Effects Of Chronic Intermittent Hypoxia On P38 MAPK / NF-κB Signaling Pathway In Hypertensive Vascular Endothelium And The Intervention Mechanism Of Bushenqinggan Prescription

Obstructive sleep apnea syndrome (OSAS), characterized by chronic intermittent hypoxia (CIH), is an independent risk factor for hypertension. The molecular response to intermittent hypoxia (IH) is not well defined. Reoxygenation after a brief period of hypoxia as experienced repetitively and systemically by OSAS patients may predispose to oxidative stress. We have hypothesized that such events favor the activation of a proinflammatory response as mediated through the P38 mitogen-activated protein kinases (P38 MAPK) and transcription factor nuclear factor-κB (NF-κB), both master regulator of inflammatory gene expression. The downstream effects of this activation include increased expression of proatherogenic factors such as tumor necrosis factor-α (TNFα), which may contribute to endothelial dysfunction and subsequently cardiovascular complications. We suggest that the selective activation of such an inflammatory pathway by IH may be an important underlying factor in the hypertension pathophysiology of OSAS. Chinese medicine Bushen Qinggan Formula (BSQG:Gouteng, Duzhong, Chuanxiong; 2:2:1) is clinically proved to have endothelial protective effection. In order to translate our findings of the clinical setting into the mechanism research, we developed in vivo and vitro models of IH that involved exposing cells and mice to increasing numbers of cycles of hypoxia/reoxygenation and investigating the mechanism profiles of BSQG Formula.This study is divided into two parts:literature review and experimental research.1. Literature reviews:Including the following two reviews:Tonifying Shen, clearing Gan, promoting blood circulation therapies and hypertensive target organ protection; chronic intermittent hypoxia and hypertension:a review of systemic inflammation and Chinese Medicine.2. Experimental research:Including the following two parts: Study 1:Establishment of intermittent hypoxia model:Including the following two studiesPart 1:Establishment of an intermittent hypoxic cell model Objective:To establish a human umbilical vein endothelial cells (HUVECs) model of IH Methods:HUVECs were treated with cycles of hypoxia/reoxygenation. And they were divided into 11 groups:normal control group (CON group), intermittent hypoxia 2,4,6,8,10,12,14,16 cycles groups (IH groups), IH+P38 MAPK inhibitor group (INH group) and persistent hypoxia group (SH group). After IH stimulation, tested cell adhesion function. Cell adhesion molecules (ICAM-1), E-selectin (E-selectin), interleukin-1 (IL-1), interleukin 6 (IL-6) were detected by inter-quantitative PCR (QPCR). Immunofluorescence and Western-blot were used to measure expression of P38 MAPK/NF-κB pathway essential protein.Results:After IH, as one of the MAPK family proteins, p-P38 MAPK specific increased and was positively correlated to hypoxia/reoxygenation cycles. Compared with CON group, HUVECs adhesion capacity of IH group enhanced (P<0.05), INH group and SH group had no significant change (P>0.05). Inflammatory factor IL-1, IL-6, TNF-a in IH group was significantly higher than CON group (P<0.05). NF-κB pathway target protein p-IκB, NF-κB P65 nuclear translocation in IH group were higher than CON group, the expression of IKK-a, IKK-β, IκB, p-P65 did not change (P>0.05). Meanwhile, significant nuclear translocation of NF-κB P65 in IH group was observed.Conclusion:In vitro environment, IH caused endothelium inflammation damage through P38 MAPK/NF-κB signaling pathway.Part 2:Establishment of a chronic intermittent hypoxic mouse modelObjective:To establish a CIH mouse model, observe the blood pressure and vascular endothelial function in mice.Methods:Mice were treated with cycles of hypoxia/reoxygenation. And they