| Objective1.Constructing a model of oxidative stress(OS)injury to the reproductive system of male rats in a simulated plateau environment using a simulated plateau hypobaric hypoxic animal experiment chamber.2.The microscopic(pathomorphological)and submicroscopic(ultrastructural)changes of rat testes were observed under the influence of hypobaric hypoxic environmental factors and after protective intervention with levocanidine(LC)in simulated plateau.3.By detecting the degree of DNA damage,apoptosis level and mitochondrial apoptosis pathway-related(Cyt C-Caspase-9-Caspase-3)gene expression in testicular tissues,we explored the mechanism of the effects of factors simulating the hypobaric hypoxic environment of plateau and the related effects on testicular tissues after applying LC protective intervention,and provided the theoretical basis for related clinical studies.Methods1.Forty 10-w-old male Wistar rats were randomly divided into plains group(Control),plateau group(Model),plateau + normal saline(NS)group(Model & NS)and plateau + LC group(Model & LC),10 rats in each group.Model,Model & NS and Model & LC rats were housed in a hypobaric hypoxic animal chamber at a simulated altitude of 6000 m,and Control rats were housed outside the chamber.Control and Model did not impose intervention,Model& LC were injected intraperitoneally with 100 mg/kg of LC injection daily,1/d for 14 d,and Model & NS were injected intraperitoneally with the same volume of NS as LC daily,1/d for14 d.Testicular tissues of rats in each group were collected by autopsy after 14 d for experiments.2.Microscopic and sub-microscopic changes of nucleus,mitochondria and endoplasmic reticulum in rat testis under the influence of simulated hypoxic environment of plateau and after LC protective intervention were observed by HE staining and transmission electron microscopy.3.Single cell suspensions of rat testicular tissues from each group were prepared and DNA damage in each group was directly detected by comet assay.4.The levels of 8-hydroxy-2 deoxyguanosine(8-OHDG),a product of oxidative DNA damage,were measured in each group of rat testis tissues using the kit,as well as the expression of H2 ax m RNA and phosphorylated H2ax(γ-H2ax)protein by q PCR and Western Blot.γ-H2 ax protein expression to indirectly reflect the level of DNA damage.5.Terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling(TUNEL)assay was used to detect testicular tissue apoptosis in rats under the influence of simulated hypoxic environment factors and after LC protective intervention.6.q PCR to detect the m RNA expression of genes related to the mitochondrial apoptotic pathway such as Cyt-C,Caspase-3,Caspase-9,Bax and Bcl-2 in the testis tissue of each group of rats.7.The expression levels of proteins related to the mitochondrial apoptotic pathway,such as Cyt C,Caspase-3,Caspase-9,Bax and Bcl-2 proteins,were analyzed by Western Blot in testis tissues of rats in each group.Results1.The HE results showed that the testicular tissue of Control rats had normal microstructure,intact basement membrane,neatly arranged and numerous cells;In Model and Model & NS rats,the testicular tissue structure was more obviously damaged,with thinning of the basement membrane of the seminiferous tubules,atrophy of the external mesenchyme,and a decrease in the number of internal spermatogonia,etc.The internal and external structures of the varicocele in the testicular tissue of Model & LC rats were significantly improved,and the basement membrane was thickened and basically restored to normal.2.The results of transmission electron microscopy showed that the testicular tissue of Control rats was in relatively good condition,with no obvious swelling,abundant organelles and fair structure,no obvious swelling of mitochondria,no expansion of cristae and laminar arrangement;Model rats had a relatively high degree of testicular tissue cell damage,cellular edema,mitochondrial swelling and vacuolization;The degree of damage of Model & NS was basically the same as that of Model,with cell edema and incomplete organelle structure;The degree of damage of Model & LC was low,the cell membrane was locally depressed and still intact,the organelles were abundant,the mitochondria were not swollen,and the cristae were slightly dilated.3.Comet assay and 8-OHDG results showed a statistically significant increase in the degree of DNA damage in testicular tissue of Model rats compared to Control(P < 0.05,P <0.01);Compared with Model & NS,the degree of DNA damage was significantly lower in Model & LC,and the difference was statistically significant(P < 0.05,P < 0.01).4.The TUNEL results showed that the apoptotic index of testicular tissue was significantly higher in Model rats compared with Control,and the difference was statistically significant(P < 0.01);The apoptotic index of Model & LC was significantly lower compared with Model & NS,and the difference was statistically significant(P < 0.01).5.The q PCR results showed that the expression of H2 ax m RNA,a DNA damage product,was changed in the testis tissue of Model rats compared with Control,but the difference was not statistically significant(P > 0.05);The expression of H2 ax m RNA was slightly reduced in Model & LC compared to Model & NS,but the difference was also not statistically significant(P > 0.05).Compared with Control,the m RNA expression of pro-apoptotic proteins Cyt-C,Caspase-3,Caspase-9 and Bax related to mitochondrial apoptotic pathway was significantly higher in testis tissue of Model rats,while the m RNA expression of anti-apoptotic protein Bcl-2 was significantly lower,and the difference was statistically significant(P < 0.05,P < 0.01);Compared with Model & NS,the m RNA expression of each of the above pro-apoptotic proteins was down-regulated in Model & LC,while the m RNA expression of anti-apoptotic proteins was up-regulated,and the difference was statistically significant(P < 0.05).6.Western Blot results showed that the expression of DNA damage product γ-H2 ax protein was significantly higher in testis tissue of Model rats compared with Control,and the difference was statistically significant(P < 0.01);The expression of γ-H2 ax protein was significantly lower in Model & LC compared to Model & NS,and the difference was statistically significant(P < 0.01).Compared with Control,the expression of pro-apoptotic proteins Cyt-C,Caspase-3,Caspase-9 and Bax related to mitochondrial apoptotic pathway was significantly higher in testis tissue of Model rats,while the expression of anti-apoptotic protein Bcl-2 was significantly lower,with statistically significant differences(P < 0.05);Compared with Model & NS,the expression of each of the above pro-apoptotic proteins was down-regulated in Model & LC,while the expression of anti-apoptotic proteins was up-regulated,and the difference was statistically significant(P < 0.05).Conclusion1.Under the influence of factors simulating the hypobaric hypoxic environment of the plateau,OS could be induced in the testicular tissue of male rats,resulting in abnormal changes in the microstructure and sub-microstructure of the nucleus,mitochondria and endoplasmic reticulum of the testicular tissue,increased DNA damage and apoptosis of the testicular tissue.2.After applying LC protective intervention,it could alleviate the abnormal changes in the microstructure and sub-microscopic structures such as nucleus,mitochondria and endoplasmic reticulum of rat testis tissue under the influence of factors simulating the low-pressure hypoxic environment of plateau,reduce the degree of DNA damage in testis tissue,and decrease the abnormal apoptosis.3.Under the influence of hypobaric hypoxic environmental factors simulating plateau,ROS may directly or indirectly activate the mitochondrial apoptotic pathway(Cyt C-Caspase-9-Caspase-3)in rat testis tissue,inducing increased m RNA and protein expression levels of the pro-apoptotic genes Cyt C,Caspase-3,Caspase-9 and Bax,and decreased m RNA and protein expression levels of the anti-apoptotic gene Bcl-2.4.Application of LC protective intervention down-regulated the m RNA and protein expression levels of the pro-apoptotic genes Cyt C,Caspase-3,Caspase-9 and Bax,while up-regulated the m RNA and protein expression levels of the anti-apoptotic gene Bcl-2,improving the anti-apoptotic ability of rat testicular tissue cells. |
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