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ObjectiveTo quantitatively investigate the changes in the total volume and contour density of hepatic oval cells （HOC） in hepatic lobules of rats with carbon tetrachloride （CCl4）-induced hepatic fibrosis. MethodsA total of 11 healthy male Sprague-Dawley rats were randomly divided into control group with 5 rats and hepatic fibrosis group with 6 rats， and CCl4 and olive oil suspension were injected subcutaneously twice a week， 3 mL/kg each time. After five weeks of hepatic fibrosis modeling， five liver tissue blocks with a size of about 1 mm3 were randomly selected from the liver of each rat to prepare one Epon812 epoxy resin-embedded ultrathin section， and the stereological method and transmission electron microscopy were used for the quantitative analysis of the total volume and contour density of HOC in the hepatic lobules of rats. In addition， four liver tissue blocks with a thickness of 2 mm were randomly selected from the remaining liver of each rat to prepare two paraffin-embedded Masson staining sections， and the degree of liver fibrosis in each rat was qualitatively evaluated according to the Metavir staging criteria for liver fibrosis. The independent-samples t test was used for comparison of continuous data between groups. ResultsThe quantitative stereological analysis showed that the total volume of HOC in hepatic lobules was 15.40±7.63 mm3 in the control group and 146.80±114.00 mm3 in the liver fibrosis group， and compared with the control group， the total volume of HOC in hepatic lobules of rats in the liver fibrosis group was significantly increased by 8.53 times （t=-2.551， P=0.031）； the contour density of HOC in hepatic lobules was 56.20±40.40 in the control group and 566.50±317.00 in the liver fibrosis group， and compared with the control group， the contour density of HOC in hepatic lobules of rats in the liver fibrosis group was significantly increased by 9.08 times （t=-3.539， P=0.006）. Qualitative observation showed that liver fibrosis stage of rats reached stage Ⅱ-Ⅲ according to the Metavir scoring criteria， and massive proliferation of HOC was observed around the proliferation site of hepatic stellate cells in the perisinusoidal space of rats. ConclusionCCl4 induces significant proliferation of HOC in hepatic lobules of rats with liver fibrosis.
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Objective To investigate the effect of Yinchenhao decoction on renal oxidative stress injury in rats with obstructive jaundice and its association with the regulation of the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear translocation. Methods A total of 32 male Sprague-Dawley rats were randomly divided into sham-operation group (S group), model group (O group), low-dose Yinchenhao decoction group (LY group), and high-dose Yinchenhao decoction group (HY group), with 8 rats in each group. For the rats in the S group, the upper common bile duct was isolated without ligation, and for those in the other groups, double ligation of the middle and upper 1/3 of the common bile duct was performed to establish a model of obstructive jaundice. After 7 days, the rats in the LY group and the HY group were given Yinchenhao decoction by gavage at a dose of 6.3 and 18.9 mL/kg, respectively, while those in the S and O groups were given an equal volume of distilled water by gavage every day for 7 consecutive days, and the rats were treated on day 14. ELISA was used to measure the serum levels of total bilirubin (TBil), direct bilirubin (DBil), alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (GGT), blood urea nitrogen (BUN), and creatinine (Cr); spectrophotometry was used to measure the activity of the oxidative stress factors superoxide dismutase (SOD) and malondialdehyde (MDA) in renal tissue; quantitative real- time PCR and Western blotting were used to measure the mRNA and protein expression levels of Nrf2, Kelch-like ECH-associated protein 1 (Keap1), and NAD(P)H quinone dehydrogenase 1 (NQO1) in renal tissue; immunohistochemistry was used to measure observe the nuclear translocation of Nrf2 protein in renal tissue. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t -test was used for further pairwise comparison within groups. Results Compared with the S group, the O group had significant increases in the levels of TBil, DBil, ALT、GGT, BUN, and Cr, a significant reduction in the activity of SOD, and a significant increase in the level of MDA (all P 0.05). Compared with the S group, the O group had a significant reduction in the positive rate of Nrf2 in cell nucleus in renal tissue ( P < 0.05), and compared with the O group, the LY group and the HY group had a significant increase in the positive rate of Nrf2 in cell nucleus ( P < 0.05). Conclusion Yinchenhao decoction can effectively alleviate renal injury caused by obstructive jaundice, possibly by upregulating the protein expression of Nrf2 in renal tissue and regulating the nuclear translocation of Nrf2 protein, so as to mediate the protein expression of downstream NQO1, regulate oxidative stress response caused by obstructive jaundice, and thereby alleviate renal injury in rats.
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Objective To investigate the therapeutic effect of Dange Jiecheng decoction on a rat model of alcoholic liver disease (ALD) and the anti-oxidative stress mechanism of Dange Jiecheng decoction. Methods A total of 96 Sprague-Dawley rats were randomly divided into blank group with 13 rats and ALD group with 83 rats, and the rats in the ALD group were given liquor by gavage to establish a model of ALD. Then the ALD group was randomly divided into model group, high-dose Dange Jiecheng decoction group (24 g/kg), low-dose Dange Jiecheng decoction group (6 g/kg), and Yiganling tablet group (21 mg/kg), with 17 rats in each group. The rats in the blank group and the model group were given normal saline by gavage, and those in the other groups were given corresponding drugs by gavage, for 4 consecutive weeks. HE staining was used to observe the pathological changes of liver tissue; Western blot was used to measure the contents of Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) in liver tissue; quantitative real-time PCR was used to measure the mRNA expression levels of Keap1 and HO-1 in liver tissue. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t -test was used for further comparison between two groups. Results Compared with the blank group, the model group had disordered arrangement of hepatocytes with necrosis, massive inflammatory cell infiltration, and a large number of lipid droplet vacuoles, significant increases in the protein and mRNA expression levels of Keap1 ( P < 0.05), and significant reductions in the protein expression levels of Nrf2 and HO-1 and the mRNA expression level of HO-1 ( P < 0.05). Compared with the model group, the high- and low-dose Dange Jiecheng decoction groups and the Yiganling tablet group had ordered arrangement of hepatocytes, reductions in hepatocyte necrosis and inflammatory cells, and occasional lipid droplet vacuoles, as well as significant reductions in the protein and mRNA expression levels of Keap1 ( P < 0.05) and significant increases in the protein expression levels of Nrf2 and HO-1 and the mRNA expression level of HO-1 ( P < 0.05). Conclusion By regulating the Keap1/Nrf2 signaling pathway, Dange Jiecheng decoction can promote the nuclear import of Nrf2, upregulate the expression of HO-1, and alleviate oxidative stress response, thereby exerting a protective effect on ALD rats.
