Study On The Intervention Of Skin Damage Caused By UVB By Regulating HOXA11-AS/EZH2 Axis Through HIF-1?

Skin photodamage caused by ultraviolet rays is a common and frequently seen skin disease in clinics.The application of natural medicine components as photoprotective agents and the exploration of its mechanism of action has been the main research directions in recent years at home and abroad.Haoqin-Huaban Decoction was proposed by Zhao Bingnan,a well-known Chinese medicine veteran doctor of dermatology surgery based on the theory of "phototoxicity and depression".It adopts"clearing heat,detoxification,and cooling blood" as a method to treat light-sensitive skin diseases and has achieved good results in clinical practice.In the preliminary basic experiments,it was found that Haoqin-Huaban Decoction can protect the skin from photodamage caused by ultraviolet radiation by regulating the immune-inflammatory response through antioxidants,but its specific internal molecular mechanism is still unclear.Long non-coding RNA was once considered to be the transcription noise in the genome,but now more and more studies have shown that the expression difference of long non-coding RNA is closely related to the occurrence of diseases.Therefore,based on network pharmacology,this study predicted the drug action pathway of Haoqin-Huaban Decoction through long non-coding RNA and explored its mechanism of action through experiments.Purpose:To predict and experimentally verify the molecular mechanism of HaoqinHuaban Decoction.Research methods:1.The prediction of the mechanism of Haoqin-Huaban Decoction in the treatment of UVBinduced skin damage based on network pharmacology:utilize databases including TCMSP and STITCH to predict the main active ingredients and function targets of artemisia annua,which is the substance that has the main therapeutic effect on the main disease or syndrome in HaoqinHuaban Decoction,use ChIPBase to predict the long-chain non-coding RNA interaction of the target,and use RNAInter database to predict the mode of action of the long-chain non-coding RNA.2.Evaluation of the effect of Haoqin-Huaban Decoction on the skin of mice with UVB injury:Divide mice into the blank control group,UVB radiation model group,and high,medium,and low dose groups of Haoqin-Huaban Decoction,feed medication,and make ultraviolet radiation intervention,observe the skin surface of the mouse back and HE stained sections,measure the changes in mouse epidermal thickness,detect the expression of Bcl-2 and Bax by immunohistochemistry,apply TUNEL staining to detect skin cell activity,measure the expression levels of ROS,MDA,SOD,GSH-Px,and nucleus and cytoplasm NRF2 of mouse skin tissue,to determine the level of oxidative stress on the back skin of mice.3.The effect of Haoqin-Huaban Decoction on HIF-1α and HOXA11-AS in the skin of mice damaged by UVB:Divide mice into the blank control group,UVB radiation model group,and high,medium,and low dose groups of Haoqin-Huaban Decoction,feed medication and make ultraviolet radiation intervention,use Western blotting to detect the expression and phosphorylation level of HIF-1α in mouse skin tissue,and use qRT-PCR to measure the relative expression level of HOXA11-AS in mouse back skin.4.Intervention effect of HOXA11-AS high expression on UVB ray damage of HaCat cells:Divide HaCat cells into blank control group,model group,control group(UVB+lncRNANC),and high expression group(UVB+HOXA11-AS).Transfect the control cells with a negative empty plasmid,reprint the high expression group with the HOXA11-AS overexpression plasmid.Apply CCK8 cell activity detection,EdU-488 cell proliferation detection,Annexin V cell apoptosis detection,TUNEL staining to detect cell activity and measure expression levels of ROS,MDA,SOD,GSH-Px,nucleus and cytoplasm NRF2,to determine the level of oxidative stress.5.Preliminary exploration of the mode of action of HIF-1α/HOXA11-AS/EZH2 intervention pathway:Use Western bolting to detect the expression level of EZH2 in HaCat cells,use qRT-PCR to detect the relative expression level of HOXA11-AS in HaCat cells.Apply dual-luciferase reporter gene detection and chromatin immunoprecipitation detection to verify the interaction between HIF-1α and HOXA11-AS.Carry out RNA pull-down test and immunoprecipitation test to verify the interaction between HOXA11-AS and EZH2.Research result:1.The results of network pharmacology studies show that Haoqin-Huaban Decoction adopts HIF-1α as a regulatory element to increase the expression level of HOXA11-AS by regulating the transcription of HOXA11-AS.The increased expression of HOXA11-AS reduces the expression level of EZH2,thereby alleviating cell damage caused by ultraviolet radiation.2.The experiment proved that Haoqin-Huaban Decoction can reduce the oxidative stress and cell apoptosis caused by UVB radiation,alleviate the skin damage caused by UVB,and has a certain dose dependency.3.The experiment confirmed that Haoqin-Huaban Decoction can reverse the effects of UVB radiation to a certain extent,increase the phosphorylation level of HIF-1α in skin cells,and increase the expression level of HOXA11-AS.4.Up-regulation of HOXA11-AS expression in skin cells can reduce oxidative stress and apoptosis caused by UVB radiation,which is consistent with the drug effect of Haoqin-Huaban Decoction.5.HIF-1α can increase its transcription level by binding to the promoter of HOXA11-AS.HOXA11-AS promotes its ubiquitination degradation by binding to EZH2 to negatively regulate its level.Conclusion:In summary,it can be inferred that the drug regulation mechanism of Haoqin-Huaban Decoction is as follows.Haoqin-Huaban Decoction can promote the phosphorylation level of HIF-1α;after phosphorylation activation,HIF-1α binds to the transcription promoter region of the lncRNA-HOXA11-AS expression gene to increase the expression level of HOXA11-AS and reverse the low expression of HOXA11-AS caused by UVB radiation;HOXA11-AS enters the cytoplasm after transcription and expression,and combines with EZH2 in the cytoplasm,which further causes the ubiquitinated proteasome degradation of EZH2 and reduces the level of EZH2 in the lower envelope,thus relieving cell apoptosis caused by oxidative stress and endoplasmic reticulum stress and relieving skin damage caused by UVB.