| Background and objectivesNonalcoholic steatohepatitis (NASH) is a concept of clinical pathology which shows fatty degeneration, inflammatory cell infiltration, glucose nuclear and Mallory's body on liver cells, and it can involve various degrees of fibrosis. NASH patients haven't a clear history of chronic alcohol liver disease, it is one important reason for cryptogenic cirrhosis, and its performance is also one of the metabolic syndrome. The pathogenesis of NASH is currently recognized as the two-hit theory: the first is insulin resistance, which, by promoting lipolysis and peripheral hyperinsulinemia causes fatty degeneration of liver cells, fatty degeneration cells provide sufficient matrix and result in the sensitivity to inside and outside damageing factors enhanced; the second is the increase of oxidative metabolism, which causes lipid oxidation, cytokines, mitochondrial uncoupling protein-2 and Fas ligand are activated, and at last inflammation, necrosis or fibrosis.Up to now, there lack effective drugs in the treatment of NASH. The causes of NASH include obesity, type 2 diabetes and hyperlipidemia. The treatment for these diseases is the basis for the treatment of NASH. In addition, according to two-hit theory, oxidative stress and lipid peroxidation in the pathogenesis of NASH plays an important role, suggesting that antioxidant treatments are important measures to prevent NASH. Vitamin E, betaine, matrine, taurine can significantly reduce the serum transaminase levels, decreased liver steatosis, inflammation and fibrosis.Sophocarpine (SP) is a kind of sophorae alkaloids derived from foxtail-like sophora herb and seed. Its structure is similar with that of Kushen alkaloids. It has been reported that SP, with low cost, has little poisonous side effect within safe dose and it has high distribution in organization in vivo. So it has good prospects for clinical application. Recently, studies have proved that SP can inactivate endotoxin in vitro and control the single nuclear phagocyte system in the body which caused by the activation of inflammatory response, reduce endotoxin cause serious damage to the body, such as systemic inflammatory response syndrome and multiple organ dysfunction syndrome and so on. In addition, an intensively inhibitive effect of SP has been observed on the secretion of both HBsAg and HBeAg in HepG2.2.15 in vitro, it is a high-performance and low toxicity of effective anti-HBV drugs. Our previous studies have shown that sophocarpine can significantly reduce the level of experimental hepatic fibrosis and improve liver function. Whereas, in literature there is few report about anti-NASH of SP in liver. This study was to investigate the mechanism of SP in preventing NASH in rats.Methods(1) SD rat model of NASHEight male SD rats of about 160g were randomly divided into 2 groups. One group was normal group which was fed with normal diet for 12 weeks; the other was model group which was fed with high fat diet (basic diet with 100g/kg lard and 20g/kg cholesterol) for 12 weeks. At the 12th weekend, rats were fasted for 12 hours, and after weighing body weight, were intraperitoneally injected 20% urethane anesthesia. Then we opened the abdominal cavity, cut out all the liver and observed appearance of the liver. The right lobe of liver was cut down, washed with normal saline, and soaked in neutral buffered 10% formalin solution. Formalin immersing organization will be used in paraffin-embedded, serial sections, the HE staining was to assess the degree of fatty degeneration and inflammatory response of liver tissues. Compared the results of two groups, we assessed the preparation of NASH model.(2) The preventive and therapeutic effect of SP on NASHThirty-two male SD rats of about 180g were randomly divided into 4 groups. The first group was normal group, fed with normal diet for 12 weeks; the second was model group, fed with high fat diet (basic diet with 100g/kg lard and 20g/kg cholesterol) for 12 weeks; the third was SP prevention group, fed with high fat diet for 12 weeks and subcutaneously injected SP(20mg/kg body weight/d) at the same time; the fourth was SP treatment group, fed with high fat diet for 12 weeks and subcutaneously injected SP from the fifth week.At the 12th weekend, rats were fasted for 12 hours, and after weighing body weight, were intraperitoneally injected 20% urethane anesthesia, opened the abdominal cavity. We got blood from the inferior vena cava and tested serum lipid (Tch, TG, HDL, LDL) and liver function (ALT, AST). We cut out the whole liver, observed its appearance, weighted the liver, and calculated liver index. Then we cut down the right lobe of liver and washed it with normal saline, and soaked it in neutral buffered 10% formalin solution, cryopreservate the others in -70℃liquid nitrogen. Formalin immersing organization will be used in paraffin-embedded, serial sections. HE staining and V-G staining were used to assess the degree of inflammatory response and fiber deposition; SudanⅢstaining was used to observe the degree of fatty degeneration. IL-6, TNF-αand TGF-β1 protein expression in liver tissue were detected by immunohistochemistry. We also used RT-PCR technology to detecte mRNA expression of adiponectin, leptin, IL-6, TNF-α, TGF-β1, a-SMA and procollagen I in liver tissue.