| Objective To observe and study the changes of body weight,mental status,liver injury,expression of Tumor necrosis factor a(TNF-?),Interleukin-6(IL-6)and intestinal microecology in rats with liver cirrhosis induced by carbon tetrachloride(CCL4)complex factors.The preventive effect and mechanism of Piper longum extract on liver cirrhosis and intestinal flora imbalance in rats.Methods Animal grouping 60 SD rats purchased from Inner Mongolia Medical University were randomly divided into blank control group,cirrhosis model group,high,medium and low dose groups of Piper longum extract,12 in each group.Weight measurement All five groups of rats were weighed twice a week and their weight was recorded.Modeling Except blank control group,rats in other groups were induced with 0.35g/l phenobarbital sodium solution for 1 week.In the 2nd and 3rd weeks,40% and 50% of the carbon tetrachloride rapeseed oil solution were injected subcutaneously at multiple points.At the end of the 4th and 14 th weeks,60% of the concentration was injected subcutaneously at multiple points.The volume of each injection was 0.3ml/100 g,two times a week,and the dosage was adjusted according to the change of body weight.At the same time,except for the blank control group drinking tap water,the other groups were given 10% of edible alcohol as the only drinking water,and the model period was 14 weeks.Oral administration Rats in each group were given orally at the same time and under the same feeding environment;rats in cirrhosis model group were given orally normal saline once a day;rats in high dose group(66.7 mg/kg),rats in medium dose group(33.3 mg/kg),rats in low dose group(16.6 mg/kg): rats in each group were given orally once a day with extract of Piper longum.After 14 weeks of continuous intragastric administration,1ml/100 g volume was administered to the stomach.Daily status of rats The changes of body weight,mental state and colour of feces were observed during the experiment.Morphological observation At the end of the 14 th weekend,rats in each group were sacrificed and their livers were collected at the 15 th week.The liver pathological changes were observed by hematoxylin-eosin staining(HE staining)and Masson staining.The distribution and expression of TNF-? and IL-6 proteins in the liver of rats in each group were detected by immunohistochemical staining.Intestinal flora sequencing The structure and abundance of intestinal flora of rats in each group were detected by second generation sequencing.Results The first part 1.Daily status of rats The blank group had glossy coat,good appetite,sensitive response to external stimuli,normal colour of feces,sparse coat,dry and dull coat,decreased appetite and activity,delayed reaction,and began to excrete black feces at the 11 th week.The hair and mental state of rats in high,middle and low dose groups were improved to varying degrees.2.Weight change of rats There was significant difference in weight between blank group and model group and treatment group(P<0.05).There was significant difference between each treatment group and cirrhosis model group(P<0.05).There was little difference in weight among treatment groups,but there was significant difference between high-dose group and low-dose group(P<0.05).3.HE staining results showed that the hepatocytes in the blank group were normal,the hepatocytes were arranged neatly,and the lobules were well-formed.In cirrhosis model group,a large number of hepatocytes showed degeneration and vacuolar necrosis.Fatty lesions were more serious,accompanied by inflammatory cell infiltration,disordered arrangement of hepatocyte cord,a large number of fibrous tissue proliferation,and the destruction of hepatic lobule structure was replaced by pseudolobule formation,suggesting that the model was successfully established.Compared with the model group,the degree of liver injury in the treatment group was significantly reduced,and the fatty degeneration of hepatocytes gradually decreased with the increase of piper longum extract concentration.4.The results of Masson staining showed that the hepatocytes in the blank group were normal in shape and arranged radially around the central vein without fatty lesions or fibrosis.In model group,degeneration and vacuolar necrosis of hepatocytes,fatty lesions and fibrosis were serious,and the structure of hepatic lobules was destroyed and pseudolobules were formed.Compared with the model group,the degree of liver fibrosis in the treatment group was generally improved,and with the increase of piper longum extract concentration,the fatty lesions and fibrosis of hepatocytes were gradually reduced.The second part 1.RT-PCR results showed that the expression of TNF-alpha and IL-6 genes in the liver was significantly increased in the model group compared with the blank group(P<0.01),compared with the model group(In the high-dose group,the expression of TNF-alpha and IL-6genes in the liver was significantly decreased(P<0.01).2.Immunohistochemical staining showed that the positive expression of TNF-alpha and IL-6 was brown granules in the cytoplasm of hepatocytes.Compared with the blank group,the expression of TNF-alpha and IL-6 protein in the cytoplasm of hepatocytes in the model group was significantly increased(P<0.01);compared with the model group,the expression of TNF-alpha and IL-6 protein in the cytoplasm of hepatocytes in the treatment group was significantly decreased(P<0.05),and the expression of TNF-alpha and IL-6 protein increased with the concentration of extract of Piper longum.The level gradually decreased.The third part 1.The sequencing results of intestinal flora showed that the appropriate dose of alfalfa extract can play a role in anti-liver fibrosis by promoting the growth of beneficial bacteria in the intestine of rats and preventing the destruction of intestinal mucosal barrier.Conclusions1.The rat model of liver cirrhosis was successfully established by subcutaneous injection of CCL4 and induction of low concentration alcohol.2.piper longum extract can alleviate hepatocyte injury and hepatic fibrosis induced by CCL4 and low-concentration alcohol in rats,and the protective effect of piper longum extract on liver is gradually enhanced with the increase of drug concentration.3.Piper longum extract can protect liver by reducing the expression of TNF-alpha,IL-6 genes and proteins in liver,which can promote liver fibrosis,thus inhibiting the further development of liver fibrosis to cirrhosis.4.Proper dosage of Piper longum extract can promote the growth of beneficial bacteria in rat intestinal tract,prevent the damage of intestinal mucosal barrier,and indirectly provide protection for liver. |
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