HJ11 Improves Endothelial Dysfunction In ASO By Inhibiting TLR4/MyD88/NF-κB Pathway-mediated Inflammatory Response

Objective:To reveal the efficacy and molecular regulation mechanism of HJ11,which was created based on the theory of "heat retention in blood vessels",in improving the endothelial dysfunction of arteriosclerosis obliterans from the pathway of "TLR4/My D88/NF-κB signaling pathway mediates inflammatory response".The results will help to identify the targets and links of the formula and lay the foundation for the development of new drugs in Chinese medicine.Methods:1.In vivo and in vitro study of HJ11 to improve endothelial dysfunction in arteriosclerosis obliterans.For in vivo experiments,a rat model of arteriosclerosis obliterans was prepared by high-fat diet feeding with distilled water perfusion and intervened with HJ11.Hematoxylin-eosin staining and macroscopic oil red staining were used to observe the pathological changes in the femoral arteries of rats,and serum TC,LDL-C and HDL-C levels were detected by biochemical analyzer,and serum NO,ET-1,VCAM-1 and ICAM-1 levels were detected by ELISA.In vitro experiments were performed to construct an endothelial injury cell model by ox-LDL-induced HUVECs.The cell viability of HUVECs was detected by CCK-8method,and the appropriate concentrations of ox-LDL and HJ11 were screened.The migration and invasion ability of HUVECs cells were detected by cell scratching assay and Transwell assay.2.Transcriptomics-based techniques were used to explore the mechanism of endothelial dysfunction improvement by HJ11.Firstly,transcriptome sequencing technology was used to screen the differentially expressed genes of ox-LDL-induced HUVECs before and after intervention with HJ11.Then,functional enrichment analysis,including GO enrichment analysis and Pathway analysis,was performed to explore the mechanism of HJ11 to improve endothelial damage.3.In vitro and in vivo studies on the improvement of endothelial dysfunction by inhibiting inflammatory response with HJ11.In vivo experiments,the levels of IL-6,IL-1β,IL-8 inflammatory factors in serum of rats with arteriosclerosis obliterans were detected by ELISA,and the expression of TLR4,My D88,NF-κB protein in rat femoral artery was detected by immunohistochemistry.In vitro experiments,the levels of IL-6,IL-1β,IL-8 inflammatory factors in the supernatant of HUVECs were detected by ELISA,and the expression of TLR4,My D88,NF-κB protein in HUVECs were detected by Western Blot.4.A study of HJ11 to improve ox-LDL-induced endothelial dysfunction in HUVECs by inhibiting TLR4/My D88/NF-κB signaling pathway-mediated inflammatory response.First,the expression of TLR4 m RNA in HUVECs after transfection with three different sequences of TLR4 si RNA was detected by q RT-PCR using si RNA transfection technique,and the sequence with the lowest TLR4 m RNA expression level was selected for subsequent experiments.Then,the cell migration and invasion ability of HUVECs were detected by scratch assay and Transwell assay.The levels of IL-6,IL-1β and IL-8 inflammatory factors in the supernatant of HUVECs were detected by ELISA,and the expression of TLR4,My D88 and NF-κB proteins in HUVECs were detected by Western Blot.Results:1.The results of in vivo experiments showed that,compared with the model group,the endothelial pathological damage in the femoral artery of rats in each dose group of HJ11 was significantly improved,LDL-C(P<0.01)and ET-1(P<0.05,P<0.01)levels were significantly decreased,HDL-C levels were significantly increased(P<0.01),serum TC(P<0.01)and VCAM-1(P<0.05,P<0.01)levels were significantly decreased,ICAM-1 levels were significantly decreased in the high-dose group of HJ11(P<0.01),and NO levels were significantly increased in the high and low-dose groups of HJ11(P<0.05).The results of in vitro experiments showed that HJ11 could improve the cell viability(P<0.01),cell migration(P<0.01)and invasion(P<0.01)ability of ox-LDL-induced HUVECs.2.Transcriptome sequencing results showed that there were 481 differentially expressed genes of HUVECs induced by ox-LDL,248 with up-regulated expression and 233 with down-regulated expression.There were 499 differentially expresse d genes of HUVECs after treatment with HJ11,with 167 up-regulated and 332down-regulated expressions.The differentially expressed genes in the HJ11 group and the ox-LDL group were mainly involved in biological processes such as immune response,regulation of smooth muscle cell proliferation,negative regulation of apoptotic process,negative regulation of programmed cell death,hypoxic response,etc.They were mainly enriched in apoptotic signaling pathway,CCKR signaling pathway,angiogenesis,p53 pathway,Toll receptor signaling pathway,VEGF signaling pathway,Notch signaling pathway,TGF-β signaling pathway,etc.The core targets of HJ11 to improve endothelial dysfunction are TNF,FOS,HSP90AA1,CXCR4 and EGR1.3.The results of in vivo experiments showed that compared with the model group,the levels of serum IL-6 and IL-1β were significantly lower in the rats of each dose group of HJ11(P<0.01),the levels of IL-8 were significantly lower in the high and medium dose groups of HJ11(P<0.01),the expression of TLR4 and NF-κB protein in the femoral artery of rats was significantly reduced in the high-dose group of HJ11(P<0.05),and the expression of My D88 protein was significantly decreased in the high and medium-dose groups of HJ11(P<0.05).The results of in vivo experiments showed that HJ11 could effectively reduce the levels of IL-6,IL-1β and IL-8 in ox-LDL-induced HUVECs(P<0.01),as well as the expression of TLR4,My D88 and NF-κB proteins(P<0.05,P<0.01).4.si RNA transfection results showed that TLR4 m RNA expression was significantly lower in the si TLR4 group compared with the blank group(P<0.01).The cell migration and invasion ability of ox-LDL+si TLR4+ HJ11 group was significantly increased(P<0.01)and the expression of IL-6,TLR4,My D88 and NF-κB was significantly decreased(P<0.01)compared with the ox-LDL+ HJ11 group.Conclusion:1.HJ11 could reduce the pathological damage of femoral artery in arteriosclerosis obliterans rats,regulate the balance of vasodilation and contraction,and inhibit the release of adhesion molecules.At the same time,HJ11 could improve ox-LDL-induced cell viability,cell migration,and cell invasion of HUVECs.Th e results at both animal and cellular levels collectively suggest that HJ11 could improve endothelial dysfunction and protect endothelium.2.The transcriptome sequencing results revealed that HJ11 may improve endothelial dysfunction by regulating inflammation,immunity,and apoptosis,of which TLR4/My D88/NF-κB may be a key pathway.3.HJ11 could effectively reduce the expression of IL-6,IL-1β,IL-8 inflammatory factors and inhibit the expression of TLR4/My D88/NF-κB signaling pathway in rats with arteriosclerosis obliterans.Meanwhile,the same experimental results were obtained at the cellular level,and the results at the animal and cellular levels together indicated that HJ11 could inhibit the inflammatory response,and the mechanism might be related to the inhibition of TLR4/My D88/NF-κB pathway.4.After TLR4 gene silencing,HJ11 could continue to inhibit the expression o f inflammatory factors and TLR4/My D88/NF-κB signaling pathway in ox-LDL-induced HUVECs and promote cell migration and cell invasion in HUVECs,indicating that in addition to TLR4/My D88/NF-κB pathway,HJ11 also had a broader range of action targets as well as action.It could improve the endothelial dysfunction of arteriosclerosis obliterans with multi-pathway and multi-target therapy.