The Study On The Mechanism Of Up-Regulation Of Respiratory Immunity By Miaoyao Fanggan Sachet Through TLR-MyD88-NFκB Pathway

Objective: MyD88 knockout mice and human normal lung epithelial cells(BEAS-2B)were used as models to elucidate the mechanism of up-regulation of respiratory immunity by Miaoyao Fanggan Sachet(MFS)through TLR-MyD88-NFκB pathway.Methods: 1.In vivo study: 16 male C57BL/6J mice,SPF grade,were randomly divided into 2 groups,8 mice in each group: blank control group(control group),sachet group(MFS group).16 MyD88 gene knock mice(purchased from Experimental Animal Center of Nanjing University),male,SPF grade,were randomly divided into 2 groups,8 in each group: MyD88 gene knockout group(MyD88-KO group),MyD88 gene knockout + sachet Group(MyD88-KO+MFS group).The mice in the sachet intervention group were continuously inhaled for 30 days(8:00 am-8:00 pm for 12 hours,inhaled sachets,and sachets were replaced once every 6 days).The mice were sacrificed 30 days later.Detection of routine biochemical indicators such as blood routine,liver and kidney function.The histopathological changes of lung tissue were observed by hematoxylin-eosin staining.The expressions of TAK1,NFκB p65,IκB and IL6 in lung tissue of mice were detected by immunohistochemistry,immunofluorescence,Western Blot and qRT-PCR.2.In vitro study: The extract of Miao Fanggan Sachet is provided by the pre-test of the Miao medicine anti-allergic sachet.The BEAS-2B cells are purchased from the Kunming cell bank of the Chinese Academy of Sciences.The CCK8 method was used to explore the optimum concentration and time of the herbicide-sucking sachet extract on BEAS-2B cells.The experiment was divided into 6 groups: blank control group(Control group),MyD88 siRNA group(MyD88 SI group),MyD88 siRNA-negative control group(MyD88 SI-NC group),sachet stimulation group(Sachet group),MyD88 siRNA group + sachet stimulation group(MyD88 SI+ Sachet group),MyD88 siRNA-negative control + Sachet stimulation group(MyD88 SI-NC+ Sachet group).Western Blot and qRT-PCR were used to detect the expression of MyD88,TAK1,NFκB p65,IκB and IL6 in each group of BEAS-2B cells.Results: 1.In vivo study:(1)Pathological results :In blank control group,MFS group,MyD88-KO group and MyD88-KO + MFS group the lung tissue of mice had complete contour,clear structure of organs,bronchi and capillaries,and no obvious exudation of alveolar wall.In MFS group,a small amount of monocytes and neutrophils infiltrated in alveolar cavity of some samples of mice.Mikawa lung injury score was counted.Compared with blank control group and MFS group,there was no statistical difference.(2)Blood routine: Leukocyte level of mice in MFS group increased(P < 0.05).Biochemical indicators: There was no significant difference in liver and kidney function of mice in each group(P > 0.05).(3)Gene and protein expression of TAK1,NFkB p65,IkB and IL6: Those were showed that compared with the blank control group,the gene and protein expression levels of the above-mentioned molecules in lung tissue of mice in MFS group increased(P < 0.05),while those in MyD88-KO group and MyD88-KO+MFS group decreased(P < 0.05)by Immunohistochemical,RT-PCR and Western Blot results.Compared with MyD88-KO group,the expression level of lung tissue in MyD88-KO + MFS group increased slightly(P < 0.05).2.In vitro study:(1)When the concentration was 0.003125g/ml,MFS extract had no affect on the cell viability of BEAS-2B cells,and the expression levels of TAK1,NFκB p65,IκB and MyD88 mRNA were the highest(P<0.05).(2)Compared with the blank control group,the expression of MyD88,TAK1,NFκB p65,IκB and IL6 gene and protein were significantly decreased in MyD88 SI group and MyD88 SI +MFS group(P<0.05),but significantly increased in Sachet group and MyD88 SI-NC + Sachet group(P<0.05).Compared with MyD88 SI group,the expression of MyD88,TAK1,NFκB p65,IκB and IL6 gene and protein in MyD88 SI + Sachet group slightly increased(P<0.05).Conclusion: MFS can improve respiratory immunity,and MyD88 may be a key factor.Its mechanism may be related to enhance MyD88 expression,regulation of TLRMyD88-NFκB signaling pathway and stimulate the release of downstream cytokines to play an immunomodulatory role by MFS.In addition to blocking the MyD88 signaling pathway,the MFS may also stimulate the expression of downstream cytokines through other alternative pathways,and play a role in regulating respiratory immunity.