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ObjectiveTo investigate the therapeutic effect of the frozen and fresh preparations of human umbilical cord mesenchymal stem cells （hUC-MSC） on a rat model of liver cirrhosis after transplantation via the portal vein or the caudal vein. MethodsA total of 70 specific pathogen-free healthy male Sprague-Dawley rats were randomly divided into normal group （13 rats fed with ordinary tap water and rat food） and liver cirrhosis model group （57 rats given subcutaneous multi-point injection of mixed carbon tetrachloride/olive oil solution）. At week 8， the growth of rats was observed for both groups， and 3 rats were selected from each group for histopathological examination to confirm the formation of liver cirrhosis. A total of 50 rats were selected from the liver cirrhosis model and were divided into model group， portal vein group+fresh cell preparation group， portal vein+frozen cell preparation group， caudal vein+fresh cell preparation group， and caudal vein+frozen cell preparation group using a random number table， with 10 rats in each group. Fresh or frozen hUC-MSC were transplanted via the portal vein or the caudal vein， and after 4 weeks of administration， the different groups were compared in terms of the changes in liver function parameters and liver fibrosis degree. Continuous data were expressed as mean±standard deviation， and the independent-samples t test was used for comparison between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsAt week 8 of modeling， the model group showed the formation of pseudolobules of different sizes in the liver and met the diagnostic criteria for liver cirrhosis， with significant increases in the levels of alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， total bilirubin （TBil）， and alkaline phosphatase （ALP） compared with the normal group （all P<0.001）， suggesting that the rat model of liver cirrhosis was established successfully. There were significant differences in the levels of ALT， AST， TBil， and ALP between the five groups （F=232.00， 177.10， 112.30， 121.70， all P<0.001）. Further comparison between two groups showed that the model group had significantly higher levels of ALT， AST， TBil， and ALP than the normal group （all P<0.01）， and the portal vein group+fresh cell preparation group， the portal vein+frozen cell preparation group， the caudal vein+fresh cell preparation group， and the caudal vein+frozen cell preparation group had significantly lower levels of ALT， AST， TBil， and ALP than the model group （all P<0.01）. ConclusionThere are significant improvements in liver function and liver fibrosis degree in a rat model of liver cirrhosis at week 4 after the transplantation of hUC-MSC， and frozen or fresh cell preparation and different transplantation approaches have no significant influence on treatment outcome.
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Objective:To investigate the role of signal lymphocyte activating molecule family member 5 (SLAMF5) in liver transplantation rejection in SD rats.Methods:Forty-five male SD rats without special pathogens, weight 260-300 g, aged 10-12 weeks were included. Among them, forty male SD rats (20 donors and 20 recipients respectively) were established with reference to the " two cuff" method. 15 liver transplantation model rats were randomly divided into 1 week (LT-1W) group, 2 weeks (LT-2W) group and 3 weeks (LT-3W) group, with 5 rats in each group, and 5 normal rats were taken as the normal control group. The expressions of SLAMF5, CD4 and CD8 were detected by polymerase chain reaction (PCR), Western blot and immunohistochemistry. The correlations between SLAMF5 expression in the lymphocyte infiltration area and the rejection activity index was analyzed.Results:The levels of alanine aminotransferase, aspartate aminotransferase and total bilirubin were significantly higher in LT-1W group, LT-2W group and LT-3W group than those in the normal control group (all P<0.05). PCR results showed that the relative expression of SLAMF5 mRNA were (5.44±1.11), (4.69±1.12), (2.18±0.68) respectively, which were increased in LT-1W group, LT-2W group and LT-3W group than those in normal control group (1.01±0.23), and the differences were statistically significant (all P<0.05). Immunohistochemical staining showed that SLAMF5 and CD4, CD8 positive T cells were mainly distributed in the portal area, hepatic lobule area and around the proliferative bile duct, and there was a certain overlap. Correlation analysis showed that there was a positive correlation between the expression of SLAMF5 in the lymphocyte infiltration area and the rejection activity index ( r=0.519, P=0.048). Conclusion:The expression of SLAMF5 is increased after liver transplantation in SD rats, and there is a correlation between SLAMF5 expression and liver transplantation rejection in rats.
