| Research background and purposeIn recent years,although China’s air pollution has been improved under continuous and vigorous control,the situation is still grim.The concentration of fine particles in the atmosphere is still much higher than the standards of the World Health Organization.Fine particulate matter is an important air pollution pollutant.Studies have shown that the smaller the particulate matter is,the greater the harm to human health is.PM2.5(fine particulate matter,PM2.5)is a fine particle with aerodynamic diameter ≤ 2.5μm.It is a mixture with complex components,which is easy to absorb toxic heavy metals,acid oxides,organic pollutants,bacteria and viruses into the human body,which is seriously harmful to health.Because of its small particles,PM2.5 can reach the end of respiration through airflow and precipitate under the action of diffusion,and then enter other parts of the body through the exchange of gas and blood in the lungs,thus causing various injuries to the tissue system of the human body.Therefore,PM2.5 can not only directly cause respiratory diseases,but also cause damage to other human tissues,especially cardiovascular diseases.Studies have shown that toxic substances carried by PM2.5 can directly cause inflammation and oxidative stress in blood vessels and myocardium,aggravate myocardial ischemia and other symptoms,increase the morbidity and mortality of cardiovascular disease,and seriously threaten human health and life safety.Some studies have shown that PM2.5 is closely related to the occurrence and development of atherosclerosis(AS),but its mechanism is not completely clear.Phenotypic alteration of vascular smooth muscle cells(HVSMC)is an important link in the development of AS.In the normal physiological state of the body,vascular smooth muscle cells are differentiated(also known as contractile type),and a variety of differentiated marker genes are highly expressed in the cells,such as α-actin(α-SMA).During the occurrence of AS,the vascular smooth muscle cells in the middle layer of the artery will undergo a series of changes,which will dedifferentiate from the differentiated type to the secretory type,that is,phenotypic transformation.At this time,vascular smooth muscle cells in the body will highly express secretory marker genes,such as vascular smooth muscle cells converted from Osteopontin(OPN),to secretory vascular smooth muscle cells enhance the ability of proliferation and migration,and secrete more extracellular matrix,resulting in the formation of AS plaque.Therefore,this study starts from the effect on the phenotypic changes of vascular smooth muscle cells,to explore the mechanism of PM2.5-induced AS,and to study its mechanism from the PI3K/Akt pathway.Resveratrol(RES)is one of the polyphenols in traditional Chinese medicine,which is rich in Polygonum cuspidatum,grapes,peanuts and red wine.Studies have shown that RES has good anti-AS activity and has less side effects when used in large quantities for a long time.The anti-atherosclerotic effect of resveratrol has been confirmed by many studies.We used network pharmacology technology to explore the potential targets of resveratrol in the treatment of atherosclerosis and found that PI3K/Akt is one of the important targets for its anti-atherosclerotic effect.Therefore,resveratrol was selected to prevent the toxicity of PM2.5 on vascular smooth muscle cells.On the one hand,we explore the methods of traditional Chinese medicine to prevent and cure the cardiovascular toxicity of PM2.5,on the other hand,we use experimental methods to verify whether the predicted target enrichment pathway PI3K/Akt signal pathway is involved in the anti-AS effect of RES.By verifying that resveratrol plays a protective role through PI3K/Akt signal pathway,it provides a reliable experimental basis for the prevention and treatment of atherosclerosis and the clinical application of resveratrol.Methords1.Timeliness study on the effect of PM2.5 on HVSMC phenotypic markers(12h,18h): the experimental groups were divided into blank control group and PM2.5 group(25μg/ml).The expression of phenotypic transformation markers OPN and α-SMA was detected by qRTPCR.2.The effect of PM2.5 on the phenotypic markers of HVSMC: the experiment was divided into blank control group and different concentration of PM2.5 group(the final concentration was 6.25μg/ml,12.5μg/ml,25μg/ml,50μg/ml,100μg/ml).After vascular smooth muscle cells were treated with PM2.5 for 12 hours,the expression of phenotypic transformation markers OPN and α-SMA were detected by qRT-PCR and Western blot techniques.3.The role of PI3K/Akt in the phenotypic transformation of HVSMC induced by PM2.5: the experiment was divided into three groups: blank control group,PM2.5 group(25μg/ml)and LY294002 pretreatment group with different concentrations(10μmol/L,20μmol/L,30μmol/L).The phenotypic transformation of HVSMC was observed.QRT-PCR and Western blot techniques were used to detect the changes of phenotypic transformation markers OPN and α-SMA.4.The expression of PI3K/Akt signal pathway protein after PM2.5 acting on HVSMC: the experiment was divided into blank control group,PM2.5 group(25μg/ml)and PM2.5 + LY group(30μmol/L).Western blot technique was used to detect the expression of PI3K/Akt signal pathway phosphorylated protein Akt after PM2.5 acting on vascular smooth muscle cells.5.Network pharmacological analysis: the targets of RES and AS were collected from different databases,and then their common targets were screened out,and the targets were further screened by String database,and the data were imported into Cytoscape software for PPI network construction and topology analysis,and then the data were imported into DAVID database to obtain GO biological process,molecular function,cell composition enrichment data,and KEGG pathway enrichment data.6.The protective effect of resveratrol on HVSMC: the experimental groups were divided into blank control group,PM2.5 group(25μg/ml)and