ABSTRACT
Objective To investigate the role of transient receptor potential melastatin 7(TRPM7) in sevoflurane preconditioning for inhibiting hippocampal neurons apoptosis and inflammation response induced by oxygen-glucose deprivation(OGD).Methods Fifty SD rats of postnatal 1 d were selected for extracting hippocampal neurons and randomly divided into 5 groups,including the control group(C),sevoflurane preconditioning group (Sev),OGD group,Sev preconditioningt OGD group (Sev + OGD) and Sev preconditioning+ bradykinin+OGD group(combined group).After 1.5 h oxygen-glucose deprivation,reintroduction was performed,and then the normal culture was performed again for preparing the OGD model.Hippocampal neurons in the control group were normally cultured only;which in the Sev group conducted 2 ％ Sev preconditioning for 1 h;which in the OGD group only prepared the OGD model;which in the SEv+OGD conducted 2％ Sev preconditioning for 1 h,and prepared the OGD model after 24 h;which in the combined group was simultaneously added with bradykinin(final concentration 200μmol/L) in Sev preconditioning,other treatment was same to that in the Sev+OGD group.After 24 h normal culture,the mRNA and protein levels of TRPM7,apoptosis rate,survival rate,mRNA and supernatant protein levels of IL-1β and TNF-α of the hippocampal neurons were detected.Results Compared with the control group,hippocampal neurons mRNA and protein levels of TRPM7,apoptosis rate,mRNA and supernatant protein levels of IL-1β and TNF-α in the OGD group were significantly increased(P＜0.05),whereas the survival rate was significantly decreased (P ＜ 0.05).Compared with the OGD group,hippocampal neurons mRNA and protein levels of TRPM7,apoptosis rate,mRNA and supernatant protein levels of IL-1β and TNF-α in the Sev group were significantly decreased(P＜0.05),whereas the survival rate was significantly increased(P＜0.05).Compared with the Sev group,hippocampal neurons mRNA and protein levels of TRPM7,apoptosis rate,the mRNA and supernatant protein levels of IL-1β and TNF-α in the combined group were significantly increased(P＜0.05),whereas the survival rate was significantly decreased(P＜0.05).Conclusion Sev preconditioning can attenuate hippocampal neurons apoptosis and inflammatory response after OGD via alleviating the overexpression of TRPM7.
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Objective To investigate the effects of transient receptor potential melastatin (TRPM) 7 on dexamethasone (Dex)-mediated cytoskeleton remodeling in human trabecular meshwork.Methods Human trabecular meshwork cells (HTMs) were primarily cultured and the cells of generation 3 to 6 were used in this study.The expression of TRPM7 protein in the cells was located using immunofluorescence technology.Dex at the dose of 0.2 mg was added into culture medium for 4 days with the final concentration of 1×10-5,1×10-6 and 1×10-7 mol/L,respectively.Western blot assay was employed to detect the relative expression level of TRPM7 protein.Cultured cells were divided into non-transfected group,siRNA transfected group,TRPM7-siRNA1 transfected group and TRPM7-siRNA2 transfected group,and the expressions of TRPM7 protein and p-cofilin protein in the cells were assayed by Western blot method.Cultured cells were divided into normal control group,Dex-treated group,siRNA transfected group and TRPM7-siRNA transfected group,and the expression of phalloidin (a cytoskeletal protein) and Vinculin (focal adhesion protein) was detected by immunofluorescence staining.In addition,cultured cells were divided into normal control group,Dex-treated group,2-APB (a Ca2+ inhibitor) treated group,ethylene glycol tetraacetic acid (EGTA) (a calcium chelator)-treated group,TRPM7-siRNA transfected group and TRPM7-siRNA+Dex group,and the [Ca2+] i in the cells was observed by Fluo-3AM immunofluorescence staining.Western blot assay was used to detect the expression of p-cofilin in the cells.Results TRPM7 was positively expressed on the cell membrane.The relative expression of TRPM7 was gradually reduced with an increase of Dex dose (F=4.210,P＜0.05),and the expression of TRPM7 was significantly decreased in 1 × 10-5 mol/L Dex group compared with the normal control group (P＜ 0.05).Western blot assay revealed that the relative expression levels of TRPM7 in the TRPM7-siRNA1 and TRPM7-siRNA2 group were significantly lower than those of non-siRNA transfected group and siRNA transfected group (all at P＜0.05).In the Dex-treated group and TRPM7-siRNA transfected group,the cells were enlarged in size with the lessened processes in comparison with the normal control group.Immunofluorescence staining showed that the actin fiber and vinculin increased in the Dex-treated group,and more spread but depolymerized actin fiber was seen in the TRPM7-siRNA transfected group.Compared with the normal control group,the fluorescence intensity of [Ca2×] i was weak in the Dex-treated group and TRPM7-siRNA transfected group.The relative expression levels of p-confilin protein was lower in the TRPM7-siRNA transfected group than that in the siRNA transfected group (0.317 ±0.031 vs.0.092±0.071) (t =5.030,P =0.007).Conclusions Dex induces the downregulation of TRPM7 expression in HTMs.The downregulation of TRPM7 probably participates in Dex-induced cytoskeletal remodeling by causing the depolymerization of actin cytoskeleton and reduction of [Ca2+] i in HTMs.