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Objective To investigate the mechanism of action of integrin α4 (ITGA4) in liver fibrosis based on the anti-liver fibrosis effect of sticky sugar amino acid (SSAA) in rats. Methods A rat model of liver fibrosis was induced by intraperitoneal injection of CCl 4 , and then colchicine and low-, middle-, and high-dose SSAA were used for intervention, with blank control group and SSAA group as control. After 12 weeks of experimental intervention, serum and liver samples were collected to measure the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and HE staining and Sirius Red staining were used to observe the pathological conditions of liver tissue; quantitative real-time PCR was used to measure the transcriptional level of ITGA4, integrin β1 (ITGB1), transforming growth factor-β1 (TGFβ1), alpha-smooth muscle actin (α-SMA), and TIMP2 in liver tissue; Western blot was used to measure the relative protein expression levels of ITGA4, ITGB1, TGFβ1, α-SMA, MMP2, TIMP1, and TIMP2; immunohistochemistry was used to observe the protein expression of TGFβ1 and α-SMA. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t -test was used for comparison between two groups. Results There were significant increases in AST and ALT in the CCl 4 model group, and intervention with colchicine or low-, middle-, and high-dose SSAA reduced the levels of AST and ALT, with a significant difference between the CCl 4 model group and the other groups (all P < 0.05). HE staining and Sirius Red staining showed disordered structure of hepatic lobules and an increase in collagen fibers in the CCl 4 model group, and the structure of hepatic lobules was improved after intervention with colchicine or low-, middle-, and high-dose SSAA. The CCl 4 model group had significantly higher transcriptional levels of ITGA4, TGFβ1, α-SMA, and TIMP2 than the other groups, and there were significant reductions in the transcriptional levels of each factor after intervention with colchicine or SSAA, with a significant difference between the CCl 4 model group and the other groups (all P < 0.05). The CCl 4 model group had significantly higher protein expression levels of ITGA4, TGFβ1, α-SMA, TIMP2, and TIMP1 and a significantly lower protein expression level of MMP2 than the other groups, and intervention with colchicine or SSAA inhibited the expression of ITGA4, TGFβ1, α-SMA, TIMP2, and TIMP1 and promoted the expression of MMP2. Immunohistochemistry showed that the CCl 4 model group had significantly higher expression levels of TGFβ1 and α-SMA than the other groups, which was inhibited by intervention with colchicine or SSAA. The high-dose SSAA group had the most significant effect in reducing aminotransferases, improving lobular structure, and inhibiting the protein expression of liver fibrosis factors. Conclusion The high expression of ITGA4 in the liver is associated with the development of liver fibrosis, which is consistent with the increases in the expression of TGFβ1 and α-SMA. Inhibiting the expression of ITGA4 can provide more therapeutic targets for liver fibrosis and expand the anti-liver fibrosis mechanism of SSAA.
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Objective To investigate the effect of Huatan Qushi Huoxue prescription on the ultrastructure of hepatocyte mitochondria in a rat model of nonalcoholic steatohepatitis (NASH). Methods A total of 48 male Sprague-Dawley rats were randomly divided into blank group, model group, Yishanfu group, and Huatan Qushi Huoxue prescription group, with 12 rats in each group. The rats in the model group and the drug groups were administered and modeled since week 2; the rats in the blank group were given normal diet, and those in the other three groups were given high-fat diet. Based on dose conversion between human and animal, the equivalent dose of Huatan Qushi Huoxue prescription was 1.26 g/100 g body weight, and the equivalent dose of polyene phosphatidylcholine capsules (Yishanfu) was 0.014 18 g/100 g body weight. The rats in the model group were given 0.9% sodium chloride by gavage, those in the Yishanfu group were given polyene phosphatidylcholine suspension by gavage, and those in the traditional Chinese medicine group were given the granules of Huatan Qushi Huoxue prescription by gavage, once a day for 10 consecutive weeks. A transmission electron microscope was used to observe liver ultrastructure and perform a quantitative analysis. A one-way analysis of variance was used for comparison of continuous data between multiple groups; for further pairwise comparison, the least significant difference t -test was used for data with homogeneity of variance, and the Dunnett's T3 was used for data with heterogeneity of variance. Results The model group had a large number of lipid droplets accumulated in hepatocytes, changes in mitochondrial morphology and structure, and reductions in the number of mitochondria and endoplasmic reticulum. The Huatan Qushi Huoxue prescription group had a significant reduction in lipid droplets in hepatocytes and significant increases in the number of mitochondria and endoplasmic reticulum compared with the model group, with intact mitochondrial membrane and structure. The Yishanfu group had a reduction in lipid droplets in hepatocytes, an increase in the number of mitochondria, and a reduction in the number of endoplasmic reticulum, with relatively intact mitochondrial membrane and structure. The quantitative analysis showed that compared with the blank group, the model group had a significant increase in the area of lipid droplets and a significant reduction in mitochondria, with a significant difference in mitochondrial density between the two groups (all P < 0.01); after drug intervention, the Yishanfu group had a significant reduction in the area of lipid droplets and a significant increase in the number of mitochondria, with a significant difference in mitochondrial density between the Yishanfu group and the model group (all P < 0.01); compared with the Yishanfu group, the traditional Chinese medicine group had a significantly greater reduction in the area of lipid droplets and a significant increase in the number of mitochondria, with a significant difference in mitochondrial density between the two groups (all P < 0.05). Conclusion Huatan Qushi Huoxue prescription can improve lipid accumulation, increase mitochondrial density, and protect mitochondrial structure and function, with a better clinical effect than Yishanfu.
