Prevention Of Saikosaponin D Dimethylnitrosamine Induced Hepatic Fibrosis Associated With Renal Injury In Experimental Research

Objective:Hepatic fibrosis is a pathological process which is due to the unbalanced production, degradation and deposition of extracellular matrix when persistent liver injury exists, and also is a common characteristic of the chronic liver disease. Advanced hepatic fibrosis can lead to liver cirrhosis and hepacarcinoma. Dimethylnitrosamine(DMN) has hepatotoxicity, genotoxicity and immunotoxicity, whose active metabolites can cause methylation of nucleic acid and protein.DMN can cause hepatocyte necrosis, extracellular matrix accumulation, finally hepatic fibrosis by given low doses many times. Hepatocyte doesn't develop fatty degradation in this model, and the lesion of hepatic fibrosis is relatively stable. Large doses of DMN can result in renal carcinoma. Therefore, the aim of this experiment was to study the major pathological mechanisms in this model, and investigate the mechanism of SSd preventing hepatic fibrosis from two aspects. Kidney injury was also observed in this model induced by DMN, and the prevention effect on kidney was also been further explored.Methods:1.model:Twenty-four male Sprague-Dawley rats, 4 weeks old and weighing 70-90 g, were randomly divided into three groups including normal, model, and SSd, with 8 rats per group. Rats in liver fibrosis model group and SSd group were given an intraperitoneal injection of DMN (10 mg/kg body weight) one time for two consecutive days per week, lasting for 4 weeks. Normal group was given saline in the same way.2.drug:Rats in SSd treated group received SSd(1.8mg/kg body weight) by intraperitoneal injection daily for four weeks. Rats in normal group and model group received PBS in the same patern.3.materials:After 4 weeks, all rats were sacrificed by intraperitoneal injection of chloral hydrate(3.5ml/kg body weight). The blood was taken from abdominal aorta and stored in -20℃after having been centrifuged. The liver and kidney were partly removed and fixed with 4 % paraform phosphate buffer. The remnant of the liver was stored in liquid nitrogen and -80℃.4.examination:Serum:ALT, AST, BUN, Scr in serum were measured by biochemical analyses and MDA,SOD by spectrophotometric methods and HA , LN, C-IV by radioimmunological methods.The Liver:The liver was embedded and fixed with paraffin and prepared for HE,Masson,sirius red staining,and immunohistological examinations of TGF-β1,α-SMA. The remnant of the liver was stored in liquid nitrogen for detection of TGF-βRⅠ,TGF-βRⅡ,Smad3,Smad7 mRNA by RT-PCR and the liver stored in -80℃was used to examine MDA,SOD by spectrophotometric methods.The Kidney: The kidney was embedded and fixed with paraffin and prepared for HE,PAS,sirius red staining,and immunohistological examinations of TGF-β1,α-SMA.Results:1.Compared with the normal group, the increase of the consentrations of ALT,AST,BUN,Scr in model group were significantly higher(P<0.01,P<0.05,P<0.05,P<0.05,respectively),and the level of HA,LN,C-IV also reached a statistical significence(P<0.01,P<0.01,P<0.01). Histopathological observation found that kidney injury also occurred in this hepatic fibrosis animal model, the major pathological changes was infiltration of large number of inflammation cells around blood vessels.2.The consentrations of ALT,AST,BUN,Scr in SSd group were significantly lower than model group(P<0.01,P<0.01,P<0.01,P<0.01),and the level of HA,LN,C-IV also reduced in SSd group(P<0.01,P<0.05, P<0.05). Histopathology observation found that SSd can reduce the collagen accumulation both in the liver and the kidney(P<0.01,P<0.01).Immunohistological examinations also revealed that SSd can decrease the expression of TGF-β1 andα-SMA both in the liver and the kidney(P<0.01,P<0.01,P<0.01,P<0.01).3.DMN could reduce the activity of SOD both in serum and the liver tissue(P<0.01,P<0.05) and increase the levels of MDA both in serum and liver tissue(P<0.05,P<0.05). The results disclosed that traditional Chinese medical monomer SSd could significantly elevate the enzyme activity of SOD in liver homogenate(P<0.01)and reduce the consentrations of MDA both in serum and liver homogenate(P<0.01,P<0.01).4.The mRNA expression of TGF-βRI,Smad3 were increased(P<0.01,P<0.01) in liver tissue in model group while the mRNA expression of TGF-βRII,,Smad7 were decreased(P<0.05,P<0.01).SSd could significantly reduce the mRNA expression of TGF-βRI,Smad3(P<0.01,P<0.05)and increase the TGF-βRII,Smad7( P<0.05,P<0.01).This evidence indicated that SSd may prevent hepatic fibrosis by influencing TGF-βsignal transduction pathway.Conclusions:1.Kidney injury occurred in hepatic fibrosis model induced by DMN. The major pathological changes were infiltration of large number of inflammation cells around the blood vessels in interstitium.2.SSd also can prevent the hepatic fibrosis and kidney injury induced by DMN .TGF-β1 andα-SMA may be the common targets of its effects.3.Lipid peroxidation is involved in the fibrosis progression in rats, and SSd can reduce the injury, protect hepatocytes, and finally prevent the progress of the hepatic fibrosis.4. TGF-βRI, TβRII, Smad3 and Smad7 participates in hepatic fibrosis. Another Possible mechanism of SSd in prevention and treatment of liver fibrosis and cirrhosis is that SSd generally regulate of TGF-beta1 signal transduction pathway through many target proteins and genes.