Effects Of Hippocampus On HPA Axis Dysfunction In Rats Under High Temperature And High Humidity Stress Combined Condition And Its Drug Intervention

ObjectiveHigh temperature and high humidity is a common stress environment,which will seriously affect human health.To elucidate the functions and mechanisms of hippocampus in HPA axis dysfunction caused by high temperature and high humidity stress,and provide theoretical basis for finding a potential therapeutic target for high temperature and high humidity stress,in this study we investigate the effects of the high temperature and high humidity stress simulated by artificial climate chamber,on hypothalamus-pituitary-adrenocortical in rats,using various methods and technologies,which include animal behavioral experiments,histopathology and several molecular biology,observe the structure and function of hippocampus and HPA axis and the therapeutic effects of drugs on combined high temperature and high humidity stressed rats intervened with glucocorticoid receptor antagonists and heat-clearing and dampness-eliminating herbs.MethodsMale SD rats were randomly divided into control group,model groups(medium temperature and high humidity group,high temperature and high humidity group),intervention groups(western medicine group,traditional Chinese medicine group),20 in each group.Each group was further tested according to the experiment time(3w,6w),10 rats in each subgroup.Rats in the control group were kept in a temperature(24±1)°C and humidity(50±5)% without any stimulation.The remaining groups were exposed daily to a high humidity environment simulated by artificial climate chambers with a relative humidity of(85±5)%.The temperature of medium temperature and high humidity groups were simulated at(30 ±1)°C,while the high temperature and high humidity group,western medicine group and traditional Chinese medicine group were simulated at(35±1)°C,4h per day,the rest of the feeding environment as same as the control group,3 weeks as one stage,the experiment for a total of 6 weeks.The traditional Chinese medicine group was given Huang Qin Hua Shi Decoction by gavage,each rat had a daily dose of 8g/kg,western medicine group was given a 25mg/kg mifepristone solution,and the administration volume was 1ml/100 g.The Morris water maze test was used to test spatial learning and memory,open field tests were used to detect exploration-related and anxiety-related behaviors.The levels of CRH,ACTH and CORT in rats were determined by ELISA.Morphological changes of adrenal glands,pituitary and hypothalamus were observed by HE staining.The RT-PCR was used to detect the expression of CRF mRNA in the hypothalamus.Apoptosis of hippocampus was detected by TUNEL apoptosis staining.The protein content of MR,GR,11?-HSD1,iNOS,TRPV1 in hippocampus were detected by immunohistochemistry.In situ hybridization was used to detect the gene expression of MR,GR,11?-HSD1,iNOS and TRPV1 in hippocampus.Results1.Effects of hippocampus on HPA axis in rats under high temperature and high humidity stress combined condition(1)At week 3 of Morris water maze test,the total distance of swimming and the latency to platform increased significantly in the medium temperature and high humidity group and high temperature and high humidity group compared with the control group.In the frequency of access to the platform quadrant,the high temperature and high humidity group was significantly reduced contrasted to the control group and medium temperature and high humidity group.At week 6,in the total distance of swimming and the latency to platform,the medium temperature and high humidity group and high temperature and high humidity group increased significantly compared with the control group.In the frequency of access to the platform quadrant,the medium temperature and high humidity group was significantly reduced compared with the control group and high temperature and high humidity group.(2)At week 3 of the open field test,the ratio of resting time to exercise time was significantly lower in the medium temperature and high humidity group than in the control group.In the number of standing,the medium temperature and high humidity group was significantly increased than the control group and high temperature and high humidity group.At week 6,the medium temperature and high humidity group and high temperature and high humidity group was significantly reduced compared with the control group in the ratio of resting time to exercise time.(3)At week 3 in ELISA,serum CRH,ACTH and CORT levels in the medium temperature and high humidity group and high temperature and high humidity group were significantly higher than those in the control group.Compared with the medium temperature and high humidity group,ACTH concentrations in the serum of the high temperature and high humidity group were significantly higher.At week 6,CRH,ACTH and CORT in the serum of the medium temperature and high humidity group and high temperature and high humidity group showed the same trend as at week 3.(4)Morphological changes of the hypothalamus,pituitary and adrenal glands were observed by HE staining.At week 3,for hypothalamus,3 cases of microglial hyperplasia were observed in the medium temperature and high humidity group.There were 2 cases of microglial proliferation and abnormal changes of nerve cells in the high temperature and high humidity group and the medium temperature high humidity group at week 6,respectively.There was no significant change in the pituitary at 3 weeks in each group,and the vacuolar degeneration in the high temperature and high humidity group was obvious at week 6.For adrenal,the hypertrophy and disorder of cortical cells and the expansion of medullary telangiectasia were found in 3 cases at week 3.It was aggravated at week 6.(5)RT-PCR showed that CRF gene expression in hypothalamus increased significantly in rats in the high temperature and high humidity group compared with the control group at week 3.At week 6,the CRF gene expression in the hypothalamus of rats in the high temperature and high humidity group exhibited the same trend as at week 3.(6)A t week 3,apoptosis staining of TUNEL detected the apoptosis rate of hippocampal cells was significantly higher in the high temperature and high humidity group compared with the control group.At week 6,the apoptosis rate of hippocampal cells in the high temperature and high humidity group showed the same trend as at week 3.(7)At week 3 of immunohistochemistry,compared with the control group,the protein levels of MR and iNOS increased significantly in the medium temperature and high humidity group and high temperature and high humidity group,and the GR protein content in the hippocampus increased significantly in the high temperature and high humidity group.At week 6,contrasted to the control group,the protein levels of MR and GR and iNOS of hippocampus were significantly increased in the medium temperature and high humidity group and high temperature and high humidity group.There was no significant difference between the week 3 and the week 6 in rat hippocampal TRPV1 and 11?