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Objective To investigate the effect of Shuganning injection (SGN) in alleviating drug-induced cholestasis and the possible mechanisms involved. Methods The liver of Sprague-Dawley rats was decellularized to prepare collagen scaffolds, and then the scaffolds were recellularized with human HepG2 cells to obtain the tissue-engineered liver (normal control group). The tissue-engineered liver was perfused with 10 μmol/L chlorpromazine (CPZ) and bile salt mixture to establish a model of drug-induced cholestasis (CPZ group), and the model was further treated with Shuganning injection (10 3 -fold dilution) as the injury protection group (SGN+CPZ group). The markers for hepatocellular injury [alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP)] and the antioxidant and oxidative stress markers [glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), and reactive oxygen species (ROS)] were measured for all groups, and the normal control group, the CPZ group, and the SGN+CPZ group were compared in terms of the mRNA and protein expression levels of the enzymes associated with liver bile salt metabolism and the enzymes associated with hepatic cholestasis. HE staining was performed to observe liver pathology. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t -test was used for further comparison between two groups. Results Compared with the CPZ group, the SGN+CPZ group had significant reductions in the markers for hepatocellular injury ALT, AST, LDH, and ALP (all P < 0.000 1), significant increases in the oxidative stress markers GSH and SOD ( P < 0.000 1 and P < 0.001), and significant reductions in the markers MDA and ROS ( P < 0.000 1 and P < 0.001). Compared with the CPZ group, the SGN+CPZ group had significant reductions in the mRNA expression levels of cholesterol 7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CPY8B1) in hepatocytes (all P < 0.001) and significant increases in the mRNA expression levels of farnesoid X receptor (FXR), small heterodimeric partner (SHP), bile salt export pump (BSEP), and multidrug resistance-associated protein 2 (MRP2) ( P < 0.000 1, P < 0.01, P < 0.000 1, and P < 0.000 1). HE staining showed that compared with the CPZ group, the SGN+CPZ group had a significant reduction in hepatocyte injury and a significant increase in the number of cells. Conclusion Shuganning injection can alleviate drug-induced cholestatic liver injury caused by chlorpromazine, and it exerts a protective effect by activating FXR in hepatocytes and increasing the expression of SHP to regulate bile salt balance. It also inhibits CYP7A1 and CYP8B1 to reduce the synthesis of hydrophobic bile acids and upregulates the expression of BSEP and MRP2 to promote the excretion of bile salts.
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OBJECTIVE@#To study the effects of buckwheat-oat-pea (BOP) composite flour [buckwheat ∶ oats ∶ peas=6 ∶ 1 ∶ 1 (quality ratio)] on blood glucose in diabetic rats.@*METHODS@#In this study, 64 male Sprague-Dawley rats were divided into 8 groups by fasting blood glucose (FBG) and body weight: normal control group, model control group, metformin group, buckwheat group, oats group, BOP low-dose group (BOP-L), medium-dose group (BOP-M), and high-dose group (BOP-H). The rats in the normal control group were fed with normal diet, the rats in the model control group and metformin group were fed with a high-fat diet (HFD), and the rats in the buckwheat group, oats group, and BOP-L, BOP-M, BOP-H groups were fed with HFD containing 10% buckwheat flour, 10% oat flour, 3.3% BOP, 10% BOP, 30% BOP, respectively. The HFD in all the groups had the same percentage of energy from fat (45%). After 30 days, the rats fed with HFD received intraperitoneal injection of streptozotocin (30 mg/kg, once a week for two weeks) to establish diabetes mellitus. After the model was successful established, the rats were fed for another 28 days. During the study, the body weight, food intake/body weight (FI/BW) and water intake/body weight (WI/BW), food utilization rate, 24 h urine volume, FBG, glucose area under curve (GAUC) of oral glucose tolerance test were measured regularly. At the end of the study, the fasting serum glucose and insulin were measured, and homeostasis model assessment of insulin resistance (HOMA-IR) was calculated.@*RESULTS@#With the inducing of HFD and streptozotocin, compared with the normal control group, the rats in the model control group had higher FI/BW, WI/BW, 24 h urine volume, FBG, GAUC, HOMA-IR (P < 0.05), and lower body weight, food utilization rate (P < 0.05). Compared with the model control group, the rats in the three BOP groups all had higher body weight, food utilization rate (P < 0.05), and lower WI/BW, HOMA-IR (P < 0.05); the rats in the BOP-L and BOP-M groups had lower FI/BW, 24 h urine volume, FBG (P < 0.05), and the rats in the BOP-M group also had lower GAUC (P < 0.05). After the establishment of diabetes, there was no significant difference in blood glucose and the other indicators between the rats in the three BOP groups and the buckwheat group or the oats group (P>0.05).@*CONCLUSION@#The BOP had the effects of reducing blood glucose, insulin resistance and diabetic symptoms on diabetic rats, and had the value for further development and utilization.
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Animals , Male , Rats , Avena , Blood Glucose , Diabetes Mellitus, Experimental , Diabetes Mellitus, Type 2 , Diet, High-Fat/adverse effects , Fagopyrum , Insulin , Insulin Resistance , Peas , Rats, Sprague-DawleyABSTRACT
ObjectiveTo investigate the protective effect and mechanism of Artemisia capillaris Thunb. decoction (YCHT), a classic heat-clearing and cholagogic traditional Chinese medicine (TCM) prescription, on rats with severe acute pancreatitis (SAP) induced by sodium taurocholate. MethodsA total of 30 Sprague-Dawley rats were randomly divided into sham-operation (SO) group, SAP model group, and YCHT (4.0 g/kg) treatment group, with 10 rats in each group. At 24 hours after successful modeling, pancreatic tissue and plasma samples were collected for analysis. HE staining was used to observe pathological injury of the pancreas; ELISA was used to measure the plasma levels of amylase, tumor necrosis factor-α (TNFα), and interleukin-1β (IL-1β); immunofluorescent staining was used to measure the fluorescence intensity of LC-3 protein, and TUNEL was used to measure cell apoptosis. Western blot was used to measure the protein expression of LC-3, Beclin-1, X-linked inhibitor of apoptosis protein (XIAP), caspase-3, and nuclear factor-kappa B (NF-κB) in the pancreas, and quantitative real-time PCR was used to measure the expression levels of lncRNA PVT1 and miRNA-30a-5p. A one-way analysis of variance and the Tukey’s test were used to analyze the differences between multiple independent samples. ResultsYCHT significantly alleviated the pathological injury of the pancreas of SAP rats, such as edema, necrosis, hemorrhage, and inflammatory cell infiltration. Compared with the SO group, the SAP group had significant increases in the plasma levels of amylase and the inflammatory factors TNFα and IL-1β, and there were significant reductions in the plasma levels of amylase, TNFα, and IL-1β after YCHT treatment (all P＜0.05). Compared with the SO group, the SAP group had significant increases in LC-3II/LC-3I ratio and the protein expression of Beclin-1, XIAP, caspase-3, and NF-κB, and compared with the SAP group, the YCHT group had significant reductions in LC-3II/LC-3I ratio and the protein expression of Beclin-1, XIAP, and NF-κB (all P＜0.05). Compared with the SO group, the SAP group had a significant increase in the expression of lncRNA PVT1 and a significant reduction in the expression of miRNA-30a-5p in the pancreas (both P＜0.05), and compared with the SAP group, the YCHT group had a significant reduction in the expression of lncRNA PVT1 and a significant increase in the expression of miRNA-30a-5p (both P＜0.05). ConclusionCell autophagy and apoptosis mediated by lncRNA PVT1/miRNA-30a-5p may be a drug target for YCHT treatment of SAP, which provides experimental and theoretical bases for further development of the TCM prescription YCHT for the treatment of SAP.
