| Part Ⅰ Effects of intermittent heat stress on BMAL1 autophagy and apoptosis in thoracic aorta of SHR and RTAECsObjective With global warming,the rise of temperature has become a hot topic of public health.Heat stress has been shown to be associated with autophagy and apoptosis.Moreover,autophagy and BMAL1 are highly related to cardiovascular disease.However,it is still unknown about the effects of intermittent high temperature on spontaneous hypertensive rats.Therefore,this part aims to explore whether intermittent heat stress can lead to BMAL1,autophagy and apoptosis changes in thoracic aorta of SHR and Rat Thoracic Aorta Endothelial cells(RTAECs).Methods In vivo,to establish the rat model,WKY and SHR were divided into control group(WKY-CN,SHR-CN),intermittent heat stress group(WKY-8,SHR-8)and continuous heat stress group(WKY-24,SHR-24).The changes of blood pressure and body weight of rats were measured before and after heat stress;HE was used to observe the morphological changes of rat thoracic aorta;The expressions of BMAL1,autophagy related genes(Beclin1,LC3,P62),apoptosis related genes(Caspase3,Bax,Bcl-2)and AMPK/m TOR/ULK1 pathway related factors were detected by immunohistochemistry,immunofluorescence and Western blot.In vitro,establishing the intermittent heat stress model according to that in vivo,and the time of intermittent heat stress was established.The control group(CN)was cultured in an incubator at 37℃;the continuous heat stress group were exposed to 40℃;the intermittent heat stress group was cultured alternately for 3 or 6 cycles under normal(37℃)and high temperature(40℃),and the heat stress and recovery were carried out alternately.Firstly,WB was used to detect the expression of autophagy proteins(Beclin1,LC3,P62),apoptosis related proteins(Caspase3,Bax,Bcl-2)and AMPK/m TOR/ULK1 pathway related factors under intermittent heat stress and continuous heat stress.and then WB or IF were used to detect the changes of BMAL1,autophagy,apoptosis and pathway factors under defined intermittent heat stress period.Results1.Compared with the control group,SBP and DBP of WKY and SHR increased,the body temperature fluctuated,and the differences were statistically significant(P<0.01).HE staining showed heat stress caused morphological changes in WKY and SHR.The arrangement and distribution of aortic intima in SHR-8 were incomplete and the cell distribution was ruptured.2.WB,IHC and IF were used to detect the expression changes of BMAL1 in spontaneously hypertensive rats(SHR)and RATECs.After intermittent heat stress,the expression level of BMAL1 increased in vivo and vitro.and the difference was statistically significant(P < 0.001).In vivo,the red fluorescence intensity and brown particles of BMAL1 increased.3.In vivo,IHC showed the increase of brown particles of Beclin1 and LC3 and P62 brown particles decreased significantly in intermittent heat stress group.IF showed that the fluorescence intensity of P62 decreased significantly in SHR-8.The results in WB were consistent with those in IHC,and the difference was statistically significant(P<0.05).In vitro experiment,compared with the control group,intermittent heat stress and continuous heat stress can both increase the expression levels of Beclin1 and LC3-II/LC3-I.After 36(6)h heat stress,there were significant differences in Beclin1 and LC3-II/LC3-I(P<0.001).In the continuous heat stress group,the expression levels of Beclin1 and LC3-II increased with time,and gradually increased after 36 h of heat stress,reaching the peak at 72 h,with a statistically significant difference compared with the control group(P<0.05).After intermittent heat stress for 36(6)h,the expression of Beclin1 and LC3-II/LC3-I was the highest,and the difference was statistically significant(P<0.05).4.In vivo,TUNEL assay showed that the number of apoptosis positive cells in SHR-8 was significantly higher than that in SHR-CN.Compared with the control group,Western blot showed that,heat stress increased the expression of Bax and decreased the expression of Bcl-2.The difference was statistically significant(P<0.05).In