Preparation Of Schisandra Chinensis Polysaccharide And Its Mechanism Of Attenuating Liver And Kidney Toxicity Induced By Cyclosporin A

ObjectiveExtraction and purification of Schisandra Chinensis Polysaccharide(SCP),to explore the effect of SCP by cyclosporine A(CsA),which cause acute liver and kidney injury in mice,and the influence of the liver and kidney cell proliferation.The action of the SCP and its primary mechanism were discussed.Methods1.Preparation and component analysis of SCPBy optimizing the traditional process,high purity SCP was prepared and its components were analyzed.Firstly,the fruit of schisandra chinensis was dried,crushed and sifted,and degreased with petroleum ether.Water extraction and alcohol precipitation was used to extract crude polysaccharide,and then deproteinize.After concentrate,remove the small molecular impurities by dialysis bag.Molecular weight of SCP was determined by Gel chromatography differential optical multi angle laser light scattering(GPC-RI-MALS).The monosaccharide components and contents were obtained by High performance anion-exchange chromatography with pulsed amperometric detection(HPAEC-PAD).The information of functional groups was obtained by Fourier transform near infrared spectroscopy(FT-IR).The chemical bonding structure of SCP was obtained by gas chromatography-mass spectrometry(GC-MS).2.Protective effect of SCP on acute liver and kidney injury in mice induced by CsACsA was gavaged for 15 days,and different concentrations SCP solutions were given at the same time.Detect the liver function indexes--alanine aminotransferase(GPT),aspartate aminotransferase(AST)and renal function indexes--urea nitrogen(BUN)and creatinine(CRE)from the serum of mice.Hematoxylin-eosin staining(HE)was used to observe liver and kidney damage in mice.Masson staining was used to observe the degree of hepatic and renal fibrosis in mice.3.Effect of SCP on the proliferation of liver and kidney cellsReal-Time Live-Cell Imaging System(Essen Bioscience,IncuCyte ZOOM)is used to determine the time and concentration of CsA and SCP,observe the effect of SCP on the cell proliferation-human hepatic stellate cells(LX-2)and renal tubular epithelial cells(HK-2).With the scratch experiment,EdU,immunofluorescence staining of Ki67 as well as the detection of cell cycle,further determine the effect of SCP on the proliferation of LX-2 and HK-2 induced by CsA.4.Study on the primary mechanism of SCP on the proliferation of liver and kidney cells under CsAThe protein chip technology was used to screen 40 growth factors related to proliferation,and which with differences were selected.The expression of related molecules in cells and serum of mice was verified by ELISA.The preliminary mechanism of SCP effect the proliferation of liver and kidney cells under CsA was discussed.Results1.The content of SCP was as follows:(65.51±2.39)%polysaccharide,(22.48±4.40)%uronic acid and(15.00±0.86)%protein.The mean molecular weight(Mn)of SCP measured by GPC-RI-MALS was 1.164×104(±4.018%)g/mol,the weight average molecular weight(Mw)was 2.916×104(±1.494%)g/mol.HPAEC-PAD measured the monosaccharide composition of SCP:glucose(Glc),mannose(Man),galactose(Gal),arabinose(Ara),xylose(Xyl),fucose(Fuc),galacturonic acid(Gal-AC)and glucuronic acid(Glc-AC),the percentage(%)respectively were 51.61,29.93,8.26,3.58,1.06,0.04,4.64,0.88.With the results of FT-IR and GC-MS,it can be inferred that SCP is mainly dextran heteropolysaccharide containing galacturonic acid.2.The reduction levels of AST,GPT,BUN and CRE in serum of mice were detected.The liver and kidney tissue pathology revealed that SCP can improve the damage of CsA to liver and kidney tissue and fibrosis degree.3.Within a certain time,Real-Time Live-Cell Imaging System showed that 250 μg/mL SCP had protective effects on cell proliferation about LX-2 and HK-2 under 15 μM CsA.Cell scratch and EdU test suggested the protective effect of SCP.Immunofluorescence of Ki67 also showed that SCP promoted the proliferation of liver and kidney cells.And SCP also affects the cell cycle.4.Protein chip technology was used to screen relevant differential growth factors,and BDNF,PDGF-AA and VEGF in the cell supernatant were selected with significant differences.Verified by ELISA in cells and serum of mice,suggested that BDNF is differentially expressed during SCP-regulated CsA liver and kidney cell proliferation.Conclusion1.SCP is a glucan heteropolysaccharide containing galacturonic acid.2.SCP can improve the damage and fibrosis degree of liver and kidney tissues caused by CsA.It can protect and repair the cell proliferation of LX-2 and HK-2 under CsA.3.BDNF is differentially expressed during SCP-regulated CsA liver and kidney cell proliferation.