(3)The statistical treatmentAll the datas were expressed with mean±standard deviation ( x ? s), variance analysis was used for inter-group comparision; rank sum test was used for pathological and immunohistochemical classification. SPSS11.0 package was used for the results, P<0.05 meaned that the difference was significant.Results (1) SD rat model of NASHAt the end of 12 weeks, livers of the model group were bigger than those of the normal group, with tense capsule, blunt and round edge, and yellow surface. HE staining showed severe hepatocyte steatosis, and Mallory bodies, inflammatory cells infiltration, focal and confluent necrosis can also be seen. But the normal group hadn't characters as above. We got the conclusion that fat diet can be used to copy the NASH model successfully.(2) The prevention and therapeutic effect of SP on NASH1. Influence of SP on liver weight and liver index. In normal group, liver weight was 14.885±1.591g, liver index was 0.0319±0.0019; in model group, liver weight was 18.905±2.110g, liver index was 0.0381±0.0038; in SP prevention group, liver weight was 14.440±1.997g, liver index was 0.0312±0.0020; in SP treatment group, liver weight was 15.766±1.777g, liver index was 0.0328±0.0026. Liver weight and liver index in model group were significantly higher than those in normal group(P<0.05). Liver weight and liver index in intervention group were decreased significantly than those in model group(P<0.05). But there was no significant difference between prevention group and treatment group(P>0.05).2. Influence of SP on liver function index of serum. ALT of serum in normal group was 35.38±5.19U/L; in model group was 73.00±19.54U/L; in SP prevention group was 52.13±10.53U/L; in SP treatment group was 54.86±8.42U/L. ALT was significantly higher in model group than that in normal group(P<0.05). ALT in intervention group decreased significantly than that in the model group(P<0.05). But there was no significant difference between prevention group and treatment groups(P>0.05). AST wasn't significantly different between intervention group and model group(P>0.05).3. Influence of SP on lipids of serum. Tch, TG, HDL and LDL of serum in normal group were 0.56±0.239mmol/L, 0.26±0.071mmol/L, 1.10±0.150 mmol/L, 0.25±0.063mmol/L; in model group were 4.28±0.572mmol/L, 0.40±0.149mmol/L,2.04±0.193mmol/L, 2.85±0.512mmol/L; in SP prevention group were 3.07±0.846mmol/L, 0.27±0.082mmol/L, 1.55±0.254mmol/L, 2.31±0.669mmol/L; in SP treatment group were 3.95±0.906mmol/L, 0.33±0.067mmol/L, 1.78±0.266mmol/L, 2.81±0.832mmol/L. Tch, TG, HDL and LDL in model group were significantly higher than that in normal group(P<0.05); Tch, TG and HDL in prevention group were decreased significantly than that in model group(P<0.05), LDL in prevention group wasn't significant decrease than that in model group(P>0.05); HDL in treatment group was decreased significantly than that in model group(P<0.05), Tch, TG and LDL in treatment group weren't decreased significantly compared with those in model group(P> 0.05).4. Influence of SP on histopathology. HE staining showed inflammation in SP intervention group decreased significantly compared with that in model group(P<0.05); Sudan III staining showed fatty degeneration in SP intervention group decreased significantly compared with that in model group(P<0.05); V-G staining showed fibrosis in SP intervention group decreased significantly compared with that in model group(P<0.05). For all the above, there was no significant difference between prevention group and treatment group(P>0.05).5.Influence of SP on protein and cytokine expression. Immunohistochemistry showed that IL-6, TNF-αand TGF-β1 protein expression of liver tissues in SP intervention group was significantly lower than that in model group(P<0.05). RT-PCR showed that IL-6, TNF-αand TGF-β1 mRNA expression in SP intervention group were significantly lower than those in model group(P<0.05), leptin,α-SMA and procollagen-I mRNA expression in SP intervention group were also significantly lower than those in model group(P<0.05), but adiponectin mRNA expression in SP intervention group was significantly higher than that in model group(P<0.05). For all the proteins and cytokines, there was no significant difference between prevention group and treatment group(P>0.05).Conclusions(1) NASH model can be made successfully with SD male rats fed with high fat diet for 12 weeks.(2) Sophocarpine can inhibit NASH process significantly by reducing serum transaminase levels, liver steatosis, inflammation and fibrosis level, and toxic cytokine production.(3) Reduce inflammatory cytokine production and increase protective cytokine synthesis may be one important mechanism of anti-NASH for sophocarpine.
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