Study On The Antihypertensive Effect Of Qingda Granule From TLR4/MyD88/NF-κB Pathway In Rostral Ventrolateral Medulla

Objective:The purpose of this study was to investigate the effects of Qingda granule(QDG)on neuroinflammation in the rostral ventrolateral medulla of spontaneously hypertensive rats(SHR),and analyze its effect on the key inflammatory pathway TLR4/My D88/NF-κB.Methods:1.In vivo experiment : A total of 60 4-week-old SPF male WKY(Wistar-Kyoto)and SHR rats were randomly divided into 4 groups according to body weight : normal group(WKY,normal saline),model group(SHR,normal saline),Qingda granule low-dose group(SHR+QDG-L,0.9g/kg/d)and Qingda granule high-dose group(SHR+QDG-H,2.7g/kg/d),15 rats in each group,continuous intervention for 8 weeks,weekly monitoring of blood pressure and body weight,and blood collection at 0,4 and 8 weeks of the intervention cycle.Cardiac function was evaluated by echocardiography;the level of norepinephrine(NE)in serum of each group was detected by enzyme-linked immunosorbent assay(Elisa).The activation of microglia was detected by immunofluorescence(IF).Western blot was used to detect the expression of microglia marker IBA1,M1(pro-inflammatory)state marker CD86,M2(anti-inflammatory)state marker CD206,and TLR4,My D88,NF-κB p65,p-NF-κB p65 protein in TLR4/ My D88/NF-κB pathway.2.In vitro experiment : Neuron microglia(BV-2)cell line was used.Cell proliferation assay(CCK-8)was used to detect the optimal concentration of QDG,lipopolysaccharide(LPS)and TAK-242 in BV-2 cells.The activation of BV-2 cells and the nuclear translocation of NF-κB p65 subunit were detected by IF.The secretion levels of inflammatory factors TNF-α,IL-1βand IL-6 in cell supernatant were detected by Elisa.Western blot was used to detect the expression of IBA1,CD86,CD206 and TLR4,My D88,NF-κB p65,p-NF-κB p65 protein in TLR4/My D88/NF-κB pathway.Results:1.In vivo experiments :(1)Blood pressure monitoring results showed that QDG intervention could significantly inhibit the increase of systolic blood pressure(SBP),diastolic blood pressure(DBP)and mean arterial pressure(MAP)in SHR(P<0.05);(2)The results of serum NE detection showed that the increase of serum NE level was significantly inhibited after 4weeks of QDG intervention(P<0.05).(3)Echocardiography results showed that QDG intervention could significantly improve the decrease of left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS)in SHR(P<0.05).(4)Immunofluorescence and Western blot quantitative results showed that compared with WKY group,microglia activation and M1 polarization were significantly increased in SHR group;QDG intervention significantly inhibited the activation of microglia and promoted M2polarization(P<0.05).(5)Western blot showed that QDG intervention could significantly reduce the expression of TLR4,My D88 and p-NF-κB p65 in the RVLM of SHR(P<0.05).2.In vitro experiments :(1)CCK8 results showed that the optimal intervention concentration of QDG was 5×10-3 mg/m L,the optimal intervention concentration of LPS was 10 μg/m L,and the optimal intervention concentration of TAK-242 was 0.1 μM.(2)Immunofluorescence results and protein quantification showed that BV-2 cell activation and M1 polarization were significantly increased under LPS stimulation.QDG and TAK intervention could significantly inhibit the increase of BV-2 and promote M2 polarization(P<0.05).At the same time,p65 nuclear translocation increased significantly under LPS stimulation(P<0.05),and p65 nuclear translocation decreased significantly after QDG and TAK-242 intervention(P<0.05).(3)Elisa results showed that compared with the Control group,the secretion levels of TNF-α,IL-1βand IL-6 in the cell supernatant stimulated by LPS were significantly increased(P<0.05);TAK-242 intervention can significantly reduce the secretion of IL-1β and IL-6;QDG intervention could significantly reduce the secretion of TNF-α,IL-1β and IL-6(P<0.05).(4)Western blot results showed that compared with the Control group,the expression of TLR4,My D88 and p-NF-κB p65 in BV-2 cells was significantly increased under LPS stimulation(P<0.05).The expressions of TLR4,My D88 and p-NF-κB p65 were significantly down-regulated after TAK-242 and QDG intervention(P< 0.05).Conclusion:Qingda granules can attenuate the inflammatory response in the rostral ventrolateral medulla of spontaneously hypertensive rats,reduce the release of norepinephrine,and inhibit the increase of peripheral blood pressure.The mechanism may be related to its regulation of TLR4/My D88/NF-κB signaling pathway.