Effects of liraglutide on adiponectin and insulin resistance in rats with non-alcoholic fatty liver disease

AIM：To investigate the effects of glucagon-like peptide 1 analog, liraglutide, on adiponectin and insulin resistance in the rats with diet-induced non-alcoholic fatty liver disease (NAFLD). METHODS：Male rats were randomly divided into 3 groups:normal diet (ND) group (n=10), high-fat diet (HFD) group (n=10), and HFD with intraperitoneal injection of liraglutide group (n=10, first 12 weeks with HFD, later 4 weeks with liraglutide). All treatments continued for 16 weeks, and then the rats were killed ethically and the blood samples and liver tissues were collected. The levels of alanine aminotransferase (ALT), fasting blood glucose (FBG), total cholesterol (TC) and triglyceride (TG) were detected by a biochemical automatic analyzer. The levels of free fatty acids (FFAs), fasting insulin (FINS) and adiponectin were measured by RIA and ELISA. RESULTS：Compared with HFD group, the body weight, liver index, homeostasis assessment-insulin resistance (HOMA-IR), the serum levels of TG, TC, ALT and FBG, and the liver levels of TG, TC and FFAs in the rats in liraglutide group were apparently lower, the degree of hepatic steatosis and inflammatory activity significantly decreased (P<0.05), and the level of adiponectin in the serum and liver homogenate increased ob-viously (P<0.05). The level of adiponectin in the liver homogenate was negatively correlated with the levels of FFAs in the liver homogenate. CONCLUSION:Liraglutide is beneficial for NAFLD rats to improve insulin resistance and reduce hepatic steatosis by increasing the level of adiponectin in the serum and liver tissues.     

Ginsenoside Rg1 promotes growth and extracellular matrix synthesis in degenerative human lumbar nucleus pulposus cells by inhibiting Wnt/β-catenin pathway

AIM: To explore the role of ginsenoside Rg1 in the growth of degenerative human lumbar nucleus pulposus cells (HNPCs). METHODS: Cultured HNPCs were subjected to oxygen-glucose deprivation (OGD) to mimic the micro-environment of degenerative HNPCs. The morphological changes of the cells in control group and OGD group were observed under optical microscope. The cells were treated with ginsenoside Rg1 at concentrations of 25, 50 and 100 μmol/L. The expression of collagen Ⅱ and aggrecan at mRNA and protein levels was determined by real-time PCR and Western blot analysis. The cell viability was measured by CCK-8 assay. The mRNA level of Ki67 was detected by real-time PCR. The apoptosis was analyzed by flow cytometry. The activity of caspase-3 was measured by a caspase-3 kit. The expression of Wnt/β-catenin pathway-related proteins was determined by Western blot. Furthermore, the expression of Wnt/β-catenin pathway-related proteins, the cell viability and apoptosis, and the expression of extracellular matrix synthesis proteins were assessed after the cells were co-treated with LiCl and 100 μmol/L ginsenoside Rg1. RESULTS: Normal HNPCs attached on the cell culture plate faster, and were almost round with rich cytoplasm. However, the cell adherence was slower, and the cells were long fusiform with decreased cytoplasm after OGD treatment, indicating that the model of degenerative HNPCs was successfully established. Compared with normal HNPCs, the expression of collagen Ⅱ and aggrecan at mRNA and protein levels was decreased in OGD group (P<0.05), which was then increased after the cells were treated with ginsenoside Rg1 at 25, 50 and 100 μmol/L (P<0.05). Compared with normal HNPCs, the cell viability and Ki67 expression were decreased in OGD group (P<0.05), which were increased after treatment with ginsenoside Rg1 (P<0.05). Meanwhile, the apoptotic rate and caspase-3 activity were significantly increased in OGD-treated cells (P<0.05), which were decreased after treatment with ginsenoside Rg1 (P<0.05). In addition, the activation of Wnt/β-catenin pathway was also inhibited by ginsenoside Rg1 treatment at dose of 100 μmol/L (P<0.05). LiCl, a Wnt/β-catenin pathway agonist, obviously decreased the protective effects of ginenoside Rg1 on OGD-induced cells (P<0.05), indicating that the Wnt/β-catenin pathway was involved in the protective effects of ginenoside Rg1 on degenerative HNPCs. CONCLUSION: Ginsenoside Rg1 promotes growth and extracellular matrix synthesis of degenerative HNPCs through inhibiting Wnt/β-catenin pathway. This study will provide a new idea for prevention and treatment of degenerative HNPCs.     