vitro,compared with the control group,after intermittent heat stress for 36(6)h,the expression of Caspase3 and Bax was the highest,and the difference was statistically significant(P<0.01).5.In vivo,WB,IHC and IF were also used to detect the expression of AMPK/m TOR/ULK1 pathway related genes.The results showed that heat stress caused the expression changes of pathway factors and their phosphorylation.Compared with SHR-CN,the expression of pm TOR decreased significantly,and the expression of p-AMPK and p-ULK1 increased in SHR-8(P<0.05).In vitro,compared with the control group,the changes of intermittent heat stress group were fluctuating,the expression of p-AMPKα(Thr172)increased significantly from36(3)h,and p-AMPK/AMPK reached the peak at 36(6)h,and the p-ULK1(Ser556)/ULK1 increased significantly at 24(6)h.ConclusionIntermittent heat stress can lead to the increase of blood pressure and body temperature and the morphological changes of thoracic aorta in SHR.At the same time,intermittent heat stress increased the expression of BMAL1 autophagy and apoptosis,and activated AMPK/m TOR/ULK1 pathway in vivo and vitro.Part Ⅱ Effects of BMAL1 on vascular endothelial autophagy and apoptosis in SHR and RTAECs under intermittent heat stressObjective To investigate whether autophagy in thoracic aorta was mediated by AMPK/m TOR/ULK1 pathway under intermittent heat stress,the relationship between autophagy and apoptosis,and the effect of BMAL1 sh RNA on autophagy and apoptosis in thoracic aorta.Methods Under intermittent heat stress,SHR was intraperitoneally injected with AMPK inhibitor CC to detect the expression of pathways and autophagy proteins.After injection of 3-m A,CQ and Rapa,autophagy was inhibited and activated respectively.The expression of AMPK/m TOR/ULK1 pathway related factors,autophagy and apoptosis related factors were observed by IHC.In vitro,CC was also used to inhibit AMPK,3-MA and Rapa in RTAECs inhibited and activated autophagy respectively.WB was used to detect the expression of autophagy and apoptosis related factors.Finally,BMAL1 was interfered with sh RNA,and the knockdown efficiency was test by RT-q PCR,autophagy and apoptosis were detected after sh BMAL1.Results1.IHC showed that compared with SHR-8,after intraperitoneal injection of CC,the brown particles of AMPK decreased significantly,the brown particles of p-m TOR(ser2448)increased,and the expression of p-ULK1(ser556)was opposite to that of p-m TOR(ser2448),indicating that AMPK was inhibited,affected the expression of p-m TOR and p-ULK1,and decreased the expression of LC3.In vitro,after the admission of CC in RTAECs exposed to heat for 36(6)h,WB showed that the ratio of p-AMPK / AMPK decreased,p-ULK1 /ULK1 showed the same trend,while p-m TOR / m TOR increased,the difference was statistically significant(P <0.001).At the same time,Beclin1 and LC3-II/LC3-I decreased,indicating that the transformation from LC3-I to LC3-II decreased and autophagy decreased.2.In vivo,compared with SHR-8,after administrating of Rapa,IHC showed that Caspase3 brown particles increased significantly and Bcl-2 decreased.Fluorescent double labeling results showed that compared with SHR-CN,the immunofluorescence brightness of LC3 B and Caspase3 in SHR-8 were enhanced;Compared with SHR-8,the administration of Rapa further enhanced the fluorescence expression intensity of LC3 and Caspase3,but the administration of3-MA showed the opposite trend.In vitro,apoptosis was attenuated and enhanced after autophagy was inhibited and activated by 3-MA and Rapa respectively.3.In vitro,BMAL1 was silenced with sh RNA.The results of RT-q PCR showed that compared with neo group,AMPK/m TOR/ULK1 in sh2 group decreased,and the difference of ULK1 was statistically significant(P<0.01).At the same time,Beclin1,Caspase3 and Bax m RNA decreased,and the difference was statistically significant(P<0.05).ConclusionIntermittent heat stress induced autophagy and apoptosis in thoracic aorta of SHR.BMAL1 affects autophagy and apoptosis induced by intermittent exposure through AMPK/m TOR/ ULK1. |
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