| Objective:Sepsis,a global healthcare problem,with high clinical incidence,mortality and poor prognosis,is a serious challenge for critically ill epidemiology.Sepsis seriously affects the patients’ quality of survival,as well as brings heavy economic burden.As a lethal toxicity factor,High mobility group protein B1(HMGB1)can be secreted into serum in late phase of endotoxin shock,which could aggravate the inflammatory cascade reaction.Therefore,to control the release of HMGB1 may be the key to control the spread of inflammatory cascade reaction.HMGB1 can be used as an important target for the prevention and control of sepsis.Shenfu decoction is a typical representative prescription of Fu-Yang theory in Chinese Medicine.Shenfu Injection,an injection from Shenfu decoction,is used treat with endotoxin shock.However,the research reports of the influences of SF to HMGB1 are less at present.In this study,we explored the effects of SF to HMGB1 through Tet-on-HMGB1 gene transferred RAW264.7 cell induced by Lipopolyscaachride(LPS),in order to explore the molecular mechanism of SF in the treatment of sepsis and the theory of traditional Chinese medicine.Methods1.We builded a luciferase(Luc)reporter gene system drived by HMGB1 gene promoter,and transferred into 293T cells to research the influence of SF injection to the activity of HMGB1 promoter.2.Through western blot to determine the amount of HMGB1 protein as well as the quantitative expression of the cytoplasm and nucleus,according to the above group extract groups of proteins of cultured cells and cytoplasmic protein and nucleoprotein,western blot test total protein and HMGB1 proteins in the cytoplasm and nucleus.3.Using ELISA method to detect cell supernatant of HMGB1 secretion,according to the above group,collecting supernatant on cells,mice HMGB1 ELISA kit operating methods HMGB1 amount of secretion expression in cell supernatant.4.The qRT-PCR method to detect SF of HMGB1 mRNA expression,the influence of the amount of to HMGB1 expression and HMGB1 mutant of RAW264.7 cell line as the research object,the experiment is divided into normal group,LPS group,sodium butyrate(SB)group,SF8,12,14 ul/mL,a total of six groups,above each delivery before joining tetracycline(Tet)start the exogenous gene expression,don’ t do any deal with normal group,LPS group and drug use 0.2 mu g/mL LPS stimulation 0.5 h,grouping 24 h after dosing to extract mRNA by PCR method for testing,analysis of SF of HMGB1 mRNA expression.Results1.HMGB1 promoter luciferase reporter gene detection results show that the LPS cell HMGB1 promoter activity of model group was obviously enhanced,and with injection HMGB1 promoter activity in each dose group of cells decreases,prompt and attached fluid can inhibit HMGB1 promoter activity.2.Western blot,according to the results of HMGB1 overexpression of cell lines of HMGB1 LPS model group cells significantly increased the total amount of protein expression,and attached the HMGB1 total protein concentration group compared with model group significantly decreased;HMGBI mutant cell lines between groups was no significant difference.Prompt and liquid could inhibit HMGB1 protein expression.3.The ELISA test results show that HMGB1 overexpression of cell lines of HMGB1 in the cell culture supernatant content in the model group increased significantly,and attached to the liquid concentration groups each culture supernatant of HMGB1 content compared with model group content decreased obviously;HMGB1 mutant cell lines between groups was no significant difference.Prompt and attached to the liquid may inhibit HMGB1 secrete cell.4.The PCR results showed that HMGB1 overexpression of cell lines in the model group HMGB1 mRNA expression significantly increased the amount,and attached injection every dose group of HMGB1 mRNA expression decreased obviously;HMGB1 mutant cell lines between groups was no significant difference.Tip with fluid can prevent the transcription of HMGB1 refs.Conclusion1.SF may inhibit HMGB1 promoter activity of wild-type cells.2.SF may reduce the expression of LPS-induced HMGB1 overexpression cell line HMGB1 mRNA,the expression of total protein,the secretion of HMGB1 content in the cell culture supernatant,and SF may inhibit HMGB1 protein from the nucleus to the cytoplasmic transfer,inhibit HMGB1 secrete from cell. |
