Study On The Mechanism Of Liver Injury Caused By Polygonum Multiflorum Based On Different Kidney Deficiency Syndrome

Objectives:To study the toxicity mechanism and relativity with TCM kidney deficiency syndrome of liver injure induced by Polygonum multiflorum on the basis of symptom-based prescription theory. And verify the hypothesis of the relevance among toxicity, target syndrome and mitochondrial energy metabolism.Methods:1. Crude Polygonum multiflorum and prepared Polygonum multiflorum were taken as the research objects to establish the pathological animal model of deficiency of kidney-yin and kidney-yang using different methods. Through observing behavioral indexes of rats and detecting the content of T3、T4、CAMP、CGMP、E2、 T、Cort in serum of rats to determine the optimum method for establishment of deficiency of kidney syndrome animal model.2. The rats of deficiency of kidney-yin and kidney-yang were orally administered with different Polygonum multiflorum decoction solution to prepare the medicated serum. Observe the morphological changes of L02 hepatocytes in each group by means of inverted microscope, transmission electron microscope and fluorescence microscope. Observe the results of medicated serum of Polygonum multiflorum inducing apoptosis of L02 hepatocytes for different times by means of flow cytometry and mtt assay. And the contents of GSH, GSSG and MDA in hepatocyte were assayed by DTNB method and 2-TBA method to study the mechanism of Polygonum multiflorum inducing apoptosis of L02 hepatocytes.3. Establish the integrated animal model of deficiency of kidney-yin and kidney-yang using different methods.The contents of ALT、AST、ALP、TBIL、DBIL in rat serum were detected by automatic biochemistry analyzer To observe the morphological changes of liver by HE staining. The activity of GSH-Px and the content of MDA in liver were respectively assayed by colorimetry and 2-TBA method. And check the apoptotic index by TUNEL method. The mitochondria of hepatocytes were prepared to measure the activity of SDH and respiratory chain complex Ⅰ. Detected the protein expression of Fas/FasL in liver by by immunohistochemistry and mesured the expression of Bcl-2 and Bax mRNA by real-time quantitative PCR to make further study on the mechanism of liver injure induced by Polygonum multiflorum.Results:1. The animal model of deficiency of kidney-yin induced by subcutaneous injection of hydrocortisone (50mg/kg) appeared less activity in daytime, loss of energy, more activity at night, increase in water and food intake, but loss of weight, tail flush, hairs withered and irritability, and moreover, the content of T3 and the ratio of CAMP/CGMP increased. The symptoms of successful model of deficiency of kidney-yin are consistent with clinical symptoms. The animal model of deficiency of kidney-yang induced by subcutaneous injection of hydrocortisone (25mg/kg) appeared less activity, loss of weight, urine increased, sparse hairs, aversion to cold, huddle up, testicular atrophy and irritability, and moreover, the content of CAMP and T, the ratio of CAMP/CGMP decreased obviously. The symptoms of successful model of deficiency of kidney-yang are consistent with clinical symptoms.2. The medicated serum of low dose group (10.8 g/kg) of prepared Polygonum multiflorum in the state of deficiency of kidney-yin could promote L02 hepatocyte proliferation might related to the characteristic of replenishing the kidney-yin. When the dosage of prepared Polygonum multiflorum lower than a certain degree, the body might produce more chemical constituents which should promote L02 hepatocyte proliferation. It may be related to the liver protection of prepared Polygonum multiflorum. When the rats were orally administered with 10.8 g/kg dosage of prepared Polygonum multiflorum, the body might less produce some chemical constituents which should inhibit L02 hepatocyte proliferation. But when the rats were orally administered with 43.2 g/kg prepared Polygonum multiflorum decoction solution in the state of deficiency of kidney-yang or the rats were orally administered with 10.8g/kg crude Polygonum multiflorum, the body might produce some chemical constituents which should obviously inhibit 102 hepatocyte proliferation.3. The liver function of rat was not obviously damaged when the dosage of prepared Polygonum multiflorum between 10.8 g/kg and 21.6g/kg treating the deficiency of kidney-yin for 3 months, meanwhile the levels of ALT and AST have no significant differences by comparing with normal group. But when the dosage of prepared Polygonum multiflorum more than 43.2g/kg treating the deficiency of kidney-yin for 3 months, the levels of ALT in the serum of rats significantly increased which suggested that the liver function of rat was not obviously damaged with the results of hepatocyte membrane destruction. The liver function of rat was damaged when the dosage of prepared Polygonum multiflorum more than 21.6g/kg treating the deficiency of kidney-yang for 3 months, meanwhile the levels of ALT and AST significantly increased. After 43.2 g/kg prepared Polygonum multiflorum treating the deficiency of kidney-yang for 3 months, antioxidant capacity of hepatocyte decreased significantly and lipid peroxidation of liver increased significantly, then hepatocytes were damaged, the apoptosis index of hepatocytes and expression of Fas, FasL rised obviously, and the expression of Bax mRNA and the ratio of Bcl-2/Bax also rised obviously, but the expression of Bcl-2 mRNA reduced obviously. And after 10.8 g/kg crude Polygonum multiflorum treating the deficiency of kidney-yang for 3 months, antioxidant capacity of hepatocyte decreased significantly and lipid peroxidation of liver increased significantly, then hepatocytes were damaged obviously than prepared Polygonum multiflorum group and focal necrosis of liver tissue were observed.Conclusions:1. The liver injure induced by prepared Polygonum multiflorum is related to TCM kidney deficiency syndrome. When treating the animal model of deficiency of kidney-yang, prepared Polygonum multiflorum induce more obvious hepatotoxicity than treating the animal model of deficiency of kidney-yin. But if treating the animal model of deficiency of kidney-yin with excess dosage, prepared Polygonum multiflorum aslo may injure the liver.2. The mechanism of Polygonum multiflorum-induced hepatotoxicity:(1) It may be related to the increase of MDA in hepatocytes and the reduced activity of GSH and ability of scavenge oxygen free radicals. (2) The metabolic disturbance may be caused by affecting respiratory chain through reducing the activity of SDH and respiratory chain complex I. (3) The apoptosis of L02 hepatocytes may be induced by upregulating the protein expression of Fas/FasL in liver. (4) The apoptosis of L02 hepatocytes may be induced by upregulating Bax and downregulating Bcl-2.