Effect Of Ginkgo Biloba Polysaccharides On Promoting Growth And Study On Its Anti-inflammatory Mechanism

Objective:To explore the separation and purification technology of ginkgo biloba polysaccharides,analyze the hair growth promoting effect of Ginkgo biloba L.Polysaccharides（GBP）on alopecia areata mice,identify the bioactive polysaccharides in ginkgo biloba leaves,and clarify its anti-inflammatory mechanism,which lays a theoretical and practical foundation for the development of ginkgo biloba products.Methods:Ginkgo biloba polysaccharides（WGBP）were extracted by water decocting method.The acidic polysaccharides of ginkgo biloba leaves and their homogeneous fractions were further isolated and purified by DEAE cellulose ion exchange chromatography and CL-6B agarose gel chromatography.Their physicochemical properties were determined and their structures were analyzed by monosaccharide composition and nuclear magnetic resonance;50male C57BL/6 mice were selected and the alopecia areata model was established by sodium sulfide（Na₂S）depilation.The mice were randomly divided into normal control group（CON）,model group（MOD）（6%Na₂S+distilled water）,blood nourishing hair capsule group（PC）（6%Na₂S+300mg/kg/d）and ginkgo biloba polysaccharide group（6%Na₂S+600mg/kg/d）.After continuous intragastric administration for 30 days,the mice were killed.Ten new hairs were randomly selected from the newborn hair area of mice,and the hair length and weight were measured.The contents of VEGF and HGF in skin tissue of mice were determined by ELISA,and the contents of TNF-α,IL-1βand IL-12 in serum were determined,and the histological changes of skin in newborn hair area were observed by HE staining;Differential expression of HUVEC gene in acid polysaccharides of ginkgo biloba leaves（WGBP-A2b）analyzed by transcriptome sequencing（RNA-seq）;Ten differential genes were randomly selected for qRT-PCR verification;The inflammatory model of HUVEC cells induced by LPS was established,and the anti-inflammatory effects of WGBP-A2b were further evaluated.They were divided into five groups:Con,Mod（1μg/ml LPS）,ginkgo biloba acidic polysaccharides low dose group（WGBP-A2b-L）（1μg/ml LPS+50μg/m L）,ginkgo biloba acidic polysaccharides medium dose group（WGBP-A2b-M）（1μg/ml LPS+100μg/m L）,high dose ginkgo biloba acidic polysaccharides（WGBP-A2b-H）（1μg/ml LPS+200μg/m L）.The proliferative ability of HUVEC was detected by tetrazolium（MTT）assay.The protein expression levels of p-p65,TNF-α,IL-1βin HUVEC cells were detected by,Western blot.Result:The WGBP-A2 component was obtained,and the WGBP-A2 was further separated into three homogeneous polysaccharides with molecular weight of 44 KDa,WGBP-A2b,with molecular weight of 13 KDa,WGBP-A2c,with molecular weight of 15 KDa and WGBP-A2d with molecular weight of 15 KDa.It mainly contains HG type pectin and a small amount of RG-I type pectin;Animal experiment results show that compared with the CON group,the hair growth of the MOD group is slower.Compared with the MOD group,the VEGF in the skin tissue of the WGBP-A2 group is P<0.05 and HGF content significantly increased,and the serum TNF-α（P<0.05）,IL-1β（P<0.05）and IL-12 content of WGBP-A2 group mice were significantly reduced.The results of HE staining showed that the number of hair follicles in the newborn hair area of the MOD group decreased,and the number of hair follicles in the newborn hair area of the WGBP-A2 group increased;RNA-seq results showed that the WGBP-A2b group had different expression genes compared with the blank control group.There are 317genes,including 214 up-regulated genes and 103 down-regulated genes.Bioinformatics analysis results show that this study has enriched 36 related signal pathways,among which the significant changes are the TNF signaling pathway,the interaction of cytokines and cytokine receptors,the IL-17 signaling pathway,and the NF-κB signaling pathway.And other inflammatory pathways.The qRT-PCR verification results showed that compared with the blank control group,the WGBP-A2b group has HLA-B,PLA2G4C,ICAM1（P<0.01）,CCL2（P<0.01）,CXCL1（P<0.01）,IL32,CXCL8（P<0.05）and MMP1（P<0.05）expression increased,SELP and CCL14（P<0.05）expression decreased.The up-regulation and down-regulation trends of qRT-PCR and RNA-Seq results are the same;cell experiment results show that the expression of p-p65,p-IκBα,TNF-α,and IL-1βprotein in the Mod group increased（P<0.01）,and the MOD group In comparison,the expressions of p-p65,p-IκBα,TNF-α,and IL-1βprotein in each group of WGBP-A2b cells were all down-regulated（P<0.01）.Conclusion:WGBP-A2 can promote the hair growth of alopecia areata mice and reduce the inflammation around the hair follicles,which may be achieved by regulating the expression of inflammatory factors.WGBP-A2b can improve the inflammatory response of HUVEC cells induced by LPS,the specific mechanism is by regulating the expression of p-p65、p-IκBα、TNF-α、IL-1β proteins in the inflammatory signal pathway.