The Study Of The Role Of Hydrogen Sulfide In Adrenal Insufficiency Induced By Hypoxia And Its Mechanism

When the body is exposed to stimulus,the hypothalamus pituitary adrenal(HPA)axis is activated,and the adrenal cortex release a large amount of glucocorticoids to maintain the homeostasis of the internal environment.However,in some cases,such as sepsis,relative adrenal insufficiency will occur.Further statistics showed that the prognosis of patients with relative adrenal insufficiency was also poor.However,the mechanism of relative adrenal insufficiency has not been fully elucidated.Hypoxia is a very common pathological process in various clinical diseases.In the early experiment of our laboratory it was found that the resistance of mice to anesthetics after hypoxia was significantly reduced.So,whether hypoxia leads to relative adrenal insufficiency,and then significantly reduce the body’s ability to resist stress deserves further research.Hydrogen sulfide is the third gas signal molecule discovered in recent years.A large number of articles have reported that H2 S plays an important role in many physiological and pathological processes.Previous studies in our laboratory have found that H2 S can improve the adrenocortical hyporesponsiveness of endotoxemia mice by improving mitochondrial function.So,whether H2 S is involved in the hypoxia-induced adrenocortical hyporesponsiveness and the related mechanism need further research.Therefore,this study will focus on the relationship between hypoxia and adrenal reactivity and H2 S.ACTH stimulation was applied to measure the adrenocortical responsiveness of mice and the we detect the expression of related proteins by Western Blot to explore the mechanism of this process.Due to the limitations of in vivo experiments,we completed part of the research by culturing Y1 cells in vitro.By studying the adrenocortical hyporesponsiveness and its related mechanisms under hypoxia,we can provide a theoretical basis for improving the adrenocortical hyporesponsiveness at high altitude or under hypoxia,improving the body’s resistance to external stress stimulation,and provide a new idea for clinical treatment of adrenocortical hyporesponsiveness.In the first part of the study,we found that the expression of CTH in adrenal gland decreased after hypoxia.Since glucocorticoids are mainly produced in zona fasciculata of adrenal cortex,in order to better study the role of specific genes or proteins in the adrenal cortex in related diseases,we carried out the second part of the study,constructing the transgenic mice specifically expressing Cre recombinase in zona fasciculata of adrenal cortex.We first screened out the gene CYP11B1 which specifically expresses in the adrenal cortex,and knocked in the 2A-Gfp Cre expression frame at its termination codon site,and constructed the transgenic mice specifically expressing Cre recombinase in the zona fasciculata of adrenal cortex.The success of construction of the transgenic mice were virified by immunofluorescence,m Tm G,Lac Z staining,Western Blot and other methods,which can provide the zona fasciculata of adrenal cortex relevant research with a powerful tool.Main Results:Part I: The role of hydrogen sulfide in attenuating adrenal insufficiency induced by hypoxia and its mechanism1.Hypoxia can induce cell apoptosis and pyroptosis,which leads to the decrease of adrenocortical responsiveness and the decrease of the resistance to stress stimulation1.The adrenocortical responsiveness was measured by ACTH stimulation after the mice were fed in normoxia environment or hypoxia tank for one or three days.The results showed that the increase of glucocorticoid in mice decreased after hypoxia,indicating that hypoxia induced adrenalcortical hyporesponsiveness;2.Mice in the sham operation group and the adrenalectomy(ADX)group were fed in the normoxia and hypoxia tank respectively,and LPS was injected intraperitoneally at a dose of 15 mg/kg.The mortality of mice in ADX group was significantly higher than that in sham operation group,and the mortality in hypoxia was significantly higher than that in normoxia3.After one or three days of feeding mice in normoxia or hypoxia tank,the adrenal tissues of mice were stained with HE and TUNEL,and the related indexes of pyroptosis were detected.One or three days after hypoxia,the thickness of adrenal cortex decreased significantly,apoptosis appeared,and the level of pyroptosis increased2.Hydrogen sulfide may improve the adrenalcortical responsiveness