-HSD1 protein content.(8)At week 3 of in situ hybridization,compared with the control group,the gene expression of MR,GR,iNOS,and TRPV1 in the hippocampus of the medium temperature and high humidity group and high temperature and high humidity group increased significantly.At week 6,the gene expression of MR and GR and iNOS and TRPV1 in the hippocampus of the former groups exhibited the same trend as at week 3.There was no significant difference in hippocampal 11?-HSD1 gene expression between the week 3 and week 6 in rats.2.Effects of drug intervention in rats under high temperature and high humidity stress combined condition(1)At week 3 of Morris water maze test,in the total distance of swimming and the latency to platform,the traditonal Chinese medicine group was significantly decreased compared with the high temperature and high humidity group.In the frequency of access to the platform quadrant,the western medicine group and traditonal Chinese medicine group were significantly increased compared with the high temperature and high humidity group.At week 6,in the total distance of swimming and latency to platform,the traditional Chinese medicine group decreased significantly compared with the high temperature and high humidity group.(2)At week 3 of the open field test,in terms of the ratio between the center and the periphery,the traditional Chinese medicine group significantly increased compared with the high temperature and high humidity group.At week 6,the traditional Chinese medicine group exhibited the same trend as at 3 weeks in the ratio between the center and the periphery.(3)At week 3 in ELISA,serum CRH,CORT and ACTH levels in the traditional Chinese medicine group and western medicine group were significantly lower than those in the high temperature and high humidity group.At week 6,CRH,CORT and ACTH in the serum of the western medicine group showed the same trend as at week 3.(4)Morphological changes of the hypothalamus,pituitary and adrenal glands were observed by HE staining.Hypothalamus HE staining showed no obvious lesions in the Chinese medicine group.In the western medicine group,there were 3 cases of microglia proliferation at 3 weeks.Pituitary staining mainly manifested as cell reduction,sparse arrangement,disorder,vacuoles in some cytoplasm,and cell morphology.At week 6,the adrenal gland lesions were slightly heavier than those at 3 weeks,mainly manifested in the disordered and denatured cells of cortical fascicular zone,inflammatory cell infiltration and medullary telangiectasia.(5)RT-PCR showed that CRF gene expression in hypothalamus decreased significantly in the western medicine group compared with the high temperature and high humidity group at week 3.At week 6,CRF gene expression in hypothalamus showed the same trend as at week 3.(6)TUNEL at week 3,the apoptosis rate of hippocampal cells was lower in the traditional Chinese medicine group and western medicine group compared with the high temperature and high humidity group,but there was no significant difference.At week 6,the apoptosis rate of hippocampal cells showed the same trend as at week 3,and the apoptosis rate of hippocampal cells in the western medicine group decreased significantly.(7)At week 3 of immunohistochemistry,compared with the high temperature and high humidity group,the protein content of GR in the hippocampus decreased significantly in the traditional Chinese medicine group and western medicine group.Compared with the high temperature and high humidity group,the protein content of MR and iNOS in hippocampus in the western medicine group were significantly reduced.At week 6,contrasted to the high temperature and high humidity group,the protein content of MR,iNOS in hippocampus were significantly decreased in the traditional Chinese medicine group and western medicine group.The protein content of GR in hippocampus in the western medicine group were significantly reduced compared with the high temperature and high humidity group.There was no significant difference in the protein content of TRPV1 and 11?-HSD1 in hippocampus between the week 3 and the week 6.(8)At week 3 of in situ hybridization,the gene expression of MR,GR and iNOS in the hippocampus of the traditional Chinese medicine group and western medicine group decreased significantly compared with the high temperature and high humidity group(P<0.05).At week 6,the gene expression of MR and GR and iNOS in the hippocampus of the intervention groups exhibited the same trend as at week 3.The gene expression of TRPV1 in the hippocampus of the traditional Chinese medicine group and western medicine group decreased significantly compared with the high temperature and high humidity group.There was no significant difference in gene expression of 11?-HSD1 in hippocampus between the week 3 and week 6.Conclusions1.According to behavioral changes from model group after stress,HPA axis function and pathological changes,histopathological changes in the hippocampus,and the apoptosis rate of hippocampal cells,all these performance conforms to the characteristics of persistent hyperactivity of HPA axis due to chronic stress.The establishment of a stress animal model induced by high temperature and high humidity was successful.2.High temperature and high humidity stress has significant influences in behavior in rats,and its effects are mainly reflected in the decline in learning ability,impaired spatial memory and increased anxiety.3.High temperature and high humidity stress leads to HPA axis dysfunction,which may be related to the increase of CRF mRNA gene expression under stress.The increase of CRF expression leads to the increase of CRH,ACTH and CORT,which causes further abnormal stress response.Finally,changes in HPA axis function and pathology were observed.4.The effects of hippocampus on HPA axis dysfunction induced by high temperature and high humidity stress may be the increase of iNOS.It leads to MR and GR inactivation and NO toxicity enhancement by the growth of NO.That promote apoptosis cell of hippocampus,impaired the structure of hippocampus,weaken the regulation of HPA axis and aggravate the dysfunction of the HPA axis.11?-HSD1 and TRPV1 may not be involved in the injury induced by high temperature and high humidity stress.5.HPA dysfunction and injury of hippocampus caused by high temperature and high humidity stress were aggravated with time,and glucocorticoid receptor antagonists and heat-clearing and dampness-eliminating herbs can effectively intervention by each advantage.