Role of SIRT1/AMPK pathway in early intervention of Lira alleviating non-alcoholic fatty liver disease caused by high-fat diet in rats

AIM To investigate the effect of early intervention of glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide (Lira) on oxidative stress, glucose tolerance, hepatic steatosis and insulin resistance of the rats with high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD), and to explore the role of silent information regulator 1 (SIRT1)/AMP-activated protein kinase (AMPK) signaling pathway in this process. METHODS Twenty-four male SD rats were randomly divided into normal diet (ND) group, HFD group and HFD+Lira group, with 8 rats in each group. After 1 week of adaptive feeding, the rats in HFD+Lira group were subcutaneously injected with Lira (200 μg/kg) per day at a fixed time point, while the rats in the remaining 2 groups were injected with normal saline at the same volume. During the intervention, the body weight, hair, appetite, defecation and activity of the rats were observed to adjust the dosage timely. The body weight, food intake and blood glucose were recorded weekly. Glucose tolerance test was performed at the end of the 16th week. At the end of the 18th week, hyperinsulinemic euglycemic clamp test was conducted after anesthesia. Blood was taken from the carotid artery. The liver and adipose tissues from different parts were taken after death. The serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and other indicators were detected. HE staining was used to observe the pathological changes of the liver tissue. Lipid accumulation in the liver tissues was observed by oil red O staining. Liver fibrosis was observed by Masson staining and Sirius red staining. Fluorescence staining for reactive oxygen species (ROS) was used to observe the oxidative stress in the liver. The expression of GLP-1 receptor in the liver was observed by immunofluorescence staining. The expression and localization of SIRT1 and phosphorylated AMPK at Thr172 [p-AMPK (Thr172)] were observed by immunohistochemical staining. The protein levels of AMPK, p-AMPK (Thr172), SIRT1, phosphorylated sterol regulatory element binding protein-1c at Ser372 [p-SREBP-1c (Ser372)], phosphorylated acetyl coenzyme A carboxylase at Ser79 [p-ACC (Ser79)], carnitine palmitoyltransferase 1A (CPT1A) and fatty acid synthase (FAS) in liver tissues were determined by Western blot. RESULTS The results of HE and oil red O staining of rat liver tissues in HFD group confirmed the structural disorder and serious lipid accumulation, while Masson and Sirius red staining showed severe fibrosis, suggesting the successful establishment of NAFLD rat model. Compared with ND group, the levels of total cholesterol (TC), triglyceride (TG), AST and ALT in serum, and the levels of malondialdehyde (MDA), TC, TG and ROS in liver tissues in HFD group were significantly increased (P<0.01), while the activity of superoxide dismutase (SOD) was decreased (P<0.01). The protein levels of p-AMPK (Thr172), SIRT1, p-SREBP-1c (Ser372), p-ACC (Ser79) and CPT1A in the liver tissues were significantly reduced (P<0.05 or P<0.01), while the expression of FAS was increased （P<0.01）. Compared with HFD group, lipid accumulation and fibrosis in the liver tissues of the rats in HFD+Lira group were significantly attenuated, the serum levels of TC, TG, AST and ALT, and MDA, TC, TG and ROS in liver tissues were markedly reduced (P<0.05 or P<0.01), while SOD activity was increased (P<0.05). The protein levels of p-AMPK (Thr172), SIRT1, p-SREBP-1c (Ser372), p-ACC (Ser79) and CPT1A in the liver tissues were significantly increased (P<0.05 or P<0.01), while the expression of FAS was decreased （P<0.01）. CONCLUSION Lira attenuates insulin resistance, oxidative stress and fibrosis, and improves liver lipid metabolism in the rats with NAFLD induced by HFD, which may be mediated by SIRT1/AMPK signaling pathway.     