PM2.5 + RES group(the final concentration was 5μmol/L,10μmol/L and 20μmol/L respectively).The expression of phenotypic transformation markers OPN and α-SMA was detected by qRT-PCR and Western blot techniques.7.The protective effect of resveratrol on HVSMC stimulated by PM2.5 and the expression of related signal pathway proteins: the experimental groups were divided into blank control group,PM2.5 group(25 μg/ml),PM2.5 + RES group(the final concentration was 5μmol/L,10μmol/L,20μmol/L)and PM2.5 + LY group(30μmol/L).The expression of phosphorylated protein Akt in PI3K/Akt signal pathway of HVSMC induced by RES was detected by Western blot technique.Results1.The effect of different stimulation time of PM2.5 on the phenotypic transformation of HVSMC:(1)compared with the control group,the expression of OPN mRNA,a marker of phenotypic transformation,was increased in PM2.5 group(25μg/ml)at 12 h,and the difference was statistically significant(P < 0.05).At the same time,compared with the control group,PM2.5 group(25μg/ml)decreased the expression of phenotypic transformation marker α-SMA mRNA(P < 0.05).(2)compared with the control group,the surface III of phenotypic transformation marker OPN mRNA in PM2.5 group(25μg/ml)increased significantly at 18h(P < 0.05).At the same time,compared with the control group,PM2.5group(25μg/ml)decreased the expression of phenotypic transformation marker α-SMA mRNA(P < 0.05).2.The effect of PM2.5 on the phenotypic markers of HVSMC: the effect of PM2.5 on phenotypic transition of vascular smooth muscle cells after 12 hours:(1)the expression of mRNA,compared with the control group,the expression of PM2.5(6.25μg/ml,12.5μg/ml,25μg/ml,50μg/ml,100μg/ml)OPN mRNA increased with the increase of PM2.5concentration.α-SMA mRNA decreased with the increase of PM2.5 concentration,and P <0.05,the difference was statistically significant.(2)compared with the control group,the expression of OPN protein in PM2.5 concentration group(12.5μg/ml,25μg/ml,50μg/ml,100μg/ml)was significantly higher than that in control group(P < 0.05),but the expression of OPN protein in PM2.5 concentration group(6.25μg/ml)was not statistically significant(P > 0.05).The protein results of α-SMA showed that compared with the control group,the protein of PM2.5 concentration group(6.25μg/ml,12.5μg/ml,25μg/ml,50μg/ml,100μg/ml)was significantly lower than that of the control group.3.The effect of LY294002 pretreatment on the phenotypic transformation of HVSMC induced by PM2.5: compared with the control group,the expression of OPN mRNA and protein in PM2.5 group increased,and compared with PM2.5 group(25μg/ml),the expression of OPN mRNA and protein decreased in different concentrations of LY pretreatment group and had statistical significance.Compared with the control group,the expression of α-SMA mRNA and protein in PM2.5 group decreased(P < 0.05),and the expression of α-SMA mRNA and protein in LY pretreatment group increased significantly compared with that in PM2.5 group(25μg/ml)(P < 0.05).4.The expression of PI3K/Akt signaling pathway protein after PM2.5 was applied to HVSMC: compared with the control group,the phosphorylated protein content in PM group(25μg/ml)increased,and P<0.05,the difference was statistically significant;compared with PM group(25μg/ml),the phosphorylated protein content of PM2.5 + LY group(30μmol/l)decreased,and P < 0.05,the difference was statistically significant.5.Acquisition of anti-atherosclerotic targets of resveratrol: 184 overlapping genes were identified,and 59 key targets were screened by degree sequencing.The top five targets were STAT3,AKT1,TP53,MAPK1 and PIK3 CA.GO enrichment analysis showed that its biological process were mainly regulated by chemical stress,oxidative stress,polypeptide hormone response and drug stimulation.In addition,KEGG pathway analysis showed that PI3K/Akt,AGEs/RAGE,Kaposi sarcoma-associated and other signaling pathways were involved in the regulation of AS by RES.6.The effect of resveratrol on the phenotypic transformation of HVSMC induced by PM2.5:(1)compared with the control group,the expression of OPN mRNA in PM2.5 group(25μg/ml)increased,compared with PM2.5 group(25μg/ml),the expression of RES(5μmol/L,10μmol/L and 20μmol/L)OPN mRNA groups decreased significantly and had statistical significance.The results of α-SMA mRNA expression showed that,compared with the control group,the expression of α-SMA in the PM2.5 group decreased;compared with the PM2.5 concentration group(25μg/ml),the RES pretreatment group with different concentrations all increased and was statistically significant significance.(2)compared with the control group,the expression of OPN protein in PM2.5 group increased,and compared with PM2.5 concentration group(25μg/ml),the expression of OPN protein decreased in different concentration RES pretreatment group and had statistical significance.Compared with the control group,the expression of α-SMA protein in PM2.5 group decreased.Compared with the concentration of PM2.5 group(25μg/ml),the expression of α-SMA protein in different concentration of RES pretreatment group increased and had statistical significance.7.Resveratrol protected the expression of AKT phosphorylation in HVSMC exposed to PM2.5: compared with the control group,the content of phosphorylated protein in PM2.5group increased,and compared with PM2.5 group(25μg/ml),the phosphorylated protein content in PM2.5 + RES group(the final concentration was 5μmol/L,10μmol/L,20μmol/L)and PM2.5 + LY group(30μmol/L)decreased,and the difference was statistically significant.Conclusion1.PM2.5 leads to phenotypic transformation of HVSMC.Resveratrol pretreatment ca n inhibit its occurrence and thus play a protective role.2.PM2.5 induces phenotypic transformation of HVSMC by activating PI3K/Akt signal pathway,while resveratrol may reduce phenotypic transformation induced by PM2.5stimulation by inhibiting this signal pathway. |
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