| BackgroudWith the development of industrialization and the change of people's life style,the impact of environmental factors on human health is increasingly obvious,such as polluted drinking water,persistent organic pollutants(such as pesticides),household chemicals containing phenol,extreme climate change,excessive noise(such as airport,traffic),etc.However,the biggest threat to public health comes from air pollution,in which fine particulate matter(PM2.5)is the main air pollutant,for it most easily enters the human body and poses the biggest threat to global public health.As a result,PM2.5 remains the focus of most scientific and supervision.Its complex components not only cause immune damage to the respiratory system,but also can enter the blood circulation and the circulatory system,which aggravates the incidence of cardiovascular related diseases and complications.Recently,it is estimated that 4.2 million people die of PM2.5 every year in the world.It is preliminarily estimated that the number will doubled by 2050,which makes it becomes one of the five major risk factors of global mortality.Since the 1970s,European and American countries began to effectively control the environment and formulate the world's ambient air quality standardsEnvironmental pollution in developed countries has improved significantly.PM2.5 is now disproportionately concentrated in developing countries,especially in India and China,with its daily and annual average emission exceeding international standards.In China,,cooking is the daily life of every family.(COFs-derived PM2.5)is similar to the composition of PM2.5 in the air,including heavy metals,organic carbon,and water-soluble inorganic substances.Cooking oil fume is more exposed in our daily life With its higher concentration,it is easier to be inhaled by human body,and cause a series of damages to the circulation and respiratory system.However,how COFS derived PM2.5 damages cardiomyocytes at the molecular level is still unknown to usVitamin D(VitD)is a kind of fat soluble vitamin.After two consecutive hydroxylation reactions,1,25-dihydroxyvitamin D3(1,25-dihydroxyvitamin D3 1,25(OH)2D3)is the main active form of vitamin D in human being's body.1,25(OH)2D3 can not only regulate the balance of calcium and phosphorus metabolism,but also act as an immunosuppressant to reduce inflammation.Recent studies at home and abroad have shown that sufficient 1,25(OH)2D3 may reduce the incidence of cardiovascular related diseases.However,whether 1,25(OH)2D3 can reduce the myocardial damage caused by COFs-derived PM2.5 and its mechanism is not clear.There is no relevant study on the molecular level of 1,25(OH)2D3 affecting the protective mechanism of PM2.5 myocardial damage at home and abroadJAK/STAT and NF-?B pathway,an important inflammatory pathway and apoptosis pathway,play an important role of control in the development of many diseases.It is believed that JAK/STAT and NF-?B signaling pathway are involved in many biological activities such as myocardial ischemia-reperfusion injury,myocardial cell proliferation and apoptosis,vascular cell migration and proliferation.Therefore,Based on the study of cells,this paper mainly explores the following aspects,including the possible mechanism and pathway of PM2.5 on myocardial cell injury,the correlation between PM2.5 exposure concentration and injury;whether vitamin D can reduce the chronic injury of PM2.5 to cardiomyocytes by changing the signaling pathwayThis paper includes two partsPart ? Cooking oil fumes-derived PM2.5 promotes mouses'cardiomyocyte injury through cytotoxicity,oxidative stress and apoptosisPart ? 1,25 dihydroxyvitamin D3 reduces the damage of Cooking oil fumes-derived PM2.5 to cardiomyocytes by inhibiting the JAK/STAT and NF-?B pathwayPart ? Cooking oil fumes-derived PM2.5 promotes mousecardiomyocyte injury through cytotoxicity,oxidative stressand apoptosisObjective1.To clarify the relationship between the toxicity and survival rate of COFs-derived PM2.5 on mouse cardiomyocytes with dose change;2.To analyze the changes of IL-6 and TNF-? levels of cardiomyocytes exposed to different concentrations of COFs-derived PM2.5,and to explore whether COFs-derived PM2.5 can affect the myocardial cell injury through inflammatory response;3.To explore the possible mechanism that COFs-derived PM2.5 may damage myocardium through related apoptosis and inflammatory signaling pathwayMethods1.COFs-derived PM2.5 was collected with a collector,and 0(control group),25,50100?g/ml of different concentrations of toxic solution were respectively prepared to inoculated cardiomyocytes;2.Cell culture:the myocardial cells of suckling mice were isolated and inoculated into the DMEM medium containing 10%fetal bovine serum,and cultured in a incubator containing 37? and 5%CO2;3.The effects of COFs-derived PM2.5 on the survival and toxicity of cardiomyocytes were evaluated by CCK-8 and LDH leakage rate;4.The levels of IL-6 and TNF-? in different concentrations of COFs-derived PM2.5 were detected by ELISA,to evaluate the effect of different concentrations of COFs-derived PM2.5 on the inflammatory response of cardiomyocytes5.Western blot was used to detect the expression of p65,JAK2 and STAT3 apoptotic protein in different concentrations of COFs-derived PM2.5,and to explore the possible mechanism of the effect of COFs-derived PM2.5 on cardiomyocytes through JAK/STAT and NF-?B signaling