| Objective: Hypoxic pulmonary hypertension(HPH)is a clinically common subtype of pulmonary hypertension,and there are obvious gender differences in its incidence.The Warburg effect is related to the proliferation of pulmonary artery smooth muscle cells and endothelial cell dysfunction.The key transcription factor for the Warburg effect is HIF-1α.This experiment has passed in vivo studies,applications Hypoxia and castrated rats establish a pulmonary hypertension model to explore whether 17β-estradiol can affect the Warburg effect through the HIF-1α signaling pathway in hypoxic pulmonary hypertension,thereby reducing pulmonary hypertension.Methods: 42 female Sprague-Dawley rats aged 6-8 weeks were selected and randomly divided into 7 groups: sham operation + normoxia group,sham operation + hypoxia group,ovariectomy + normoxia group,ovariectomy +hypoxia group,ovariectomy + normoxia + 17β-estradiol(E2)group,ovariectomy + hypoxia + 17β-estradiol(E2)group,ovariectomy + hypoxia+ dichloroacetic acid(DCA)group.The ovariectomized group was sutured after removal of the ovary;the sham operation group was sutured directly after finding the ovary.The hypoxia group was hypoxic for 24 hours a day.The E2 group was injected subcutaneously with 17β-estradiol(E2)20μg/kg daily,and the other groups were injected subcutaneously with normal saline(0.1ml/d).In the dichloroacetic acid group,the dichloroacetic acid salt solution(DCA,1.6g/L,0.75g/l,PH=7.0)was intragastrically administered daily at a dose of 80mg/kg/d,and the other groups were intragastrically administered with normal saline(0.1ml/d);after 8 weeks of feeding,measured the mean pulmonary artery pressure(m PAP)and right ventricular hypertrophy index(RVHI)of rats.The morphology of pulmonary arterioles was observed by HE staining and WA% and WT% were measured.The contents of lactic acid and pyruvate in lung tissue were measured by kit method.ELISA method was used to detect the activity of pyruvate dehydrogenase kinase-1(PDK-1)lactate dehydrogenase(LDHA).RT-PCR detection of tissue LDHA and PDK-1 m RNA levels.Western blot was used to detect the protein content of tissue PDK-1,LDHA,pyruvate dehydrogenase(PDH),and HIF1-α.Results:1.Successfully established the hypoxic pulmonary artery hypertension rat model:Compared with group 1(sham operation + normoxia group),group 2(sham operation + hypoxia group),and group 4(ovariectomy + hypoxia group)with prolonged hypoxia time,the reaction becomes weak,and the coat colors darkness,and gradually reduced intake of water,the final state is mainly resting,no dead specimens,and the growth rate of group 6(ovariectomy +hypoxia + E2 group),group 7(ovariectomy + hypoxia + DCA group)was also slowed down,but the mobility was slightly better than that of group 4(ovariectomy + hypoxia group).Compared with group 1(sham operation +normoxia group),the change was no significantly changes between the group3(ovariectomy + normoxia group)and the group 5(Ovariectomy normoxic+E2 group).2.m PAP and RVHI of each group of samples:Compared with group 1(sham operation + normoxia group),group 3(ovariectomy + normoxia group),and group 5(ovariectomy + normoxia + E2group),The m PAP and RVHI were increased in group 2(sham operation +hypoxia group)and group 4(ovariectomy + hypoxia group)(P<0.05),and were consistent with the level of pulmonary hypertension;compared with group 4(ovariectomy + hypoxia group),The m PAP and RVHI of rats were reduced in group 6(ovariectomy + hypoxia + E2 group)and group 7(ovariectomy + hypoxia + DCA group)(P<0.05),suggesting that estrogen and DCA can improve hypoxia pulmonary hypertension.There was no difference in m PAP and RVHI levels in group 6(ovariectomy + hypoxia + E2 group)and group 7(ovariectomy + hypoxia + DCA group).3.Morphological changes of lung tissues in each group:Compared with group 1(sham operation + normoxia group),group 3(ovariectomy + normoxia group),and group 5(ovariectomy + normoxia + E2group),The thickened pulmonary artery wall and smooth muscle,markedly narrowed arterial lumen,severe infiltration of inflammatory cells,consistent with the morphological changes of pulmonary hypertension in group 2(sham operation + hypoxia group),group 4(ovariectomy + hypoxia group);compared with group 4(ovariectomy + hypoxia group),the pulmonary artery lumen showed different degrees of reversal,and the infiltration of inflammatory cells was relatively reduced in group 6(ovariectomy + hypoxia+ E2 group),group 7(ovariectomy + hypoxia + DCA group).However,there was no significant difference between the group 6(ovariectomy + hypoxia +E2 group)and group 7(ovariectomy + hypoxia + DCA group).Compared with group 1(sham operation + normoxia group),group 3(ovariectomy + normoxia group),and group 5(ovariectomy + normoxia + E2group),The WA% and WT% significantly increased,there was a statistical difference in group 2(sham operation + hypoxia group),group 4(ovariectomy+ hypoxia group)(P<0.05);compared with group 4(ovariectomy + hypoxia group),the WA% and WT% were significantly reducer in group 6(Ovariectomy + hypoxia + E2 group),group 7(ovariectomy + hypoxia + DCA group),there was a statistical difference(P<0.05),However,there was no significant difference between the group 6(ovariectomy + hypoxia + E2 group)and group 7(ovariectomy + hypoxia + DCA group).4.Lactic acid concentration level and pyruvate concentration level in lung tissue:Compared with group 1(sham operation + normoxia group),group 3(ovariectomy + normoxia group),and group 5(ovariectomy + normoxia + E2group),there was significantly higher in group 2(sham operation+ hypoxia group)and group 4(ovariectomy + hypoxia group)(P<0.05).Compared with group 4(ovariectomy + hypoxia group),The increase in lactic acid and pyruvic acid levels decreased relatively in group 6(ovariectomy + hypoxia +E2 group)and group 7(ovariectomy + hypoxia + DCA group)(P<0.05).There was no significant difference in group 6(ovariectomy + hypoxia + E2group)and group 7(ovariectomy + hypoxia + DCA group).It is suggested that hypoxia can increase the concentration of lactic acid and pyruvate in lung tissue.5.Elisa method detects the activity of PDK-1 and LDHA in plasma:In this experiment,the ELISA method was used to detect the activity of PDK-1 and LDHA in the plasma of SD rats.It was compared with group 1(sham operation + normoxia group),group 3(ovariectomy + normoxia group),and group 5(ovariectomy + normoxia+E2 group),the PDK-1 LDHA enzyme activity in group 2(sham operation + hypoxia group)and group 4(ovariectomy + hypoxia group)was significantly enhanced(P < 0.05),Compared with group 4(ovariectomy + hypoxia group),there had significantly lower PDK-1 LDHA enzyme activity in group 6(ovariectomy + hypoxia + E2group)and group 7(ovariectomy + hypoxia + DCA group)(P<0.05),but there was no statistical difference in enzyme activity between group 6(ovariectomy+ hypoxia + E2 group)and group 7(ovariectomy + hypoxia + DCA group),6.The PDK-1 and LDHA m RNA levels in lung tissue of SD rats in each groupIn this experiment,RT-PCR was used to detect the levels of PDK-1 and LDHA m RNA in the lung tissues of SD rats in each group,compared with group 1(sham operation + normoxia group)and group 3(ovariectomy +normoxia group)group 5(ovariectomy + normoxia + E2 group),the expression of PDK-1 and LDHA m RNA in group 2(sham operation +hypoxia group)and group 4(ovariectomy + hypoxia group)increased significantly(P<0.05).Compared with group 4(ovariectomy + hypoxia group),PDK-1 m RNA was