Effect of ginsenoside Rg1 on activity and protein expression of NOS in rats with cerebral ischemic reperfusion injury

AIM: To observe the neuroprotective effects of ginsenoside Rg1 on focal cerebral ischemia reperfusion (I/R) injury in rats. METHODS: SD rats were applied to right middle cerebral artery occlusion (MCAO) for 2 h followed by 24 h of reperfusion. The rats were randomly divided into sham-operation group, I/R group and ginsenoside Rg1 pretreatment groups. The rats in ginsenoside Rg1 pretreatment groups were pretreated with ginsenoside Rg1 at doses of 10, 20 or 40 mg/kg once a day for 7 days and then subject to MCAO. The neurological deficit score was measured by Longa's method. The neurons were observed with Nissel staining. The nitric oxide (NO) content, the activity of nitric oxide synthase (NOS) and inducible NOS (iNOS) in the brain tissues were determined. The expression of neuronal NOS(nNOS) and iNOS was detected by Western blotting. RESULTS: Compared with sham-operation group, ginsenoside Rg1 significantly reduced the neurological deficit score and increased the neuron number in the hippocampus. The activity of NOS and iNOS, and NO content were decreased. Ginsenoside Rg1 also down-regulated the expression of nNOS and iNOS. CONCLUSION: Ginsenoside Rg1 has protective effect on the brain during cerebral I/R injury in rats. The mechanism may be related to reducing the content of NO and the activiy of NOS dose-dependently.     

Expression of calpain 2 and Bax in rat fibrotic liver tissues

AIM：To observe the expression of calpain 2 and Bcl-2-associated X protein (Bax) in rat fibrotic liver tissues and to explore their effects on hepatic fibrosis.
METHODS：Forty male Wistar rats were randomly divided into four groups (each n=10): 4-week control group, 8-week control group, 4-week liver fibrosis group and 8-week liver fibrosis group. Liver fibrosis model was induced by subcutaneous injection of 40% CCl4 (3 mL/kg) every 3 days for 4 or 8 weeks. The apoptosis of hepatocytes was detected by TUNEL. Additionally, the mRNA expression of calpain 2 and bax was determined by real-time PCR, and the protein expression of calpain 2 and Bax was determined by immunohistochemistry and Western blotting. RESULTS：Real-time PCR showed that the mRNA expression of calpain 2 and bax in liver tissues was elevated in 4-week and 8-week liver fibrosis groups. The results of immunohistochemistry and Western blotting revealed that there was no difference of calpain 2 protein expression in liver tissues between 4-week liver fibrosis group and control group, but that in 8-week liver fibrosis group was obviously increased. The protein expression of Bax in 4-week and 8-week liver fibrosis groups was higher than that in control groups. Additionally, the numbers of apoptotic hepatocytes in 4-week and 8-week liver fibrosis groups were obviously increased compared with control groups.CONCLUSION：Calpain 2 and Bax may play important roles in the process of liver fibrosis.     

Effects of astragaloside IV combined with ginsenoside Rg1 on autophagy and PI3K signaling pathways of PC12 cells induced by oxygen glucose deprivation/reoxygenation

ATM: To probe the effect and the mechanism of astragaloside IV and ginsenoside Rg1 on autophagy of PC12 cells induced by oxygen glucose deprivation/reoxygenation (OGD/R). METHODS: The autophagy injury model of PC12 cells induced by OGD/R was established(PC12 cells were exposed to 2 h of OGD followed by 24 h of reoxygenation). The effects of astragaloside IV combined with ginsenoside Rg1 on autophagy of PC12 cells were observed, and the mechanism was studied through PI3K Ⅰ/Akt/mTOR and PI3K Ⅲ/becline-1/Bcl-2 signaling pathways. RESULTS: After OGD/R, LC3-Ⅱ/LC3-Ⅰin PC12 cells was increased. Astragaloside IV, ginsenoside Rg1 and astragaloside IV combined with ginsenoside Rg1 restrained the increase in LC3-Ⅱ/LC3-Ⅰ, the effect of the combination was greater than using the drug alone. Ginsenoside Rg1, astragaloside IV combined with ginsenoside Rg1 up-regulated the phosphorylation level of PI3K Ⅰ, Akt and mTOR. The effects of the combination were stronger than those of using the drug alone. Astragaloside IV, astragaloside IV combined with ginsenoside Rg1 inhibited the protein expression of PI3K Ⅲ and becline-1, the effects of the combination were better than those of single astragaloside IV and single ginsenoside Rg1. Meanwhile, the combination treatment increased Bcl-2 protein expression. CONCLUSION: The autophagy of PC12 cells induced by OGD/R is inhibited by astragaloside IV and ginsenoside Rg1. Furthermore, astragaloside IV combined with ginsenoside Rg1 plays synergitic inhibition on autophagy, the mechanism may be related to PI3K Ⅰ/Akt/mTOR and PI3K Ⅲ/becline-1/Bcl-2 signaling pathways.     