ABSTRACT
Objective To investigate the role of transient receptor potential melastatin 7 (TR PM7) in the protective role of sevoflurane preconditioning against hippocampal neuron injury caused by oxygen glucose deprivation (OGD).Methods Hippocampal neurons were harvested from postnatal day 1 SD rats,and randomly divided into 5 groups:control group (group C),sevoflurane group (group Sev),oxygen-glucose deprivation group (group OGD),sevoflurane+ OGD group (group SD) and sevoflurane+OGD+bradykinin group (group B).To build up the model of OGD,the neurons were cultured in a deoxygenated glucose-free medium and exposed to 95％ N2 and 5％％ CO2 in an anaerobic chamber equilibrated at 37℃ for 1.5 h,followed by replacement with glucose containing medium and return to a standard incubator for additional 24 h.The neurons in group C received no treatment.Group OGD was preconditioned with 2 ％ sevoflurane for 1 h.The neurons in group OGD were subjected to OGD.Group SD was preconditioned with 2％ sevoflurane for 1 h,followed by OGD at 24 h after the sevoflurane exposure.The neurons in group B was incubated in a medium supplemented with 200 μmol/L bradykinin (the selective agonist of TRPM7),followed sequen tially by the preconditioning of 2％ sevoflurane for 1 h and then OGD challenge.Twenty-four hours after re-oxygenation,The relative neuronal cell viability was determined by MTT assay,the neuronal apoptotic rate was analyzed by TUNEL assay,the protein expression of TRPM7 was detected by Western blot,the mRNA level of TRPM7 was estimated by real-time PCR,the neuronal release of IL-1β and TNF-α in the serum was measured by ELISA.Results Compared with group C,the mR NA and protein levels of TRPM7,the neuronal apoptotic rate,the mRNA and supernatant protein levels of IL-1β and TNFα were significantly up-regulated in group OGD (P＜0.05),whereas the cell viability was decreased (P＜0.05).Compared with group OGD,the mRNA and protein levels of TRPM7,the neuronal apoptotic rate,the mRNA and supernatant protein levels of IL1β and TNF-α were significantly down-regulated in group SD (P＜0.05),whereas the cell viability was increased (P＜0.05).Compared with group SD,the mRNA and protein levels of TRPM7,the neuronal apoptotic rate,the mRNA and supernatant protein levels of IL-1β and TNF-α were significantly up-regula ted in group B (P＜0.05),whereas the cell viability was decreased (P＜0.05).Conclusion Sevoflurane attenuates apoptosis and inflammatory responses induced by OGD via reduction of the overex pression of TRPM7 in the hippocampal neurons.
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Objective To evaluate the expression of transient receptor potential melastatin 7(TRPM7)in cholangiocarcinoma and its correlation with prognosis.Methods The expressions of TRPM7 were detected by SP immunohistochemical in 49 cases of cholangiocarcinoma,7 cases of benign bile duct lesions and 36 cases of adjacent histologically noncancerous bile duct tissues,and to analysis its relationship with the clinical pathological characteristics of cholangiocarcinoma.Results The positive expression rate of TRPM7 in cholangiocarcinoma was 77.6%(38/49),which was higher than that in benign bile duct lesions(0,0/7)and adjacent his-tologically noncancerous bile duct tissues(2.8%,1/36),the difference was statistically significant(P 0.05).Kaplan-Meier analysis showed that increased expression of TRPM7 was associated with shorted overall survival (P <0.05).Cox regression analysis showed that the expression level of TRPM7 was significantly associated with prognosis and an independent risk factor for prognosis(P <0.05 ). Conclusion TRPM7 plays an important role in the tumorigenesis progression,invasion,and metastasis of cholangiocarcinoma,and is an important factor for prognosis in patients with cholangiocarcinoma.
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Transient receptor potential melastatin 7 (TRPM7) is a member of the melastatin-related subfamily and contains a channel and a kinase domain. TRPM7 is known to be associated with cell proliferation, survival, and development. It is ubiquitously expressed, highly permeable to Mg2+ and Ca2+, and its channel activity is negatively regulated by free Mg2+ and Mg-complexed nucleotides. Recent studies have investigated the relationships between TRPM7 and a number of diseases. TRPM7 regulates cell proliferation in several cancers, and is associated with ischemic cell death and vascular smooth muscle cell (VSMC) function. This review discusses the physiologic and pathophysiologic functions and significance of TRPM7 in several diseases.