| Objective:(1)To explore the TCM pathogenesis of coronary microvascular dysfunction(CMD)based on the theory of qi and blood,and treatment effcts of Chinese medicine for invigorating qi and activating blood on CMD.(2)To observe the intervention effect of Tianxiangdan(TXD)on rat model of CMD and injured coronary microvascular endothelial cell model.(3)To explore the mechanism of TXD on microvascular inflammation and endothelial function through the NRF2/ARE pathway,so as to explore the new mechanism of TXD in the treatment of CMD.Methods:(1)(1)Systematically search the classical ancient books of traditional Chinese medicine and the related books and literatures of qi and blood theory,and explain the connotation of qi and blood theory.Under the guidance of the theory of qi and blood,discussed the etiology and pathogenesis of CMD,the prevention and treatment effect of Chinese medicine for invigorating qi and activating blood on CMD.(2)Literatures related to clinical randomized controlled trials on the treatment of CMD with qi-invigorating and blood-activating drugs combined with western medicine from January 2010 to October2020 were retrieved from Chinese and English databases,Meta-analysis was used to evaluate the efficacy of drugs for replenishing qi and activating blood circulation on CMD.(2)(1)CMD models were established by injecting sodium Laurate into the left ventricle of SD rats.The rats were divided into sham operation group(Sham),CMD model group(CMD),TXD low-dose group(TXD-L),TXD middle-dose group(TXD-M),TXD high-dose group(TXD-H)and nicorandil group(NCR),Low,medium and high doses of Tianxiangdan and Nicodil were given for intervention.(2)Evaluated the cardiac function,and observed the changes of myocardial microvascular structures.(3)Detected the serum levels of nitric oxide(NO),endothelin-1(ET-1)and e NOS protein expressions in myocardial tissue to evaluate the effect of Tianxiangdan on endothelial function of CMD rats.(4)Human coronary microvascular endothelial cells(HCMECs)induced by lipopolysaccharide(LPS)to establish injured HCMECs model,and HCMECs cells were divided into 4 groups: control group(Control),LPS group(LPS),TXD group(TXD),LPS and TXD co-treatment group(TXD+LPS).(5)Cell viability,e NOS protein expression level in cells,NO and LDH levels in supernatant and permeability of monolayer microvascular endothelial cells were detected,to evaluate the protective effect of TXD on HCMECs.(6)Detected serum levels of inflammatory factors,and protein expression levels of TNF-α,NF-κ B and Nrf2 in myocardial tissue,to evaluate the ameliorative effect of TXD on inflammatory injury in CMD rats.(7)Inflammatory factors in cell supernatant and ROS,Nrf2 and NF-κB expressions in cells were detected,to evaluate the protective effect of Tianxiangtan on inflammation injury of HCMECs.(3)(1)Detect the m RNA and protein expression of Nrf2/ARE pathway and IKKβ/IκBα/ NF-κB pathway in CMD rats.(2)HCMECs were pretreated with ML385(an inhibitor of Nrf2),and then co-treated with TXD and LPS.Detected the expressions of m RNA and protein of Nrf2/ARE pathway and IκKβ/ IκBα/ NF-κB pathway in HCMECs in order to clarify the mechanism of TXD on HCEMCs inflammation.(3)Detected the expression of e NOS protein and the cell viability of HCMECs to explore the protective mechanism of TXD on CMD.Results:(1)Theoretical exploration of pathogenesis and prevention of CMD:(1)Qi deficiency and blood stasis are the main pathogenesis of CMD,deficiency of Qi in the heart and obstruction of Qi and blood in the heart collaterals are the key factors leading to "Qi and blood disorders",CMD should be treated with invigorating qi and activating blood circulation.Pharmacological studies have shown that Qi-enhancing and blood-activating drugs have the function of "enriching qi",such as improving endothelial function,regulating energy metabolism and promoting microangiogenesis,and the functions of "promoting blood circulation",such as inhibiting inflammation,cell apoptosis and microthrombosis.(2)The Meta-analysis results showed that compared with the treatment of coronary microcirculation disorders with western medicine alone,the combined treatment with drugs for invigorating qi and activating blood circulation could better improve the clinical efficacy,improve ECG performance,and TCM syndromes,reduces levels of c-reactive protein and ET 1 level.