Effects of ginsenoside Rg1 of Panax notoginseng on brain tissue injury and Nrf2/HO-1 signal pathway after cerebral ischemia/reperfusion in mice

AIM:To observe the effects of ginsenoside Rg1 of Panax notoginseng on brain tissue injury after mouse cerebral ischemia/reperfusion(I/R), and to explore the mechanisms involving nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) signal pathway. METHODS:C57BL/6 mice were randomly divided into sham group, cerebral I/R group, ginsenoside Rg1+cerebral I/R group and edaravone+cerebral I/R group. Ginsenoside Rg1 was successively administered for 3 d. One hour after final administration, bilateral common carotid arteries were ligated to induce brain tissue injury for 20 min, and then reperfusion for 24 h. Edaravone, a drug for anti-oxidative stress injury in the treatment of ischemic cerebro-vascular disease, was used as a positive control. The brain tissues were obtained to determine the neural cellular pathology in hippocampal CA1 region. The mRNA expression of Nrf2 and HO-1 was detected by RT-PCR. The protein levels of Nrf2 in the nucleus and cytoplasm and HO-1 in the whole cells in the brain tissues were measured by Western blotting. RESULTS:After ischemia/reperfusion for 24 h, the pathological injury in the neural cells was obvious, and the cell survival rate decreased. Ginsenoside Rg1 and edaravone attenuated the neural cell injury, and significantly increased the cell survival rate. After ischemia/reperfusion for 24 h, the mRNA expression of Nrf2 and HO-1 significantly increased in the brain tissues. The protein levels of Nrf2 in the nucleus and cytoplasm in the brain tissues were increased, the nuclear translocaition rate and the protein expression of HO-1 also increased. Ginsenoside Rg1 and edaravone both decreased the protein levels of Nrf2 in the cytoplasm of the brain tissues, increased that in the nucleus, and also increased Nrf2 nuclear translocation rate and the protein expression of HO-1. The effect of edaravone was higher than that of ginsenoside Rg1, but they had no significant effect on the mRNA expression of Nrf2 in the brain tissues. CONCLUSION: Ginsenoside Rg1 has the effect of anti-brain tissue injury on cerebral ischemia/reperfusion. The mechanisms may be related to activating the Nrf2/HO-1 signal pathway, promoting Nrf2 synthesis and nuclear translocation, thus promoting the expression of downstream antioxidant protein HO-1.     

Effect of ginsenoside Rb1 on liver cell pyroptosis in hyperlipidemia rats and its possible mechanism

AIM:To discuss the mechanism of ginsenoside Rb1 against liver lipid deposition by observing the effect of ginsenoside Rb1 on liver cell pyroptosis in hyperlipidemia rats. METHODS:Totally 32 healthy SPF rats were randomly divided into control group, model group, ginsenoside Rb1 group and simvastatin group. The rats in control group was given the basic feed, while the others were given high-fat diet. The rats in ginsenoside Rb1 group and simvastatin group were given corresponding drugs. The rats in control group and model group were intraperitoneal injected with equal volume of saline. Eight weeks later, the serum levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were tested by the automatic biochemistry analyzer. The pathological changes of the liver tissues were observed with HE staining. The protein and mRNA expression levels of pyroptosis-related factors NLRP3, caspase-1, IL-1β, IL-18 and GSDMD were detected by Western blot and RT-qPCR. RESULTS:Compared with control group, the serum levels of TC, TG and LDL-C in model group were increased significantly (P<0.01), and the HDL-C content was decreased significantly (P<0.05). The steatotic liver cells covered the visual field. The mRNA and protein expression levels of NLRP3, caspase-1, IL-1β, IL-18 and GSDMD were increased significantly (P<0.01). Ginsenoside Rb1 significantly decreased the serum levels of TC, TG and LDL-C (P<0.05), and significantly increased the content of HDL-C (P<0.01). Ginsenoside Rb1 also significantly decreased the degree of steatosis, and the number and size of lipid droplets. The mRNA and protein expression levels of NLRP3, caspase-1, IL-1β, IL-18 and GSDMD were decreased significantly (P<0.05 or P<0.01). CONCLUSION:Ginsenoside Rb1 atte-nuates liver injury and inhibits liver lipid deposition in hyperlipidemia rats by reducing the expression of hepatic pyroptosis-related factors.     