significantly lower in group 6(ovariectomy +hypoxia + E2 group)and group 7(ovariectomy + hypoxia + DCA group)(P<0.05),but in group 6(ovariectomy + hypoxia + E2 group),group 7(ovariectomy+hypoxia+DCA group),there was no statistical difference between the two groups.7.The expression of PDK-1 and LDHA protein in lung tissue of SD rats in each groupIn this experiment,the Western blot method was used to detect the expression of PDK-1 and LDHA protein in lung tissue of SD rats in each group,compared with group 1(sham operation + normoxia group)and group3(ovariectomy + normoxia group)group 5(ovariectomy + normoxia + E2group),the expression of PDK-1 and LDHA protein in group 2(sham operation + hypoxia group)and group 4(ovariectomy + hypoxia group)were significantly increased(P<0.05),Compared with group 4(ovariectomy +hypoxia group),the PDK-1 protein expression was significantly reduced in group 6(ovariectomy + hypoxia + E2 group)and group 7(ovariectomy +hypoxia + DCA group)(P<0.05).There was no significant difference in protein expression between group 6(ovariectomy + hypoxia + E2 group)and group 7(ovariectomy + hypoxia + DCA group).8.The expression of PDH protein in lung tissue of SD rats in each groupIn this experiment,Western blot was used to detect the expression of PDH,the key enzyme of aerobic oxidation in the lung tissues of SD rats in each group,Compared with group 1(sham operation + normoxia group),group 3(ovariectomy + normoxia group)and group 5(ovariectomy +normoxia + E2 group),the expression of PDH protein were significantly reduced in group 2(sham operation + hypoxia group)and group 4(ovariectomy + hypoxia group)(P<0.05),Compared with group 4(ovariectomy + hypoxia group),the PDH protein expression were significantly increased in group 6(ovariectomy + hypoxia + E2 group)and group 7(ovariectomy + hypoxia + DCA group)(P<0.05).There was no significant difference in protein expression between group 6(ovariectomy + hypoxia +E2 group)and group 7(ovariectomy + hypoxia + DCA group).9.The expression of HIF-1α protein in the lung tissue of SD rats in each groupIn this experiment,the Western blot method was used to detect the expression of HIF-1α in lung tissue of SD rats in each group,compared with group 1(sham operation + normoxia group)and group 3(ovariectomy +normoxia group)group 5(ovariectomy + normoxia + E2 group),the expression of HIF-1α in group 2(sham operation + hypoxia group)and group4(ovariectomy + hypoxia group)were significantly increased(P<0.05),Compared with group 4(ovariectomy + hypoxia group),the HIF-1αexpression was significantly reduced in group 6(ovariectomy + hypoxia + E2group)and group 7(ovariectomy + hypoxia + DCA group)(P<0.05).There was no significant difference in protein expression between group 6(ovariectomy + hypoxia + E2 group)and group 7(ovariectomy + hypoxia +DCA group).Conclusions:1.Long-term chronic hypoxia environment can induce hypoxic pulmonary arterial hypertension,and the intervention of estrogen and dichloroacetic acid can improve hypoxic pulmonary hypertension.2.Hypoxia increases the expression and activity of PDK-1 and LDHA,increases the content of lactate and pyruvate,and reduces the expression of PDH,thereby increasing the metabolism related to the Warburg effect.3.Dichloroacetic acid can improve pulmonary hypertension by affecting the activity of PDK-1,enhancing the tricarboxylic acid cycle,reducing the Warburg effect.4.Estrogen may inhibit the activity of PDK-1 through the HIF-1αsignaling pathway through a function similar to dichloroacetic acid,thereby enhancing the glycometabolism tricarboxylic acid cycle,inhibiting the Warburg effect,reducing lactic acid production,and improving hypoxic pulmonary hypertension. |
PDF Full Text Request