were divided into 3 groups:normal control group (CON group), model group (MOD group), CIH+ P38 MAPK inhibitor group (INH group). Inflammatory cytokines were measured by QPCR. Detect the expression of P38 MAPK/NF-κB pathway essential protein in Western-blot.Results:Compared with CON group, systolic blood pressure (SBP) of MOD group is higher (P<0.05). Compared with MOD group, SBP of INH group is higher (P<0.05). Compared with CON group, inflammatory factors IL-1, IL-6, TNF-a in MOD group were significantly increased (P<0.05); p-P38 MAPK, p-IκB in aortic endothelial cells significantly increased compared with CON group, IKK-α, IKK-β, IκB, p-P65 did not change (P>0.05).Conclusion:In vivo environment, CIH caused endothelium inflammation damage through P38 MAPK/NF-κB signaling pathway, resulting in high blood pressure with mice. Study 2:Intervention mechanism of BSQG Formula in CIH-induced hypertensionPart 1:The inhibitory effect of three effective components of BSQG Formula through the P38 MAPK/NF-κB signaling pathway in IH model of HUVECs in vitroObjective:To explore the mechanism by which BSQG Formula inhibiting inflammation induced by IH through P38 MAPK/NF-κB signaling pathway in vitro.Methods:On the basement of IH model of HUVECs, three effective components of BSQG Formula (GDC:Isorhynchophylline, Aucubin, Ligustrazine) in four various concentrations (1μg/ml,0.1μg/ml, 0.001μg/ml, 0.001μg/ml) were added. After determining the optimal drug concentration, HUVECs were divided into 4 groups:normal control group (CON group), intermittent hypoxia group (IH), IH+GDC (GDC group) and IH+P38 MAPK inhibitor group (INH group). Cell adhesion function was tested. ICAM-1, E-selectin, IL-1, IL-6, TNF-a were detected by QPCR. Immunofluorescence and Western-blot were used to measure expression of P38 MAPK/NF-κB pathway essential protein.Results:Compared with IH group, GDC treatment (0.0 1μg/ml) significantly reduced the inflammatory factor IL-1, IL-6, TNF-a and the number of adherent cells (P<0.05), other concentration of GDC did not change(P>0.05). NF-κB pathway target protein p-IκB, NF-κB P65 nuclear translocation in GDC group were decreased (P<0.05) compared with IH group. Meanwhile visible GDC group nuclear translocation of NF-κB P65 is inhibited.Conclusion:GDC 0.01μg/ml attenuated inflammatory injury induced by IH through inhibiting P38 MAPK/NF-κB signaling pathway.Part 2:Effect of BSQG Formula through P38 MAPK/NF-κB signaling pathway in the CIH model of mouse/n vivoObjective:To explore the mechanism by which BSQG Formula inhibiting inflammation induced by CIH through P38 MAPK/NF-κB signaling pathway in vivo.Methods:On the basement of CIH model of mouse, C57/6J mice were randomly divided into 7 groups:normal control group (CON group), MOD group (model group), INH group (P38 MAPK inhibitor group), AB group (amlodipine group) and BSQG-H group (BSQG Formula high dose group), BSQG-M group (BSQG Formula middle dose group), BSQG-L group (BSQG Formula low dose group). Inflammatory cytokines were measured by QPCR. Detect the expression of P38 MAPK/NF-κB pathway essential protein in Western-blot.Results:Compared with MOD group, BSQG-H group, AB group, INH group significantly decreased SBP (P<0.05), AB group is lower than BSQG-H group (P<0.05). Compared with MOD group, BSQG-H treatment decreased IL-1, IL-6, TNF-a expression (P <0.01), BSQG-H group has advantages over the low-dose group, but has no significant difference (P> 0.05). Meanwhile, anti-inflammatory effect of BSQG-H is better than positive control (AB group) (P<0.05). Compared with MOD group, p-P38 MAPK, p-IκB were decreased in BSQG-H group.Conclusion:BSQG Formula can attenuated inflammatory injury induced by CIH through inhibiting P38 MAPK/NF-κB signaling pathway, reduced the blood pressure in mice CIH model, and the ability of anti-inflammatory is better than amlodipine.