Effect of insulin resistance on fatty liver in high-fat diet-fed mice

AIM: To study the influence of insulin resistance on fatty liver in the mice fed with high-fat diet (HFD).METHODS: Male 8-week-old C57BL/6J mice were randomly divided into HFD group (with 60% calories by high saturated fatty acid) and control group (with chow diet).The mice in both groups were fed for 12 weeks. The body weight, liver weight, serum triglyceride (TG) and total cholesterol (TC), and blood glucose and insulin levels were measured. Hyperinsulinemic euglycemic clamp experiment was applied to reflect insulin sensitivity. The lipid deposition in the liver was analyzed by HE staining, Sudan IV staining and measurement of liver fat content. The phosphorylation levels of IRS1 and Akt, and the protein levels of SREBP-1 and FAS were determined by Western blot to reflect the activities of insulin signaling and lipid synthesis.RESULTS: Compared with control group, the body weight and liver weight were significantly increased in HFD group. TG and TC contents in serum and liver tissues were remarkably increased in HFD group. High-fat diet induced insulin resistance, as evidenced by increased serum insulin levels, reduced glucose infusion rate and decreases in IRS1 and Akt phosphorylation levels. In livers of HFD group, HE staining showed that the cytoplasm of hepatocytes was filled with vacuoles. Sudan IV staining also displayed that many different sizes of red lipid drops existed in the hepatocytes, and the protein levels of SREBP-1 and FAS were significantly increased. In primary normal hepatocytes with exogenous oleic acid intervention for 48 h, the phosphorylation levels of IRS1 and Akt were reduced, and the protein expression of SREBP-1 and FAS was significantly increased in a dose-dependent manner.CONCLUSION: Feeding with HFD leads to insulin resistance, resulting in activation of lipid synthesis and accumulation of lipid deposition in the liver, thus inducing fatty liver.     

Changes of endoplasmic reticulum stress-related molecules CHOP and TRB3 in rat fibrotic liver

AIM: To observe the changes of endoplasmic reticulum stress-related molecules CCAAT/enhancer-binding protein homologous protein(CHOP) and Tribbles homolog 3（TRB3） in the process of liver fibrosis induced by carbon tetrachloride (CCl4). METHODS: Male Wistar rats weighing 180 g to 200 g were divided into 4-week normal control group, 8-week normal control group, 4-week liver fibrosis group and 8-week liver fibrosis group. The rats in liver fibrosis groups were induced by subcutaneous injection of 40% CCl4 for 4 weeks or 8 weeks. The pathological changes of the liver were observed under light microscope. The protein level of ATF6 was determined by Western blotting. The protein and mRNA levels of CHOP and TRB3 in the liver were analyzed by immunohistochemistry, Western blotting and real-time PCR, respectively. The apoptosis of hepatocytes was measured by TUNEL assay. RESULTS: Pseudolobuli formed in the liver tissue of hepatic fibrotic rats. Compared with the control rats, the protein level of p90ATF6 was obviously decreased, the protein level of p50ATF6 was obviously increased, and the protein and mRNA levels of CHOP and TRB3 were obviously higher in the hepatocytes of hepatic fibrotic rats. The apoptosis of hepatocytes was also increased in 4-week and 8-week fibrosis groups. CONCLUSION: In the process of liver fibrosis induced by 40% CCl4, the obviously increased expression of endoplasmic reticulum stress-related molecules CHOP and TRB3 at protein and mRNA levels indicates that endoplasmic reticulum stress may play an important role in the liver fibrosis by promoting the apoptosis of hepatocytes.     

Effect of berberine on oxidative damage and the SIRT1/p53 pathway in liver tissues of NAFLD rats

