the inflammatory infiltration and fibrotic area of the lung tissues. The inhibition trend was consistent with that of collagen content. CONCLUSION: Both pirfenidone and nintedanib have anti-inflammatory, anti-oxidative and anti-fibrosis effects in bleomycin-induced pulmonary fibrosis in mice. Pirfenidone is more effective in the early prevention study group, and nintedanib has a better effect in early therapy study group, late therapy study group and full-course therapy study group.     

Effect of taurine on kidney injury induced by paraquat poisoning in rats

AIM:To study the effects of taurine at different doses on renal oxidative stress and inflammation induced by paraquat in rats.METHODS:Male SD rats (n=48) were randomly divided into 4 groups:negative control group,paraquat group,paraquat+low-dose taurine group,and paraquat+high-dose taurine group.The serum levels of creatinine and urea nitrogen were detected by a biochemical analyzer.The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were measured by colorimetry.The plasma concentrations of IL-6 and intercellular adhesion molecule (ICAM)-1 were detected by ELISA.Renal reactive oxygen species (ROS) was checked by fluorescence probe dihydroethidium (DHE).The protein levels of renal p-P38 MAPK,p-ERK1/2 and p-JNK were determined by Western blot.The mRNA expression of TNF-α,IL-6 and TGF-β1 was detected by real-time PCR.RESULTS:Serum creatinine and urea nitrogen increased after paraquat poisoning,and decreased after feeding with taurine in poisoned rats,with better result in high-dose taurine group.Taurine reduced the oxidative stress and inflammation in the renal tissue,and also reduced the protein levels of p-JNK,p-ERK1/2 and p-P38 in the kidney of paraquat-poisoned rats.CONCLUSION:Taurine attenuates renal injury induced by paraquat poisoning in rats.The mechanism may be related to reducing renal MAPK activity,oxidative stress and inflammatory response.     

Intervention of hydrogen on memory damage induced by chronic hypoxia-hypercapnia in rats

AIM: To investigate the effects of hydrogen on the memory damage caused by chronic hypoxia-hypercapnia in rats. METHODS: Twenty-four SD rats trained by eight-arm radial maze test were randomly divided into 3 groups:normal control group (NC), hypoxia-hypercapnia+saline group (MS) and hypoxia-hypercapnia+ hydrogen group (MH). The rats in the latter 2 groups were placed in a closed cabin for 8 h/day,6 days/week and lasted for 4 weeks, in which O₂ was 9％-11％ and CO₂ was 5％-6％. In every time after the animals were out of the cabin, the MS rats were intraperitoneally injected with saline (5 mL/kg) and the MH rats were intraperitoneally injected with hydrogen solution at the same dose. The learning and memory function, the activity of superoxide dismutase (SOD), the content of malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) were examined after the cabin training. The ultramicrostructures of hippocampus were also observed. RESULTS: (1) Compared with NC group, the number of working memory errors, the total errors, the content of hippocampus 8-OHdG and serum MDA in MS and MH groups were higher and the activity of serum SOD was lower (P<0.05). The hippocampus structure was destroyed and some degree of edema and more apoptosis in the neurons were obserued in MS group and MH group. (2) Compared with MS group, the number of working memory errors(WME), the total errors, the content of hippocampus 8-OHdG and serum MDA were lower and the activity of serum SOD was higher in MH group (P<0.05). In MH group, the morphology of hippocampus structures kept nearly normal arrangement and the only mild edema and fewer apoptosis in the neurons were found. CONCLUSION: Hydrogen may attenuate chronic hypoxia-hypercapnia-induced memory damage in rats by inhibiting apoptosis of the neurons and decreasing detrimental free radicals reaction.     

