Study The Effects And Mechanisms Of Shenshao Oral Liquid On The Hepatocyte Damage Of Type 2 Diabetic Rats

Objectives To explore the effect and mechanism of Shenshao oral liquid on diabetesinduced liver injury rats,through detecting serum biochemical indexes, histomorphology and the expression of TLR4, NF-?B, TGF-?1 and other inflammatory factors, by constructing a model of liver injury in rats with type 2 diabetes mellitus, were treated with different dose of Shenshao oral liquid.Methods 1 Grouping: Fifty specific pathogen free male Wistar rats(170-200g)were randomly divided into five groups: control group, model group, shenshao oral liquid: lowdose of treatment group, medium-dose of treatment group, high-dose of treatment group.Each group has ten rats. 2 Model of type 2 diabetic rat: After adaptive feeding for 1 weeks,the control group was fed with normal diet, and the experimental group was fed with high fat diet, until 1 months after the start of modeling. The M group, T-L group, T-M group and T-H group were induced by intraperitoneal injection of Streptozotocin(25mg/kg)(concentration 1%, p H 4.5), the C group injected intraperitoneally the same dose of citrate buffer. Two successive fasting blood glucose ? 16.7 mmol/L and polydipsia, polyuria,polyphagia appear, initially confirmed the successful model of type 2 diabetes. Before the end of the experiment, measured fasting blood glucose ?16.7 mmol/L again for type 2diabetes model successfully, otherwise eliminated. 3 Drug intervention: After molding, TL, T-M and T-H group were given to 125 mg/Kg, 250 mg/Kg, 500mg/Kg of Shenshao oral liquid gavage and C, M group were given equal volume of distilled water, all groups for eight weeks. 4 Materials and processing: After eight weeks, all rats were sacrificed, blood samples and hepatic tissue were obtained. Using full automatic biochemical analytic method to measure the level of blood glucose(GLU), alanine aminotransferase(ALT),aspartate amino shift enzyme(AST), total cholesterol(TC), triglyceride(TG), low density lipoprotein cholesterol(LDL) to observe the prognosis of Shenshao oral liquid; HE staining was used to observe the morphological changes of liver tissue. Masson staining was used to observe the expression of fiber in liver tissues; Immunohistochemical staining was used to observe the location expression of TLR4?NF-?B?TGF-?1?IL-6 ?MMP-9 in rat liver tissues.Semi quantitative detection of TLR4?NF-?B?TGF-?1?IL-6 ?MMP-9 in liver tissue of rats was performed by western blot technique.Results 1 The model group rats appeared polydipsia, polyphagia, polyuria, lethargy, less body, bleak color and weight loss, long time observation and 3 fasting plasma glucose were significantly increased and stable, modeling success. Serum ALT and AST levels in model group rats were increased, HE showed diffuse swelling of liver cells, hydropic degeneration, sinusoid narrowing, spotty necrosis, inflammatory cell infiltration, that diabetic rats had significant liver injury. 2 Biochemical index:Compared with group C,body weight, liver weight decreased(P<0.05) and blood glucose, ALT, AST, TC, TG and LDL increased in group M(P<0.05). In each treatment group of Shenshao oral liquid,the above biochemical indexes were improved. Body weight and liver weight significantly increased, blood glucose, ALT, AST, TC, TG and LDL decreased obviously in rats of group T-H(P<0.05), the difference was statistically significant. 3 HE staining: Compared with group C, liver cell diffuse swelling, hydropic degeneration, spotty necrosis, hepatic sinus stenosis or occlusion, nuclear staining shallow or disappeared, nuclear were squeezed to one side, binuclear, portal area surrounded by fibrous tissue hyperplasia, inflammatory cell infiltration in group M. After the intervention of Shenshao oral liquid, liver cell edema, hydropic degeneration, inflammatory cell infiltration has improved in group T-L,further reduce in group T-M and sinus gap widened. In group T-H, no liver cell edema,sinusoid normal, portal area inflammatory cell infiltrate were reduced. 4 Masson staining: A large number of thick blue collagen fibers can be seen in the blood vessels,periportal and disse space of liver tissue in group M. In shenshao group: group T-L changes little, thick collagen fibers were still visible. Blue collagen fibers decreased and thinner in group T-M. Only a few blue collagen fibers were observed in group T-H, which was close to the normal control group. 5 Immunohistochemical Staining: The TLR-4protein was mainly expressed in the membrane of liver cells, brownish yellow granules.Compared with group C, yellow granules increased in group M. In shenshao group: yellow granules of group T-L was not obviously reduced. Group T-M and group T-H were significantly decreased, but group T-H was the most obvious. Similar expression were observed in NF-?B?TGF-?1?MMP-9?IL-6. Almost no expression of group C, group M increased significantly, group T-L decreased, group T-M reduced obviously, and there was only a small amount of expression in group T-H. 6 Western blot: Compared with group C, the expression of TLR-4, NF-?B, TGF-?1, MMP-9, IL-6 protein significantly increased in group M(P<0.05). In group of Shenshao oral liquid: the expression of TLR-4,TGF-?1, IL-6 protein was lower in group T-L than that in group M(P<0.05), while NF-?B, MMP-9 had a decreasing trend, but no significant; The expression of TLR-4, NF-?B,TGF-?1, IL-6 protein in group T-M was significantly lower than that in group M(P<0.05), but there was no significant difference between the group M in the expression of MMP-9; The expression of TLR-4, NF-?B, TGF-?1, MMP-9, IL-6 protein was obviously reduced in group group T-H(P<0.05).Conclusions 1 Shenshao oral liquid has a protective effect on liver injury in diabetic and there is dose-dependent relationship among125 mg/kg?250 mg/kg?500 mg/kg. 2 The protective effects of Shenshao oral liquid on diabetic hepatic injury may be achieved through the regulation of TLR4 and NF-?B meidiated inflammation injury.