AIM:To investigate the effect of berberine on oxidative damage and silent mating type information regulation 2 homolog 1 (SIRT1)/p53 pathway in the liver tissues of nonalcoholic fatty liver disease (NAFLD) rats and to explore the mechanism of berberine against NAFLD. METHODS:The male SD rats (n=24) were randomly divided into normal group, model group and berberine group (8 rats in each group). The rats in normal group was fed with normal diet, while the rats in model group and berberine group were fed with high-fat diet. The rats in berberine group was intragastrically administered with daily doses (100 mg/kg) of berberine for 16 weeks. The levels of liver total cholesterol (TC), triglyceride (TG), superoxide dismutase (SOD), malondialdehyde (MDA) and total anti-oxidant capatity (T-AOC) were measured. HE staining, oil red O straining and transmission electron microscopy were used to observe the histological changes of the livers. The protein levels of SIRT1, p53 and acetylated p53 (Ac-p53) were determined by Western blot. RESULTS:Compared with model group, the levels of liver TC, TG and MDA in berberine group were significantly reduced (P<0.05 or P<0.01), and the levels of SOD and T-AOC were significantly increased (P<0.01). The results of pathological observation showed that the lipid accumulation in the liver of berberine group was significantly attenuated. Compared with model group, the expression of SIRT1 was significantly increased and the expression of Ac-p53 was obviously reduced in berberine group (P<0.01). CONCLUSION:Berberine reduces hepatic steatosis and oxidative damage in NAFLD rats induce by high-fat diet, and this effect may be associated with regulation of the SIRT1/p53 signal pathway.     

Mechanism of Fuzilizhong decoction alleviating inflammatory damage of rats with non-alcoholic fatty liver disease

AIM To observe the effect of Fuzilizhong decoction on the inflammatory damage of non-alcoholic fatty liver disease （NAFLD） rats and to explore its mechanism. METHODS SPF male SD rats were randomly divided into 6 groups： control group, model group, high dose （20 mg·kg^-1·d^-1）, middle dose （10 mg·kg^-1·d^-1）, low dose （5 mg·kg^-1·d^-1） Fuzilizhong decoction group and Yishanfu （30 mg·kg^-1·d^-1）group, 8 rats in each group. A NAFLD rat modelwas established by intragastric administration of fat emulsion for 4 weeks. Then the drug was given for 4 weeks in each treatment group. HE staining was performed to observe the histopathological changes of the rat liver.The serum levels of interleukin-2（IL-2）, IL-6 and tumor necrosis factor-α（TNF-α） were measured by ELISA. The expression of toll like receptor 4（TLR4） and NF-κB p65 in liver tissues at mRNA and protein levels was determined by RT-qPCR and Western bolt,respectively. RESULTS Compared with control group, the inflammatory damage of liver tissue was more serious, the serum levels of IL-2, IL-6 and TNF-α, the mRNA expression TLR4 and NF-κB p65 in liver tissues were significantly increased in model group（P<0.05）. However, compared with model group, the liver pathological changes in each treatment group were significantly relieved, the serum levels of IL-2, IL-6 and TNF-α, the mRNA expression of TLR4 and NF-κB p65 in liver tissues were significantly reduced（P<0.05）.In addition, the changes of TLR4 and p-NF-κB p65 protein levels in liver tissue were consistent with the changes of TLR4 and NF-κB p65 mRNA. CONCLUSION Fuzilizhong decoction attenuates the inflammatory damages of NAFLD in rats by inhibiting TLR4/NF-κB p65 signaling pathway.     

Effects of IGFBPrP1 and thioacetamide on liver tissues in mice

AIM: To investigate the effects of insulin-like growth factor binding protein related protein 1(IGFBPrP1) and thioacetamide (TAA) on the liver tissues, and to identify the role of IGFBPrP1 in liver fibrosis. METHODS: Thirty-two male C57BL/6 wild-type mice were randomly divided into 4 groups (n=8 in each group): control group, recombinant murine IGFBPrP1(rmIGFBPrP1) 4 weeks group, TAA 2 weeks group and TAA 4 weeks group. The methods of hematoxylin-eosin (HE) staining, picric acid-Sirius red staining, immunohistochemistry and Western blotting were performed. RESULTS: The extensive fatty degeneration of liver cells in rmIGFBPrP1 4 weeks group was observed. The collagen deposition was found in TAA 2 weeks group. In TAA 4 weeks group, the degree of hepatic fibrosis was more serious than that in TAA 2 weeks group. The expression levels of IGFBPrP1, transforming growth factor beta 1(TGF-β₁), Smad3, p-Smad2/3, collagen Ⅲ, collagenⅠand fibronectin (FN) in liver tissues were higher in rmIGFBPrP1 4 weeks group, TAA 2 weeks group and TAA 4 weeks group than those in control group. No significant difference of the expression levels of IGFBPrP1, collagen I and FN between rmIGFBPrP1 4 weeks group and TAA 2 weeks group was observed. CONCLUSION: IGFBPrP1 plays an important role in the process of thioacetamide-induced liver fibrosis. Meanwhile, IGFBPrP1 induces excessive deposition of extracellular matrix through TGF-β₁/Smad3 pathway.     