(2)Effects of TXD on CMD:(1)Effect of TXD on cardiac function and myocardial microvascular structure in CMD rats: Compared with CMD group,the LVEF value of TXD-M and TXD-H groups were increased(P < 0.05),and the heart function was enhanced.Compared with CMD group,TXD-M and TXD-H groups could reduce microvascular thrombosis,improve the edema of vascular wall,and reduce the infiltration of perivascular inflammatory cells,can also improve the swelling of endoplasmic reticulum and fracture of basement membrane of microvascular endothelial cells,and protect the structure of myocardial fiber and mitochondria of myocardial cells;compared with CMD group,the microvessel density in the medium-dose and high-dose groups was significantly increased(P < 0.05).(2)Effect of TXD on endothelial function in CMD rats: compared with the CMD group,the serum level of NO and NO/ET-1 ratio in TXD-M and TXD-H groups increased significantly(P < 0.05).Compared with the CMD group,the e NOS protein level of myocardium tissue in TXD-M and TXD-H groups were increased(P < 0.05),and there was no statistical difference compared with the NCR group.(3)Protective effect of TXD on LPS-induced HCMECs: Compared with LPS group,the survival rate of HCMECs in LPS+TXD group was significantly increased(P < 0.01).Compared with LPS group,the expression level of e NOS protein and NO in supernatant in LPS+TXD group were increased,and the release level of LDH was decreased(P < 0.05).(4)Effect of TXD on Coronary Microvascular Inflammation: The levels of serum IL-1β and TNF-α in CMD rats were significantly lower than those in CMD group(P <0.05),and the level of IL-6 in high dose group was also lower than that in CMD group(P<0.05).The expression of TNF-a,NF-κB and Nrf2 protein in myocardium was detected by IHC method.The results showed that the expression of TNF-an and NF-κB p65 in TXD-M and TXD-H groups group was significantly lower than that in CMD group(P<0.05),while the expression of Nrf2 was significantly higher than that in TXD group(P<0.05).At the cellular level,compared with LPS alone,the expression of IL-1β,TNF-an in the supernatant and ROS in cells co-treated with TXD and LPS decreased significantly(P<0.05),and the fluorescence intensity of Nrf2 increased significantly,while the fluorescence intensity of NF-κ B decreased(P<0.05).(3)The improvement mechanism of TXD on coronary microvascular inflamma-tion and endothelial function:(1)Compared with CMD group,TXD could increase the m RNA and protein expression levels of Nrf2,HO-1 and IκBα(P < 0.05);decreased the m RNA level of NF-κB p65,the protein levels of IKKβ,IL-1β,TNF-α and phosphorylated protein level of p65(P < 0.05).(2)At the cellular level,before the application of Nrf2 inhibitor,compared with the LPS group,the m RNA expression levels of Nrf2,HO-1 and IκBα and the protein expression levels of Nrf2 and HO-1 were increased in the LPS+TXD group(P < 0.05),while the m RNA expression levels of NF-κB p65 and its phosphorylated protein were decreased(P < 0.05);After using the Nrf2 inhibitor,the promotion of IκBα expression and the inhibition of NF-κB p65 phosphorylation by TXD were partially cancelled.The inhibitory effect of TXD on IL-1β and TNF-α was weakened.the promotion effect of TXD on e NOS expression was not significantly reduced,but the improvement of the survival rate of HCMECs was somewhat reduced.Conclusion:(1)Qi deficiency and blood stasis are the main pathogenesis of CMD,deficiency of Qi in the heart and obstruction of Qi and blood in the heart collaterals are the key factors leading to "Qi and blood disorders",CMD should be treated with invigorating qi and activating blood circulation.The drugs for invigorating qi and activating blood circulation have good effect of improving CMD.(2)TXD can improve cardiac function,myocardium microvascular structure and endothelial function in rats with coronary microcirculation disturbance,can protect microvascular endothelial cells,alleviate the inflammatory damage of HCMECs and CMD rats.(3)TXD can mediate the Nrf2/ARE pathway to regulate the IκBα/NF-κB pathway,thereby inhibiting the inflammation and injury of coronary microvessels,protect microvascular endothelial cells,and improve CMD. |
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