pathway6.Statistical analysisAll statistical analyses involved used of SPSS16.0 software.Data are reported as mean± standard deviation(x ± s).P<0.05was considered statistically significantResults1.When the concentration of COFs-derived PM2.5 was 100 ?g/mL,the most significant reduction occured in CCK-8.When the concentration of COFs-derived PM2.5 was 50?g/ml,the difference was statistically significant(*P<0.05).When the concentration of COFs-derived PM2.5 reached 100?g/ml,the most obviously increasing occured in LDH leakage rate of cardiomyocytes,When the concentration of PM2.5 reached 50?g/ml,the difference was statistically significant(*P<0.05),and a dose-response relationship existed.;2.Compared with the control group,the level of IL-6 and TNF-? increased with the concentration of COFs-derived PM2.5,and the difference was statistically significant when the concentration of COFs-derived PM2.5 reached 50?g/ml(*P<0.05);3.Compared with the control group,the higher the concentration of COFs-derived PM2.5,the higher the expression of apoptosis related protein P65,P-JAK2 and P-STAT3.When the concentration of COFs-derived PM2.5 reaches 50 ?g/mL,the difference is obvious(*P<0.05)Conclusion1.COFs-derived PM2.5 can reduce the activity of cardiomyocytes and increase it's cytotoxicity.The toxicity of COFs-derived PM2.5 to cardiomyocytes is related to theconcentration.When the concentration exceeds the standard,COFs-derived PM2.5 can cause obvious cytotoxicity to cardiomyocytes;2.COFs-derived PM2.5 can induce inflammatory reaction,promote the production of inflammatory factors and increase myocardial injury.The higher the concentration was,the more obvious the inflammatory reaction occured;3.COFs-derived PM2.5 may regulate apoptosis and induce cardiomyocyte apoptosis through JAK/STAT and NF-?B signaling syste.The degree of apoptosis damage is related to the concentrationPart ? 1,25 dihydroxyvitamin D3 reduces the damage of Cooking oil fumes-derived PM2.5 to cardiomyocytes by inhibiting the JAK/STAT3 and NF-?B pathwayObjectcive1.To figure out whether 1,25(OH)2D3 has direct cytotoxic effect on rat cardiomyocytes;2.To explore whether 1,25(OH)2D3 can reduce the inflammatory damage of COFs-derived PM2.5 on cardiomyocytes by inhibiting the inflammatory response and reducing the release of inflammatory factors;3.Tomake clear whether 1,25(OH)2D3 can reduce the oxidative damage of COFs-derived PM2.5 to cardiomyocytes by inhibiting oxidative stress and reducing the release of ROS.4.To determine whether COFS derived PM2.5 can reduce myocardial apoptosis and inflammatory damage by inhibiting JAK/stat,NF-? B pathway signaling system and Bax/Bcl-2 gene regulationMethods1.Cell culture and groupingThe myocardial cells of SD suckling mice were inoculated in DMEM medium containing 10%peptide bovine serum,and the control group(COFs-derived PM2.5,VitD concentration of 0 ?gmL,VitD3 group(10nmol/L1,25(OH)2D3),COFs-derived PM2.5 group(50 ?g/mLCOFs-derived PM2.5),COFs-derived PM2.5+VitD group(50?g/mL COFs-derived PM2.5+10 nmol/L1,25(OH)2D3)were used added to 37?incubator for 24 hours2.Detection of cytotoxicityThe level of CCK-8 and LDH levels were observed after 24 hours incubation with 0,1,5,10,50,100 nmol/L VitD33.The levels of IL-6 and TNF-? in the control group,VitD3 group,COFs-derived PM2.5 group and COFs-derived PM2.5+VitD3 group were detected by ELISA4.The level of ROS in cardiomyocytes was detected by DCFH-DA fluorescence probe and the level of GSH and SOD was detected by detection kit5.The expression of Bax/bcl-2 mRNA was detected by Q-PCR,the degree of apoptosis was detected by AnnexinV/PI staining,and the expression of GRP78,Capsase-1,cracked Caspase-3,P65,P-JAK2 and P-STAT3 were analyzed by Western blot(WB)6.Statistical analysisAll statistical analyses involved used of SPSS16.0 software.Data are reported as mean± standard deviation(x ± s).P<0.05was considered statistically significant.Results1.There was no significant difference in CCK-8 and LDH levels between different concentrations of VitD3 and the control group(P>0.05);2.The levels of IL-6 and TNF-? were the highest in the COFs-derived PM2.5 group(*P<0.05).The levels of IL-6 and TNF-? were relatively lower in the control group and VitD3 group.The levels of IL-6 and TNF-? in the VitD3+COFs-derived PM2.5 group were significantly lower than those in the COFs-derived PM2.5 group;3.In the COFS derived PM2.5 group,the red fluorescence intensity and green fluorescence intensity increased significantly,while in the VitD3 co incubation group,the red fluorescence intensity and green fluorescence intensity decreased significantly;the ROS level was the highest in COFs-derived PM2.5 group(*P<0.05),and co incubation with VitD3 significantly reduced the ROS level in COFs-derived PM2.5 group;4.Bax/Bcl-2,GRP78,Caspase-1,cleaved Caspase-3,P65,P-JAK2 and P-STAT3 were significantly increasing in COFs-derived PM2.5 group,while in VitD3 group,Bax/Bcl-2 and P65,P-JAK2 and P-STAT3 were significantly decreasing.Conclusion1.1,25(OH)2D3 had no significant toxic effect on cardiomyocytes;2.1,25(OH)2D3 could inhibit the inflammatory damage of COFs-derived PM2.5 on cardiomyocytes of SD rats;3.1,25(OH)2D3 could reduce the level of ROS and oxidative stress induced by COFs-derived PM2.5;4.1,25(OH)2D3 inhibited the apoptosis induced by COFs-derived PM2.5 by inhibiting JAK/STAT and NF-?B pathway. |
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