Role of β-catenin in apoptosis of pancreatic acinar cells induced by cae-rulein

AIM: To study the role of β-catenin in the apoptosis of pancreatic acinar cells induced by cae-rulein. METHODS: Rat pancreatic acinar AR42J cells were treated with caerulein. The expression of β-catenin at mRNA and protein levels in the AR42J cells was determined by real-time PCR and Western blot. The β-catenin over-expression vector was transfected into AR42J cells. After treatment with caerulein, the over-expression effect was evaluated by real-time PCR and Western blot. The changes of cell viability were measured by MTT assay. The leakage rates of lactate dehydrogenase (LDH) and amylase (AMY) were measured by binitrophenyl hydrazine method and iodine starch colorimetry, respectively. The apoptosis was analyzed by flow cytometry. The protein levels of endoplasmic reticulum stress protein CHOP and cleaved caspase-12 in the AR42J cells were determined by Western blot. RESULTS: The expression of β-catenin at mRNA and protein levels in the AR42J cells was decreased after treatment with caerulein (P<0.05). The expression of β-catenin in the AR42J cells was significantly increased by transfection with β-catenin over-expression vector. The viability of AR42J cells after treatment with caerulein was reduced, while the leakage rates of LDH and AMY, the apoptotic rate and the protein levels of CHOP and cleaved caspase-12 in the cells were increased (P<0.05). Over-expression of β-catenin enhanced the viability of AR42J cells after treatment with caerulein, reduced the leakage rates of LDH and AMY, and decreased the apoptotic rate and the protein levels of CHOP and cleaved caspase-12 in the AR42J cells. CONCLUSION: β-Catenin significantly inhibits the apoptosis of pancreatic acinar cells induced by caerulein. The mechanism is related to the reduction of endoplasmic reticulum stress.     

Up-regulation of CD36 induced by casein-induced inflammation promotes renal injury in mice

AIM: To investigate the role of CD36 in casein-induced mouse renal injury.METHODS: Eight-week-old male C57BL/6J mice and CD36 knockout (CD36KO) mice were randomly divided into C57BL/6J saline injection group, C57BL/6J casein injection group and CD36KO casein injection group (n=8 in each group). After 14 weeks of treatment with high-fat diet, the mouse serum, 24 h urine and kidney tissue samples were collected for analysis. The serum content of tumor necrosis factor-α (TNF-α) was measured by ELISA. The renal function markers in the serum and urine were determined by an automatic biochemical analyzer. The pathological changes of the kidney were observed by HE staining and Masson staining. The expression of CD36 and cytokines/chemokines (TNF-α, IL-6 and MCP-1) at mRNA and protein levels in the renal tissues were determined by real-time PCR and Western blot. The content of tissue hydrogen peroxide (H₂O₂) was measured by a commercial kit. The protein levels of Nrf2 and TGF-β1 in the renal tissues were measured by immunohistochemical staining.RESULTS: Compared with saline injection group, casein injection increased the level of TNF-α in the serum and in the kidney tissues of C57BL/6J mice (P<0.05), suggesting that casein injection successfully induced chronic inflammation in C57BL/6J mice. Casein injection also promoted the protein expression of CD36 and TGF-β1 in the renal tissues of the C57BL/6J mice, accompanied with glomerular sclerosis, proteinuria, increased serum creatinine content, increased H₂O₂ content, and decreased Nrf2 protein level and the ability of antioxidant in the kidneys (P<0.05). Furthermore, CD36 deficiency protected the mice from casein-induced renal injury, as evidenced by improved kidney pathological changes and decreased proteinuria. The content of H₂O₂ in the kidneys of casein-treated CD36 knockout mice was also lower than that in casein-treated C57BL/6J mice.CONCLUSION: Inflammatory responses promote the oxidative stress and renal injury in a CD36-dependent manner.     

Effect of simvastatin on function of mouse pancreatic beta cells and its mechanisms

AIM: To observe the influence of simvastatin on insulin secretion function of mouse pancreatic beta cell line MIN6 and to explore its possible mechanisms.
METHODS: MIN6 cells were randomly divided into normal control group and low-, middle-and high-concentration simvastatin treatment groups, which were cultured for 48 h with high-glucose DMEM containing 15% fetal bovine serum plus 0, 2, 5 and 10 μmol/L simvastatin, respectively. The insulin secretion of MIN6 cells was measured by radioimmunoassay. The content of ATP in MIN6 cells was measured by biochemiluminescence method. The mRNA and protein expression levels of inwardly rectifying potassium channel 6.2 (Kir6.2), voltage-dependent calcium channel 1.2 (CaV1.2) and glucose transporter 2 (GLUT2) were detected by real-time fluorescence quantitative PCR and Western blotting, respectively.
RESULTS: Compared with normal control group, middle-and high-concentration simvastatin treatment markedly decreased the synthesis and secretion of insulin in MIN6 cells (P<005). The content of ATP in MIN6 cells was markedly decreased in simvastatin treatment groups (P<005). The mRNA expression level of Kir6.2 in MIN6 cells was significantly up-regulated in simvastatin treatment groups (P<001), while the mRNA expression levels of CaV1.2 and GLUT2 were significantly down-regulated in middle-and high-concentration simvastatin treatment groups (P<001). The protein expression of Kir6.2 was significantly increased but that of CaV1.2 was significantly decreased in middle-and high-concentration simvastatin treatment groups (P<001), and the protein expression level of GLUT2 was markedly decreased in high-concentration simvastatin treatment group (P<001).
CONCLUSION: Simvastatin inhibits insulin synthesis and secretion in mouse pancreatic beta cell line MIN6 via suppressing ATP production, up-regulating the expression of Kir6.2 and down-regulating the expression of CaV1.2 and GLUT2.     

