The Protective Effect Of Polysaccharide Of Grifola Frondosa On Carbon Tetrachloride-induced Liver Injury And Its Mechanism

Backgroud:Chemical liver injury has become the second-largest liver injury incentives after the viral hepatitis。There are about 500 to 1,000 kinds of Chinese and Western medicines in clinical application as well as variety of chemicals, can cause of liver disease. According to information reported at home and abroad,20% of fulminant hepatic failure caused by chemical substances. In recent years, chemical liver injury has been growing as a serious threat to people's health. But there is still no effective clinical prevention drugs, thus it is very importamce to enhance the protection of liver injury and carry out chemical protection work.Carbon tetrachloride (CC14) liver injury is the classic model of liver injury, and it can make the liver a strong injury, include degeneration, necrosis, fatty degeneration, and fibrosis. CCl4-induced liver cell injury is similar with alcohol liver damage. So CCl4-induced liver injury models are widely used in acute and chronic liver injury reseasch.Polysaccharide of Grifola Frondosa(PGF) is extracted from Maitake (Grifola frondosa) fruit bodies or mycelium of a fungus, and its activity structure with a high degree of branchingβ-1,3-glucan andβ-1,6-glucan. Pharmacological analysis confirmed that the PGF have many bio-activity,such as anti-tumor, improve the immune system activity, regulating blood sugar,blood fat and cholesterol levels, scavenge oxygen free radical etal. However, the hepatoprotection of this polysaccharide has not reported.Objective: 1,Establish the liver injury model by CCl4 in vitro and in vivo.2,Study the protective effect of Polysaccharide of Grifola Frondosa on CCl4-induced liver injury and to explore its mechanism, which afford the theory system for PGF as one clinical therapy.Methods:1,In vitro study(1) To establish a stable liver injury model induced by 60% saturated CCl4;(2) The cells were divided into different groups:normal control group, CCl4 injured group, PGF protective groups (50μg/ml, 100μg/ml,200μg/ml,400μg/ml);(3) The morphology of cells were measured by Giemsa staining;(4) Cell viability was assayed by MTT assay;(5) Intracellular MDA and SOD were measured via the biochemical assays;(6) The changes of Ca2+ in different groups, staining with Flou-3/AM, were detected, using laser scanning confocal microscopy;(7) The changes of mitochondria membrane potential were analyzed by flow cytometry;(8) Bcl-2,Bax which were related to apoptosis were detected by Western-blot;2,in vivo study(1) To build mouse acute liver injury models induced buy CCl4;(2) The mice were divided into 8 groups:a blank control group, a CC14 group, six doses of PGF groups (4.5mg/kg,9mg/kg,18mg/kg,36mg/kg,72mg/kg, 144mg/kg);(3) Observe the histopathological changes of liver tissue by HE staining;(4) ALT,AST in the serum were measured via the biochemical assays;(5) MDA and SOD in the Liver homogenate were measured via the biochemical assays;(6) Bcl-2/Bax which were related to apoptosis were detected by Western-blot;Results:1,In vitro study (1) Morphological observation:normal group cell growth in good condition; injury group cells, there are more cells undergo apoptosis; while the PGF Protection groups cell survival status are superior to injury,which 100μg/ml is the most effective protection group.(2) MTT assay:cell viability in injury group reduced markedly (32.31%), PGF can enhance cell viability evidently,and in concentration 50～400μg/ml, the protective effect with the increased concentration of PGF role in the weakening after the first increase。The best protection of the concentration is 100μg/ml group, which the relative survival rate 74.92%。The protect results in the following indicators are the results of best protection group concentration (100μg/ml)(3) AST,ALT tests:①AST:normal group 70.11IU/L, CC14 damaged group 152.16IU/L,100μg/ml PGF group 78.47IU/L;②ALT:normal group 35.26 IU/L, CCl4 damaged group 96.47 IU/L,100μg/ml PGF group 44.39 IU/L(4) SOD,MDA tests:①SOD:normal group 121.94U/mgprot, CCl4 damaged group 56.55 U/mgprot, 100μg/ml PGF group 112.67U/mgprot;②MDA:normal group 2.14nmol/mgprot, CCl4 damaged group 5.19 nmol/mgprot, 100Oμg/ml PGF group 2.55 nmol/mgprot.(5) IOD of fluorescence intensity of Ca2+:normal group 302.6, CC14 damaged group 2393.4, protective group 529.4;(6) mitochondrial membrane potentia:normal group 455.17, CCl4 damaged group 96.26, PGF protective group 248.75;(7) Bcl-2 and Bax①Bcl-2:normal group 1.53, CCl4 damaged group 0.16, PGF group 1.20;②Bax:normal group 0.45, CCl4 damaged group 1.01, PGF group 0.71 nmol/mgprot; 2,in vivo study(1) Liver morphology:normal group:the liver is red and soft texture; CCl4 damaged group:the liver shows yellow soil, texture harden, or see the size of needle-like nodules; the PGF protected group:the liver shape between the normal group and CCl4 damaged group, apparent protective effectly.(2) Pathological observation:normal group:hepatic lobule, hepatic cord arrangement rulesand structural integrity, the cells were morphologically normal, no inflammatory cell infiltration. CCl4 injury group:a large number of inflammatory cells infiltrating the hepatic sinusoids, liver cord disarray, surrounding a large central vein hepatocytes cell swelling. The PGF Protection PGF of liver cell necrosis and inflammatory cell infiltration significantly reduced,hepatic lobule structure, and cell morphology were normal;The protect results in the following indicators are the results of best protection group concentration (72mg/kg)(3) Serum enzyme tests:①AST:normal group 30.52IU/L, CC14 damaged group 142.87IU/L, PGF group 43.69IU/L;②ALT:normal group 15.18 IU/L, CC14 damaged group 218.37 IU/L, PGF group 57.42 IU/L)(4) Histochemical detection of liver homogenate:①SOD:normal group 93.44U/mgprot, CCl4 damaged group 47.28 U/mgprot, 72mg/kg PGF group 88.47U/mgprot;②MDA:normal group 3.62nmol/mgprot, CCl4 damaged group 7.83 nmol/mgprot,72mg/kg PGF group 4.51 nmol/mgprot;(5) Bcl-2 and Bax①Bcl-2:normal group 1.05, CCl4 damaged group 0.21, PGF group 0.95;②Bax:normal group 0.27, CCl4 damaged group 0.87, PGF group 0.51 nmol/mgprot; Conclusion:1,Establishment of a stable CCl4 liver cell injury model in vitro with 60% saturated CCl4;2,Make sure that PGF has a distinct protective effect on liver injury induced by CCl4. The best protection concentration in vitro is 100μg/ml;3,GPs can prevent the increasing of intracellular calcium concentration, prevent the decreasing of mitochondrial membrane potential, change the expression level of Bcl-2 and Bax, and then raise the cell survival;4,In vivo results show that PGF can effectively protect liver injury induced by CCl4, the best protection of the concentration is 72mg/kg;5,In vivo results show that the mechanism of PGF may inculde maintain the membrane stability to reduce the vitality of serum transaminasethe, increase intracellular SOD content, decreased MDA content to enhance the intracellular free radical scavenging capacity and reducing lipid peroxidation,, regulating Bcl-2/Bax protein to suppress apoptosis.