Changes of liver sinusoidal endothelial cells and basement membrane in early stage of liver fibrosis induced by carbon tetrachloride in rats

AIM：To explore the development of hepatic sinusoidal capillarization in the early stage of liver fibrosis induced by carbon tetrachloride (CCl4) in rats. METHODS：Clean SD rats were randomly divided into normal control group (group N, n=6) and liver fibrotic model group (group M, n=32). The rats in group N were intraperitoneal injected with saline and the rats in group M were intraperitoneal injected with CCl4 (2 mL/kg, twice a week for 4 weeks). At the end of the 3rd day and the 1st, 2nd and 4th weeks, all rats were killed and then the samples were collected. The pathological changes in the livers were observed by HE staining and Masson straining. The development of hepatic sinusoidal capillarization was observed by transmission electron microscopy (TEM) and immunohistochemical staining. The cell surface expression of vascular endothelium-associated marker CD31, collagen type Ⅳ (Col IV) and laminin (LN) was determined. RESULTS：HE and Masson staining showed the formation of liver fibrosis after treatment with CCl4 for 4 weeks. TEM showed that the fenestrate diameter of liver sinusoidal endothelial cells (LSECs) grew down, the fenestrate numbers of LSECs were decreased along with the development of liver fibrosis, and the consecutive basement membrane was formed at the end of the experiment. The expression of CD31 was significantly increased along with the development of defenestration, and the expression of Col IV and LN was significantly increased after the treatment with CCl4 for 2 weeks and 4 weeks, respectively. CONCLUSION：The typical hepatic sinusoidal capillarization was detected in the early stage of liver fibrosis, and the deposition of LN in the liver sinusoidal walls was the mainly factor of formation of the consecutive basement membrane.     

mRNA expression of insulin receptor and leptin receptor in liver of nonalcoholic fatty liver disease from diabetic rats

AIM: To observe the pathologic changes of liver in diabetic rats and to investigate the role of mRNA expression of insulin receptor and leptin receptor in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). METHODS: Twenty male Sprague-Dawley rats were divided randomly into two groups: normal control group and diabetic group. After fed with high-fat diet for 4 weeks, diabetic rats were injected with streptozotocin at a dosage of 30 mg/kg intraperitoneally to induce NAFLD model of type 2 diabetes mellitus. Then the diabetic animals were fed with high-fat diet continuously for 12 weeks. At the end of the experiment, the rats were sacrificed, the concentrations of blood glucose, serum lipid, ALT and AST were measured biochemically. The levels of serum leptin and serum insulin were detected by enzyme-linked immunosorbent assay (ELISA) and radio immunoassay (RIA), respectively. The pathologic changes of liver were observed under light microscopy (LM) stained with HE, Sudan Ⅲ and Masson trichrome staining, respectively. The ultra-structural changes of liver were observed under transmission electron microscopy (TEM). Additionally, the mRNA expressions of PEPCK, G6Pase, insulin R and leptin R from rat livers were assayed by semi-quantitative RT-PCR. RESULTS: The levels of blood glucose, serum insulin, serum TG, ALT and AST increased significantly (P<0.01), serum TC elevated (P<0.05), and the levels of serum leptin decreased (P<0.01) in diabetic group compared to those in normal control group. Obvious liver fatty degeneration, piecemeal necrosis with accompanying inflammatory infiltration and fibrosis were found under LM. Hepatocytes pyknosis, lots of lipid deposits in cytoplasm of hepatocytes, proliferation of collagen in space of Disse were observed under TEM in diabetic group. The expression of insulin R and leptin R mRNA in liver from diabetic rats increased significantly (P<0.01) while the expression of PEPCK and G6Pase mRNA remained unchanged. CONCLUSION: Insulin resistance plays an important role in the pathogenesis of NAFLD. Low level of serum leptin, up-regulation of mRNA expression of insulin R and leptin R in liver caused by insulin resistance may be involved in this process.     