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ObjectiveTo investigate the protective effect of Lycium barbarum polysaccharide (LBP) combined with aerobic exercise (AE) on the liver of rats with nonalcoholic steatohepatitis (NASH) induced by high-fat diet based on the p38 mitogen-activated protein kinase (p38 MAPK)-nuclear factor-κB (NF-κB) pathway. MethodsAfter 1 week of adaptive feeding, 45 Sprague-Dawley rats, aged 8 weeks, were randomly divided into control group (10 rats fed with normal diet) and high-fat group (35 rats fed with high-fat diet). At the end of week 28, the high-fat group was randomly divided into model group, LBP group, AE group, and LBP+AE group, with 8 rats in each group, and intervention was performed for 10 weeks. At the end of the experiment, fasting blood glucose was measure for all rats, and serum samples, liver tissue, and visceral fat were collected. Biochemical kits were used to measure the serum levels of triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT), and aspartate aminotransferase (AST); ELISA kits were used to measure the serum levels of fasting insulin (FINS), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemotactic protein-1 (MCP-1); quantitative real-time PCR and Western blot were used to measure the mRNA and protein expression levels of Toll-like receptor 4 (TLR4), p38 MAPK, and NF-κB in liver tissue. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the control group, the model group had significant increases in TG, TC, AST, ALT, FINS, and homeostasis model assessment of insulin resistance (HOMA-IR) (all P ＜0.05), a tendency of increases in the serum levels of the inflammatory factors MCP-1, TNF-α, and IL-6 (all P ＜0.05), and significant increases in the mRNA and protein expression levels of TLR4, p38 MAPK, and NF-κB in liver tissue (all P ＜0.05). Compared with the model group, each intervention group had significant reductions in TG, TC, AST, ALT, FINS, and HOMA-IR (all P ＜0.05), a tendency of reductions in the serum levels of the inflammatory factors MCP-1, TNF-α, and IL-6 (all P ＜0.05), and significant reductions in the mRNA and protein expression levels of TLR4, p38 MAPK, and NF-κB (all P ＜0.05). Compared with LBP group, the LBP+AE group had significant reductions in TG, ALT, FINS, HOMA-IR, MCP-1, the mRNA expression level of TLR4, protein expression levels of p38 MAPK and NF-κB(all P＜0.05). Compared with Ae group, the LBP+AE group had significant reductions in FINS, HOMA-IR, IL-6, MCP-1, the mRNA expression level of TLR4 (all P＜0.05). ConclusionLBP combined with AE may improve inflammation in NASH rats by regulating the p38 MAPK/NF-κB pathway.
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ObjectiveTo investigate the effect of pretreatment with adenosine monophosphate-activated protein kinase (AMPK) agonist on rats with hepatic ischemia-reperfusion injury (HIRI) and the possible mechanism. MethodsA total of 54 healthy specific pathogen-free male Sprague-Dawley rats were randomly and equally divided into 5-aminimidazole-4-formamide nucleotide (AICAR) treatment group (experimental group), ischemia-reperfusion group (control group), and sham-operation group. Samples were collected at 12, 24, and 72 hours after hepatic ischemia-reperfusion surgery to measure the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil), tumor necrosis factor-α (TNFα), and interleukin-6 (IL-6) and the level of adenosine triphosphate (ATP) in the liver. HE staining was used to observe liver histological changes; quantitative real-time PCR was used to measure the relative mRNA expression levels of AMPK, mammalian target of rapamycin (mTOR), glucose transporter type 4 (GLUT4), and multidrug resistance-associated protein 2 (MRP2); Western blot was used to measure the protein expression levels of phosphorylated AMPK, phosphorylated mTOR, phosphorylated GLUT4, and MRP2. The repeated-measures analysis of variance was used for comparison between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsHE staining showed that the experimental group had milder liver injury than the control group at each time point. At 12, 24, and 72 hours after surgery, the control group had significantly higher serum levels of ALT, AST, TBil, IL-6, and TNFα than the experimental group and the sham-operation group, while the experimental group had significantly higher levels than the sham-operation group (all P＜005). At 12, 24, and 72 hours after surgery, the experimental group had a significantly higher level of ATP in liver tissue than the control group and the sham-operation group, and the control group had a significantly lower level than the sham-operation group (all P＜0.05). At 12, 24, and 72 hours after surgery, compared with the control group and the sham-operation group, the experimental group had significantly higher relative mRNA expression levels of AMPK, GLUT4, and MRP2 and protein expression levels of phosphorylated AMPK, phosphorylated GLUT4, and MRP2 (all P＜0.05); compared with the sham-operation group, the control group had significantly higher relative mRNA expression level of AMPK and protein expression level of phosphorylated AMPK, as well as significantly lower relative mRNA expression levels of GLUT4 and MRP2 and protein expression levels of phosphorylated GLUT4 and MRP2 (all P＜0.05). The experimental group had significantly lower relative mRNA expression level of mTOR and protein expression level of phosphorylated mTOR than the control group and the sham-operation group (all P＜0.05), and compared with the sham-operation group, the control group had significantly higher relative mRNA expression level of mTOR and protein expression level of phosphorylated mTOR (both P＜0.05). ConclusionAICAR pretreatment can activate the AMPK signaling pathway, improve energy metabolism pathway, alleviate liver inflammation, and thus reduce the severity of HIRI.