caused by hypoxia by inhibiting the activation of NLRP31.After one or three days of feeding mice in normoxia or hypoxia tank,the adrenal tissue of mice was taken and WB results showed that the expression of CTH was down regulated;2.Mice were divided into three groups: normoxia group,hypoxia group and hypoxia with GYY4137 group.The mice in the hypoxia group were fed in the hypoxia box for 3 days,and the mice in the hypoxia with GYY4137 group were injected intraperitoneally with a dose of 50mg/kg H2 S donor GYY4137 every day on the basis of the hypoxia group.GYY4137 can reverse the decrease of adrenocortical thickness and cell apoptosis caused by hypoxia,and improve the increase of pyroptosis level caused by hypoxia;3.After hypoxia,the expression of NLRP3 and Caspase-1 p20 was significantly increased,and GYY4137 could reverse this.3.Hypoxia induced the down-regulation of CTH expression and the decrease of PSMA7 S-sulfhydration level in adrenal cortex,which resulted in the accumulation of MAVS and the activation of NLRP3 and Caspase-1.GYY4137 can improve the related changes caused by hypoxia1.The results showed that the level of PSMA7 S-sulfhydration in adrenal gland decreased significantly after hypoxia,which could be reversed by GYY4137;2.The results of Co-IP showed that PSMA7 interacted with MAVS in adrenal tissue;3.Western results showed that hypoxia could increase the expression of MAVS,NLRP3 and Caspase-1 p20 in adrenal tissues,while GYY4137 could significantly reverse the expression of these proteins.4.S-sulfhydration of Cys70 plays an crucial role in the maintenance of PSMA7 function1.IAA was used to stem the S-sulfhydration modification in Y1 cells.Western blot showed that the expression of NLRP3 was up-regulated in Y1 cells treated with IAA;Western blot showed that the expression of NLRP3 in Y1 cells was up-regulated after Cys70 was mutated.Part II: Construction of transgenic mice with specific Cre recombinase expression in zona fasciculata in adrenal cortex1.CYP11B1 expresses in zona fasciculata of adrenal cortex specificallyCYP11B1 protein expresses in zona fasciculata of adrenal cortex specifically,without expressing in zona glomerulosa,zona reticularis or medulla.2.Detect the expression of Cre recombinase by m Tm G and Lac Z staining1.Mating CYP11B1 Cre mice with Rosa26 m Tm G tool mice.Select the both Cre positive and m Tm G positive mice.Collect their adrenal gland and other main organs for fluorescence detection.The results showed that the zona fasciculata of adrenal cortex showed green fluorescence and the rest and other organs showed red fluorescence;2.Mating CYP11B1 Cre mice with Rosa26 lacZ tool mice.Select the both Cre positive and Lac Z positive mice.Collect their adrenal gland and other main organs for Lac Z staining.The results showed that the zona fasciculata of adrenal cortex was blue stained,while the rest and other organs were not blue stained.3.CYP11B1 Cre can specifically knock out the expression of protein in zona fasciculata of adrenal cortex1.The expression level of CTH had no significant difference between CTHf/f mice’s adrenal gland and that of WT mice.And in CTH/f/CYP11B1 Cre mice’s adrenal gland,the expression level of CTH was significantly decreased,and there was little CTH expressing in CTHf/f/CYP11B1 Cre mice’s adrenal gland.The expression level of CTH in other organs among different genotypes showed no significant difference.2.There was much expression of CTH in the adrenal cortex of CTHf/f/CYP11B1 Cre mice,but the positive staining of CTH in the adrenal cortex of CTH/f/CYP11B1 Cre mice decreased significantly,especially in the zona fasciculata of the adrenal cortex.Conclusions:In the first part of the study,we found that hypoxia leads to the decrease of hydrogen sulfide producing enzyme CTH expression in adrenal tissue,and decreases the level of S-sulfhydration of PSMA7 protein,leading to the accumulation of MAVS,activating NLRP3 and Caspase-1.The inflammatory causes adrenocortical hyporesponsiveness eventually.GYY4137,a hydrogen sulfide donor,can restore the S-sulfhydration level of PSMA7,and inhibit the activation of downstream pathway,thus protect the adrenocortical function.In order to study the function of related proteins in the adrenal cortex,we carried out the second part of the study.Using CYP11B1 as the target,we constructed the transgenic mice expressing Cre recombinase specifically in adrenal cortex cells,and verified the successful construction of the transgenic mice by various methods.