Effects of traditional Chinese medicine Dan-shao-hua-xian capsule on expression of miR-200s in rat fibrotic livers

AIM: To investigate the effect of Dan-shao-hua-xian (DSHX) capsule on the expression of the family of microRNA-200 (miR-200s) in rat fibrotic livers. METHODS: Forty male Wistar rats weighing 180 g to 220 g were divided into 5 groups (control group, two model groups and two interference groups). The rats in model groups and interference groups were induced by hypodermic injection of CCl4 for 4 weeks and 8 weeks. The rats in interference groups were also treated with DSHX capsule (0.5 g/kg) once daily for 4 weeks and 8 weeks at the same time. The liver index and serum activity of ALT and AST were analyzed. The liver fibrosis was observed under microscope. Additionally, the expression of miR-200a, -200b, -200c, -141 and -429 was determined by quantitative real-time PCR. RESULTS: The liver index, and serum activity of ALT and AST in model groups and 4-week interference group were obviously higher than those in normal control group. The apparent liver fibrosis was observed in 8-week model group. The expression of miR-200a,-200b, -200c, -141 and -429 in the liver of 8-week model groups was obviously higher than that in control group. CONCLUSION: In the process of liver fibrosis induced by CCl4, the obvious changes of miR-200s may play an important role in the development of liver fibrosis. The miR-200s might be the potential target that DSHX capsule inhibits the process of liver fibrosis.     

Influence of Tangshenfang on diabetic liver injury and the expression of SIRT1 and PGC-1α in the liver tissue

AIM: To observe the effect of Tangshenfang (TS) on the liver protection and the levels of silent information regulator 1 (SIRT1) and peroxisom proliferator-activated receptor γ coactivator-1α (PGC-1α) in the liver tissue. METHODS: The rat model of diabetes mellitus (DM) was established by intravenous injection of streptozotocin (STZ;30 mg/kg) after having the high fat/high glucose diets for 1 month. The diabetic rats were randomly divided into DM group, DM with high-dose TS (TS^Hi) group, medium-dose TS (TS^Med) group and low-dose TS (TS^Low)group. The normal rats were served as control group. There were 8 rats in each group. After treatment with TS for 12 weeks, the serum biochemical indices including fasting blood glucose (FBG), triglyceride (TG), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were tested. Fasting insulin (FINS) was also detected by radioimmunoassay, and homeostatic model assessment for insulin resistance (HOMA-IR) was calculated. The serum levels of tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) were measured by ELISA. The activity of SOD and content of MDA in the liver tissues were measured by the methods of hydroxylamine and thiobarbituric acid. The liver pathological changes were observed under light microscope with HE and Masson staining. The protein expression of SIRT1and PGC-1α in the liver tissues was determined by Western blot. RESULTS: In DM group, serum FBG, TG, ALT, AST, FINS, HOMA-IR, TNF-α and IL-1 were obviously increased compared with the control group (P<0.01). The fatty changes, local necrosis, inflammation and fibrosis in the liver tissues were observed. The content of MDA in liver increased, while the activity of SOD decreased markedly. The protein expression of SIRT1 and PGC-1α was decreased (P<0.05). In TS treatment groups, all these changes in DM rats were markedly reversed by TS, and the protein expression of SIRT1 and PGC-1α in the liver tissues was markedly increased. CONCLUSION: TS may protect the rats from diabetic liver injury by increasing the expression of SIRT1 and PGC-1α, and thereby improving insulin resistance and oxidative stress.     

Effect of ginsenoside Rg1 and Rh1 onthe anti-tumor activity of dendritic cell

AIM: To study the effect of ginsenoside Rg1 and Rh1 on the anti-tumor activity of dendritic cells (DC). METHODS: Effect of Rg1 and Rh1 on the production of IL-12 p40 protein was detected by ELISA, and the IL-12 p40 mRNA level of DC was monitored by RT-PCR. Anti-tumor activity of DC-LPAK was determined by neutral red staining assay. RESULTS: The results of ELISA showed that Rg1 and Rh1 significantly enhanced the production of IL-12 p40 of DC. Rg1 at 1 mg/L and Rh1 at 100 mg/L upregulated the IL-12 p40 mRNA level. Rg1 and Rh1 enhanced the anti-tumor ability of DC, induced lymphokine and PHA activated killer (DC- LPAK) on human papillate tumor cell line. Each dose of Rg1 can obviously accelerate the cytotoxity to L929 at the E∶T ratio of 5∶1(P<0.05,0.01), while only Rh1 10 mg/L enhanced the cytotoxity ability of DC-LPAK (P<0.05). CONCLUSION: Rg1 and Rh1 enhanced the production of IL-12 p40. This effect may be mediated by the increase in the mRNA level. As a result, Rg1 and Rh1 promote the ability of DC to stimulate the cytotoxitic acticity of DC-LPAK.