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ObjectiveTo investigate the feasibility of apical sodium-dependent bile salt transporter (ASBT) and asialoglycoprotein receptor (ASGPR) in the design of oral liver-targeting preparations for the treatment of hepatic alveolar echinococcosis (HAE) by measuring the expression of ASBT and ASGPR. MethodsA total of 18 male Sprague-Dawley rats were selected, among which 10 were used to establish a model of HAE (HAE group) and 8 were used as controls (normal group). Immunofluorescence assay, Western blotting, and quantitative real-time PCR were used to measure the expression distribution, protein expression level, and mRNA expression level of ASBT in the ileal tissue of HAE model rats and normal rats; the same methods were used to measure the expression level of ASGPR in the non-diseased liver tissue and the marginal zone of liver tissue lesion of HAE model rats and the liver tissue of normal rats. The t-test was used for comparison of normally distributed continuous data between two groups; a one-way analysis of variance was used for comparison between three groups, and the least significant difference t-test was used for comparison between two groups. ResultsThe results of immunofluorescence assay, Western blotting, and quantitative real-time PCR showed that compared with the normal group, the HAE group had significantly upregulated expression of ASBT in the ileal tissue (t=5309, 4.110, and 28.060, all P＜0.05) and a significantly higher expression level of ASGPR (the closer to the lesion, the higher the expression) (F=110666, 128.201, and 143.879, all P＜0.001). ConclusionASBT and ASGPR can be used as potential mediated receptors for oral liver-targeting preparations for HAE, which provides a theoretical basis for the design of oral liver-targeting preparations for the treatment of HAE.
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Objective To investigate the expression of NLRP3 inflammatory body in the process of liver fibrosis in a rat model of common bile duct ligation (BDL) and the association of NLRP3 inflammatory body with liver fibrosis. Methods A total of 65 Sprague-Dawley rats were randomly divided into sham-operation group with 15 rats and BDL model group with 50 rats. On days 3, 7, 14, 21, and 28, 10 rats in the model group and 3 rats in the sham-operation group were sacrificed. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), direct bilirubin (DBil), total bilirubin (TBil), total bile acid (TBA), and alkaline phosphatase (ALP) were measured, and HE staining, Masson staining, and sirius red-picric acid staining were performed for liver tissue to evaluate liver fibrosis degree. Immunohistochemistry was used to measure the expression levels of alpha-smooth muscle actin (α-SMA) and transforming growth factor-β1 (TGF-β1) in liver tissue, Western blot and qRT-PCR were used to measure the expression level of NLRP3 inflammatory body, and ELISA was used to measure the level of the inflammatory factor interleukin-1β (IL-1β) in liver tissue. An analysis of variance was used for comparison of continuous data between groups, and the least significant difference t -test was used for further comparison between two groups. Results Compared with the sham-operation group, the BDL model group had significant increases in the serum levels of ALT, AST, DBil, TBil, TBA, and ALP (all P < 0.05) and the level of IL-1β in liver tissue ( P < 0.05), which reached the highest level on day 3 and then decreased. Compared with the sham-operation group over time, the BDL group had a significant increase in liver fibrosis score ( P < 0.05); immunohistochemistry showed gradual increases in the expression of SMA-α and TGF-β1 ( P < 0.05), and Western blot and qRT-PCR showed a gradual increase in the protein expression of NLRP3 inflammatory body in liver tissue ( P < 0.05), which remained stable after day 14. Conclusion Liver injury exists persistently in a rat model of BDL, and liver histopathology shows the dynamic evolution of hepatitis, liver fibrosis, and liver cirrhosis. NLRP3 inflammatory body is in a state of continuous activation and may play an important role in the process of liver fibrosis.