Effect of resveratrol on autophagy and renal interstitial fibrosis in kidney of diabetic mice

AIMTo observe the effect of resveratrol (Res) on renal autophagy level and renal interstitial fibrosis in the mice with diabetes mellitus (DM), and to discuss the possible mechanism. METHODSThe wild-type C57BL/6 mice were randomly divided into 3 groups, including normal control (NC) group, DM group and Res group (8 in each group). The diabetic mouse model was established by injection of streptozotocin. After 8 weeks of successful replication of the diabetic model, Res was given to the mice in Res group by continuous gavage for 12 weeks, and then the mice in each group were sacrificed to detect the relevant biochemical parameters. The pathological changes of the kidney tissues were observed by HE staining and Masson staining. The levels of the proteins related to autophagy, epithelial-mesenchymal transition (EMT) and fibrosis were determined by Western blot. The mRNA expression of collagen type IV (Col IV), α-smooth muscle actin (α-SMA) and E-cadherin were detected by real-time PCR. RESULTSCompared with NC group, fasting blood glucose (FBG), kidney index (KI), serum creatinine, 24-hour urinary albumin excretion rate and 24-hour urine total protein were remarkably increased in DM group (P<0.05). The results of HE and Masson staining indicated that renal tissue presented fibrosis in DM group. The protein levels of E-cadherin, beclin-1, microtubule-associated protein 1 light chain 3-II (LC3-II) were reduced in DM group, while the levels of α-SMA, Col IV and Snail1 were increased (P<0.05). After intervention with Res for 12 weeks, all the relevant biochemical parameters and KI were reduced (P<0.05) except FBG (P>0.05), and renal fibrosis lesions were obviously alleviated. Compared with DM group, the protein levels of E-cadherin, beclin-1 and LC3-II were increased in Res group, but the protein expression levels of α-SMA, Col IV, Snail1 were reduced (P<0.05). Compared with DM group, the mRNA level of E-cadherin was increased in Res group , but the mRNA levels of Col IV and α-SMA were reduced (P<0.05). CONCLUSION Resveratrol significantly inhibits EMT and reduces renal interstitial fibrosis in diabetic mice, and its mechanism may be related to the promotion of renal autophagy.     

Synergistic and attenuating effects of Qiongyugao on pancreatic cancer mice with chemotherapy

AIM: To observe the synergistic and attenuating effects of Qiongyugao on pancreatic cancer mice with chemotherapy and to explore its possible mechanism. METHODS: C57BL/6 mice (n=50) were randomly divided into normal group, model group, gemcitabine treatment group, Qiongyugao treatment group, and combined treatment (gemcitabine+Qiongyugao) group. The pancreatic cancer mouse xenograft model was established by subcutaneous inoculation with pancreatic cancer Panc02 cells among the mice in model group and 3 treatment groups. Normal saline, gemcitabine, Qiongyugao and gemcitabine plus Qiongyugao were also used accordingly. The tumor growth curve, blood routine and tumor markers were observed in each group. Furthermore, flow cytometry was used to detect the expression of Th1/Th2 and Th/Treg in peripheral blood of the mice. RESULTS: Subcutaneous vaccination with Panc02 cells successfully established a transplanted tumor model of pancreatic cancer. Compared with model group, Qiongyugao, gemcitabine and the combination of the 2 drugs inhibited tumor growth (P<0.05), and decreased the concentration of carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA) (P<0.05). Qiongyugao decreased the ratio of Th1/Th2 and Th17/Treg, and increased the white blood cell (WBC), red blood cell (RBC) and platelet (PLT) counts and hemoglobin (HGB) content in gemcitabine treated mice (P<0.01). CONCLUSION: Qiongyugao may have a certain therapeutic effect on pancreatic cancer and produce certain synergistic and attenuating effects on gemcitabine chemotherapy.