Protective Effect And Mechanism Of Hippocampal Protein On Inflammatory Injury Of RAW264.7 Macrophages

The hippocampus is a bony fish known for its special medicinal ingredients.Hippocampus is one of the most famous Chinese medicine materials in China.It has been studied for its biological activities such as anti-inflammatory,anti-oxidation,anti-aging and anti-fatigue.In the invasion of external substances,such as bacteria and viruses entering the body,inflammation is a protective mechanism.However,prolongation or withdrawal of the inflammatory response may result in a variety of diseases.Such as inflammatory bowel disease,arthritis,asthma,Alzheimer’s disease,Parkinson’s disease and diabetes.This study aimed to discuss the anti-inflammatory effects of hippocampal protein extracts.The total protein was extracted by water extraction method,and the optimal extraction conditions were optimized by orthogonal test.The active protein fraction was separated by dialysis,ultrafiltration and SDS polyacrylamide gel electrophoresis.The isolated protein fractions were tested for anti-inflammatory activity by MTT assay,Griess assay and in vitro anti-inflammatory activity model.The effects of hippocampus active protein on inflammatory factors secretion were detected by ELISA and real-time fluorescent quantitative PCR.In this experiment,the total protein was extracted by water extraction method.The extraction concentration of hippocampus protein was optimized by orthogonal test.The optimal extraction conditions were extraction time 24h,salt concentration 1moL,and the ratio of material to liquid ratio was 1:20.The protein concentration was 117p,g/mL.Dialysis demineralization,SDS-PAGE electrophoresis,ultrafiltration method were used to separate protein components of less than 10KD fraction,10KD～30KD fraction and molecular weight greater than 30KD.Anti-inflammatory activity tests were performed on each ultrafiltration component of the isolated hippocampal total protein:MTT assay was used to test the RAW264.7 macrophage cytotoxicity of hippocampal ultrafiltration fraction protein.The experimental results showed that:At the same concentration,the hippocampal protein fraction of less than 10KD had a weak effect on cytotoxic activity.The 10-30KD hippocampal protein component had no effect on cytotoxic activity,and the hippocampal fraction greater than 30KD had a greater effect on cells.The effect of each ultrafiltration component protein on the NO content of mouse RAW264.7 macrophages was studied by Griess method.The experimental results showed that:The activity components of hippocampus in less than 10KD and 10～30KD inhibited the NO content of RAW264.7 macrophages,which proved anti-inflammatory effect.The 10-30KD hippocampal active protein inhibited the RAW264.7 macrophage content.The hippocampal protein fraction greater than 30KD did not significantly inhibit the secretion of NO by RAW264.7 macrophages.Detection of inflammatory factors at mRNA and protein levels was detected by ELISA and qRT-PCR,respectively.Experimental results show that:Compared with the lipopolysaccharide group,the 10-30KD hippocampal active protein(12.5,25,50μg/mL)significantly down-regulated the expression levels of TNF-α,IL-6 and IL-1β induced by LPS.The 10-30KD hippocampus active protein component significantly inhibited the expression of nitric oxide(NO),tumor necrosis factor(TNF-α)and interleukin-1β(IL-1β)protein induced by lipopolysaccharide.The results show that hippocampal active protein with a molecular weight of 10-30KD has anti-inflammatory protective effect.The research in this thesis expands the research on the medicinal ingredients of the traditional Chinese medicine hippocampus,and provides preliminary guidance for the protection and mechanism research of hippocampus in the treatment of RAW264.7 macrophage inflammatory injury.