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Resumo Fundamento Os glicocorticóides (GCs) são amplamente prescritos para o tratamento de numerosos distúrbios clínicos devido às suas propriedades anti-inflamatórias e imunomoduladoras, e um dos efeitos indesejáveis mais comuns desses medicamentos é a dislipidemia. Objetivo Avaliar o efeito da quercetina, um flavonoide derivado de plantas, no perfil lipídico de ratos tratados com glicocorticóides em altas doses. Métodos Um total de 32 ratos Sprague-Dawley foram distribuídos aleatoriamente entre quatro grupos (8 ratos por grupo) e tratados por 6 semanas com uma das seguintes opções : (i) solução salina normal; (ii) 40 mg/kg de succinato sódico de metilprednisolona (MP); (iii) MP + 50 mg/kg de quercetina; (iv) MP + 150 mg/kg de quercetina. O MP foi injetado por via subcutânea e a quercetina foi administrada por gavagem oral 3 dias por semana. No final do estudo, o perfil lipídico dos animais foi medido através de kits enzimáticos. Os dados foram analisados e a significância estatística foi estabelecida em p <0,05. Resultados Os níveis séricos médios de colesterol total (CT), triglicerídeos (TG) e LDL aumentaram drasticamente em animais tratados com GC em comparação com o grupo controle. Ambas as doses de quercetina (50 e 150 mg/kg) melhoraram o CT (43% e 45%), LDL (56% e 56%) e TG (46% e 55%, respectivamente). A razão Apo B/A1 diminuiu mais de 20% após a ingestão de Anti-Inflamatory Agents. Conclusões Esses dados sugerem que a ingestão de quercetina Quercetin; induzida por glicocorticóides. (Arq Bras Cardiol. 2020; 115(1):102-108)
Abstract Background Glucocorticoids (GCs) are widely prescribed for the treatment of numerous clinical disorders due to their anti-inflammatory and immune-modulatory properties and one of the most common untoward effects of these drugs is dyslipidemia. Objective To evaluate the effect of quercetin, a plant-derived flavonoid, on the lipid profile of high-dose glucocorticoid treated rats. Methods A total of 32 Sprague-Dawley rats, were randomly distributed among four groups (8 rats per group) and treated for 6 weeks with one of the following: (i) normal saline; (ii) 40 mg/kg methylprednisolone sodium succinate (MP); (iii) MP + 50 mg/kg quercetin; (iv) MP + 150 mg/kg quercetin. MP was injected subcutaneously, and quercetin was administered by oral gavage 3 days a week. At the end of the study, the animals' lipid profile was measured by enzymatic kits. Data were analyzed and statistical significance was set at p<0.05. Results The mean serum total cholesterol (TC), triglyceride (TG) and LDL levels were drastically increased in GC-treated animals compared with the control group. Both doses of quercetin (50 and 150 mg/kg) ameliorated TC (43% and 45%), LDL (56% and 56%) and TG (46% and 55% respectively). Apo B/A1 ratio decreased more than 20% following quercetin intake and the decline in TC/HDL, TG/HL, LDL/HDL ratios were significant. Conclusions These data suggest that quercetin intake with both doses of 50 and 150 mg/kg could be considered as a protective agent for glucocorticoid-induced dyslipidemia. (Arq Bras Cardiol. 2020; 115(1):102-108.)
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Animals , Rats , Quercetin/pharmacology , Glucocorticoids , Apolipoproteins , Triglycerides , Rats, Sprague-Dawley , LipidsABSTRACT
OBJECTIVE@#To evaluate the sub-acute oral effect of titanium dioxide (TiO2) nanoparticles on the oxidation/antioxidation biomarkers and inflammatory cytokines in blood, liver, intestine, and colon in rats.@*METHODS@#Twenty four 4-week-old clean-grade Sprague Dawley (SD) rats were randomly devided into 4 groups by body weight (n=6, control, low, middle, and high), in which the rats were orally exposed to TiO2 nanoparticles at doses of 0, 2, 10 and 50 mg/kg body weight/day for 28 consecutive days separately. Food intake, body weight and abnormal behaviors during the experiment were recorded. The rats were euthanized on the 29th day. The blood was collected via abdominal aortic method and centrifuged to collect the serum. Tissues from liver, intestine and colon were collected and homogenated. Then enzyme-linked immunosorbent assay (ELISA) and microwell plate methods were used to detect oxidation/antioxidation biomarkers including superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GSH-Px), total mercapto (T-SH), glutathione disulfide (GSSG), malomdialdehvde (MDA) and inflammatory cytokines including interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in the serum, liver, intestine and colon in the rats.@*RESULTS@#Compared with the control group, no significant differences in body weight, food intake and organ coefficients were observed in all the three groups after TiO2 gavage. No significant changes in GSH, GSH-Px, T-SH, and IL-6 were observed. Compared with the control group, significant increase of SOD activity in serum in high dose group, signi-ficant increase of GSSG concentration in intestine in middle and high dose group and significant increase of MDA concentration in liver in low and high dose group were observed. Compared with the control group, a significant increase of TNF-α in liver in middle and high dose group was observed.@*CONCLUSION@#TiO2 nanoparticle can increase antioxidant enzymes activities in blood, increase oxidative biomarkers in liver and intestine, increase inflammatory cytokines in liver in rats after a 28-day sub-acute orally administration. Among blood, liver, intestine, and colon, liver is most sensitive to the toxicity induced by TiO2 nanoparticles, followed by intestine, blood, and colon in sequence.
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Animals , Rats , Antioxidants , Biomarkers , Cytokines , Nanoparticles , Oxidative Stress , Rats, Sprague-Dawley , TitaniumABSTRACT
ObjectiveTo investigate the effect of Quzhi Ruangan decoction on the mRNA and protein expression of organic anion transporting polypeptide 2B1 (OATP2B1) in the small intestine of rats with nonalcoholic fatty liver disease (NAFLD). MethodsAfter 1 week of adaptive feeding, 36 male Sprague-Dawley rats were randomly divided into normal group, model group, simvastatin group, and high-, middle-, and low-dose Quzhi Ruangan decoction groups, with 6 rats in each group. Liver tissue was collected and HE staining was used to observe hepatic steatosis; an automatic biochemical analyzer was used to measure the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), glucose (GLU), and cholesterol (CHOL); RT-PCR and Western blot were used to measure the mRNA and protein expression of OATP2B1 in the small intestine of rats. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. Results There were significant differences in liver index, GLU, and CHOL between groups (F=10.814, 12.298, and 5.024, all P＜0.05), and there were also significant differences in the mRNA and protein expression of OATP2B1 in the small intestine (F=13.384 and 73.025, both P＜0.05). Compared with the normal group, the model group had significant increases in the mRNA and protein expression of OATP2B1 (both P＜0.05); compared with the model group, the simvastatin group and the high- and middle-dose Quzhi Ruangan groups had a significant reduction in the mRNA expression of OATP2B1 (all P＜0.05); compared with the simvastatin group, the middle- and low-dose Quzhi Ruangan groups had a significant reduction in the mRNA expression of OATP2B1 (both P＜0.05). Compared with the normal group, the model group had a significant increase in the protein expression of OATP2B1 (P＜0.05); compared with the model group, all treatment groups had a significant reduction in the protein expression of OATP2B1 (all P＜0.001); compared with the simvastatin group, the high-, middle-, and low-dose Quzhi Ruangan groups had a significant reduction in the protein expression of OATP2B1 (all P＜0.05). ConclusionQuzhi Ruangan decoction may alleviate the pathological changes of NAFLD by reducing the overexpression of OATP2B1.
