| Objective:(1)To explore the effects of subacute B[a]P exposure on cardiovascular structure and function in male Wistar rats.(2)To investigate the roles of oxidative stress and TLR4/MyD88/NF-κB signaling pathway-mediated inflammation on cardiovascular injury induced by B[a]P.Methods:Forty healthy male SPF grade Wistar rats were randomly divided into five groups,eight in each group.They were blank control group,solvent control group,low,medium and high dose group(B[a]P concentrations were 1.0,2.5,6.25 mg/kg body weight,respectively);Three groups were injected intraperitoneally with the corresponding concentration of B[a]P,the solvent control group was given 1.0mL/kg body weight of olive oil and the blank control group did not do any treatment,continuous exposure for 28 days.After the end of exposure,rats were collected for 24 hours urine.M-mode echocardiography was used to observe the change of left ventricular structural and function in rats.One week after adaptive feeding,the BL-410 biofunctional experimental system was used to detect blood pressure and left ventricular pressure.Blood samples were taken from the abdominal aorta,the rats were dissected,and the left ventricle and thoracic aorta were fixed to make pathological sections.The urine 1-hydroxypyrene content was determined by high performance liquid chromatography.Pathological changes of rat thoracic aorta and left ventricle were observed by HE staining.Serum IL-1β,IL-6 and TNF-α were detected by enzyme-linked immunosorbent assay.The serum SOD activity was determined by the water-solublenitroblue tetrazolium salt method,and the MDA content was determined by thiobarbituric acid method.Reverse transcription-polymerase chain reaction was used to determine the expression levels of TLR4,MyD88 and NF-κB genes in whole blood.Data entry and analysis were performed using SPSS 20.0 software.P<0.05 was considered statistically significant.Results:(1)Body weight change in rats after B[a]P exposure: There was a statistically significant difference in body weight gain between the different groups(H=12.108,P=0.017).The weight gain of the high dose group was lower than that of the blank group(adjusted P=0.007).(2)Cardiovascular function change after B[a]P exposure: The total difference in ejection fraction(EF)and fractional shortening(FS)between the groups was statistically significant(H=11.497,P=0.022;H=11.422,P=0.022),the ejection fraction(EF)and fractional shortening(FS)of the middle dose group was lower than the solvent group(adjusted P=0.020;adjusted P=0.020);The overall difference in left ventricular end diastolic pressure(LVEDP)was statistically significant in different groups(H=10.104,P=0.039),the left ventricular end diastolic pressure(LVEDP)of the high dose group was higher than that of the solvent group(adjusted P=0.035).(3)Results of cardiovascular pathological sections after B[a]P exposure: HE staining of the thoracic aorta in the middle and high dose groups showed partial loss of vascular intima,partial loss of endothelial cells,large gap in the middle layer;In the middle and high dose groups,the left ventricular transverse stripes were unclear,the muscle fibers were reduced or disappeared,the myocardial space was widened,and inflammatory cells or myocardial interstitial oozing were observed occasionally.(4)1-hydroxypyrene levels: There was a statistically significant difference in urine1-hydroxypyrene concentration between the groups(H=11.256,P=0.024).The1-hydroxypyrene concentration in the high-dose group was higher than that in the solvent control group(adjusted P=0.018).(5)Correlation analysis between 1-hydroxypyrene and cardiac function indexes: The ejection fraction(EF)was correlated with 1-hydroxypyrene,and the partial correlation coefficient was-0.618(P=0.001).The fractional shortening(FS)was positively correlated with 1-hydroxypyrene,and the partial correlation coefficient was 0.514(P=0.009).(6)Oxidative stress response in rats: There was no significant difference in serum SOD activity and MDA content between the groups(F=2.091,P=0.112;F=1.585,P=0.209).(7)Inflammatory response mediated by TLR4/MyD88/NF-κB signaling pathway:The overall difference of TLR4,MyD88 and NF-κB gene expression levels between the groups was statistically significant(F=4.850,P=0.005;F=6.442,P=0.001;F=9.282,P<0.001),the expression levels of TLR4,MyD88 and NF-κB in high dose group were up-regulated compared with the blank control group and the solvent control group(P<0.05).There was a statistically significant difference in serum IL-6 level between the groups(F=4.465,P=0.007),the level of IL-6 in the high dose group was higher than that in the blank group and the control group(P<0.05),the level of IL-6 in the middle dose group was higher than that in the blank group(P<0.05).There was no significant difference in serum IL-1β and TNF-α level between the groups(F=1.407,P=0.261;F=1.766,P= 0.167).(8)Correlation analysis between inflammatory response and cardiac function indexs:The ejection fraction(EF)was negatively correlated with the expression of TLR4,MyD88 and NF-κB genes,and the partial correlation coefficients were-0.787,-0.710,-0.651(P<0.001);The fractional shortening(FS)was positively correlated with the expression of TLR4,MyD88 and NF-κB genes,the partial correlation coefficients were 0.711,0.617,0.529(P<0.001;P=0.001;P=0.007).The diastolic blood pressure(DBP)was negatively correlated with the expression of TLR4 gene,and the partial correlation coefficient was-0.513(P=0.009).The ejection fraction(EF)was negatively correlated with IL-6,and the partial correlation coefficient was-0.717(P<0.001).The fractional shortening(FS)was positively correlated with IL-6,and the partial correlation coefficient was 0.651(P<0.001).(9)Correlation analysis between 1-hydroxypyrene and TLR4/MyD88/NF-κB signaling pathway genes and cytokines: The partial correlation analysis between1-hydroxypyrene and TLR4,MyD88 and NF-κB was not statistically significant,and the partial correlation coefficients were 0.137,0.217,0.192(P>0.05);1-hydroxypyrene was positively correlated with inflammatory factor IL-6 with a partial correlation coefficient of0.708(P < 0.001).(10)Multiple linear regression analysis of the factors affecting cardiac function indexes: The ejection fraction(EF)was greatly affected by 1-hydroxypyrene,and the standard partial regression coefficient b’ was-0.319.Conclusions:(1)Benzo[a]pyrene may cause cardiovascular and endothelial damage in male Wistar rats.(2)Cardiovascular structural and functional damage induced by subacute benzo[a]pyrene exposure may be associated with inflammatory response mediated by TLR4/MyD88/NF-κB signaling pathway. |
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