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ObjectiveTo investigate the effect and significance of cyclooxygenase-2 (COX-2) inhibitors on the expression of the Acsl gene family in the ileum of rats with nonalcoholic fatty liver disease (NAFLD). MethodsA total of 45 Sprague-Dawley rats were randomly divided into normal control group (15 rats given normal diet), NAFLD model group (15 rats given high-fat diet), and nimesulide group (15 rats given high-fat diet and nimesulide). All rats were sacrificed after 12 weeks of feeding, and then blood samples were collected from the inferior vena cava to measure total cholesterol (TC) and triglyceride (TG). HE staining and oil red O staining were performed for the liver to evaluate the degree of hepatic steatosis in each group, and quantitative real-time PCR was used to measure the mRNA expression of the Acsl family genes in the ileum. An analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the normal control group, the NAFLD model group had significant increases in serum TC and TG and marked hepatic steatosis (all P＜0.05); compared with the NAFLD model group, the nimesulide group had significant reductions in serum TC and TG and degree of hepatic steatosis (all P＜0.05). Compared with the normal control group, the NAFLD model group had a significant increase in the expression of COX-2 in the ileum (P＜0.05), and compared with the NAFLD model group, the nimesulide group had a significant reduction in the expression of COX-2 in the ileum (P＜005). Compared with the normal control group, the NAFLD model group had significant increases in the mRNA expression of Acsl3 and Acsl5 in the ileum (both P＜0.05), and compared with the NAFLD model group, the nimesulide group had significant reductions in the mRNA expression of Acsl3 and Acsl5 (both P＜0.05). ConclusionThe COX-2 inhibitor nimesulide can regulate the expression of the Acsl gene family in the ileum of rats with NAFLD, suggesting that COX-2 inhibitors may inhibit the progression of NAFLD through the Acsl gene.
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OBJECTIVE@#To investigate the mechanism of Chinese medicine Buyang Huanwu decoction (BYHWD) promoting neurogenesis and angiogenesis in ischemic stroke rats.@*METHODS@#Male SD rats were randomly divided into sham operation group, model group, BYHWD group, antagonist group and antagonist control group with 14 rats in each. Focal cerebral ischemia was induced by occlusion of the right middle cerebral artery for 90 min with intraluminal filament and reperfusion for 14 d in all groups except sham operation group. BYHWD (13 g/kg) was administrated by gastrogavage in BYHWD group, antagonist group and antagonist control group at 24 h after modeling respectively, and BrdU (50 mg/kg) was injected intraperitoneally in all groups once a day for 14 consecutive days. miR-199a-5p antagomir or NC (10 nmol) was injected into the lateral ventricle at d5 after ischemia in antagonist and antagonist control groups, respectively. The neurological deficits were evaluated by the modified neurological severity score (mNSS) and the corner test, and the infract volume was measured by toluidine blue staining. Neurogenesis and angiogenesis were detected by immunofluorescence double labeling method. The expression level of miR-199a-5p was tested by real-time RT-PCR, and the protein expressions of vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) were determined by Western blotting.@*RESULTS@#BYHWD treatment significantly promoted the recovery of neurological function (@*CONCLUSIONS@#Buyang Huanwu decoction promotes neurogenesis and angiogenesis in rats with cerebral ischemia, which may be related to increased protein expression of VEGF and BDNF through upregulating miR-199a-5p.
Subject(s)
Animals , Male , Rats , Brain Ischemia/drug therapy , Drugs, Chinese Herbal/therapeutic use , Ischemic Stroke/drug therapy , MicroRNAs/genetics , Neurogenesis/drug effects , Rats, Sprague-Dawley , Up-Regulation/drug effects , Vascular Endothelial Growth Factor A/geneticsABSTRACT
OBJECTIVE@#To investigate the effects of astragaloside Ⅳ (AS-Ⅳ) on microglia/macrophage M1/M2 polarization and inflammatory response after cerebral ischemia in rats.@*METHODS@#Forty eight male SD rats were randomly divided into sham operation control group, model control group and AS-Ⅳ group with 16 rats in each. Focal cerebral ischemia model was induced by occlusion of the right middle cerebral artery (MCAO) using the intraluminal filament. After ischemia induced, the rats in AS-Ⅳ group were intraperitoneally injected with 40 mg/kg AS-Ⅳ once a day for 3 days. The neurological functions were evaluated by the modified neurological severity score (mNSS) and the corner test on d1 and d3 after modelling. The infarct volume was measured by 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining on d3 after ischemia. The expression of M1 microglia/macrophage markers CD86, inducible nitric oxide synthase (iNOS) and pro-inflammatory factors TNF-α, IL-1β, IL-6, M2 microglia/macrophages markers CD206, arginase-1 (Arg-1), chitinase-like protein (YM1/2) and anti-inflammatory factors interleukin-10 (IL-10) and transforming growth factor beta (TGF-β) was detected by real-time RT-PCR. The expression of CD16/32/Iba1 and CD206/Iba1 was determined by double labeling immunefluorescence method in the peripheral area of cerebral ischemia.@*RESULTS@#Compared with model control group, AS-Ⅳ treatment improved neurological function recovery and reduced infarct volume after ischemia (@*CONCLUSIONS@#The findings suggest that AS-Ⅳ ameliorates brain injury after cerebral ischemia in rats, which may be related to inhibiting inflammation through promoting the polarization of the microglia/macrophage from M1 to M2 phenotype in the ischemic brain.