The Effect Of TRPM8 Channel On The BMMSCs Proliferation/apoptosis Of Zmpste24^-/- Mice

Hutchinson-Gilford progeria syndrome(HGPS) is a rare genetic disease,characterized by clinical features that childhood patients appear symptoms of aging and affect a variety of tissues and organs, including the skin, muscle, bone and cardiovascular.HGPS is associated with common complications of older people, and ultimately lead to death, the average life expectancy of children with the disease is about 13.5 years. HGPS is usually caused by a dominant mutation in LMNA gene that interferes with the prelamin A protein processing of post-translation, leading to abnormal accumulation of prelamin A in the nucleus. Zmpste24 is a necessary enzyme which is involved in the formation process of mature lamin A from prelamin A. Zmpste24 knockout mice with typical aging performance, is the preferred model for the study of cell senescence mechanism currently.Transient receptor potential channel is a regulator of intracellular calcium balance, widely expressed in many tissues in mammals, and involved in the regulation of cell proliferationand apoptosis, neurotransmitter release, cell differentiation, gene transcription and many other important physiological functions. According to the report, endothelial cells of progeria patients induced from pluripotent stem cells express high level of TRPV2,ultimately leading to apoptosis, associated with cell senescence. But the function of TRP channels on BMMSCs is still rarely reported, and the mechanisms how TRP channels affect cellular aging are not clear. Cellular senescence involves the accumulation of toxic metabolites, DNA damage, mitochondrial disorders and so on, which are associated with oxidative stress. Therefore, the purpose of this experiment is to explore the relationship between TRP channels and oxidative stress by comparing the cell senescence,proliferation, apoptosis and the expression of TRP channels between Zmpste24-/- and wild-type mice, and to further explore the TRP channel functions on cellular senescence and its possible mechanism.[Aim]1. To Compare the cell senescence, proliferation and apoptosis of BMMSCs between Zmpste24-/- and wild-type mice;2. To Compare the TRP channel expression in BMMSCs of Zmpste24-/- and wild-type mice, and investigate the TRP channels related to cell proliferation and apoptosis;3. To explore the possible mechanisms that TRP channels have an impact on cell proliferation and apoptosis.[Methods]1. We applied the method of whole bone marrow culture to isolate and purify BMMSCs of both Zmpste24-/- and wild-type mice. BMMSCs were identified with CFU-F assay and MSC-related monoclonal antibodies by flow cytometric analysis;2. Flow cytometry was performed to compare the proliferation ability, cell cycle and apoptosis between Zmpste24-/- and wild-type mice. The senescence level was determined by SA-β-gal staining kit. The expression of Cyclin D1, CDK1, CDK4,CDK6 and age-related P16, P21, P53 was tested by RT-PCR;3. RT-PCR was performed to test the expression of TRP channels. BMMSCs were cultured by serum-free and H2O2 medium to screen out specific TRP channels thatassociated with cell proliferation and apoptosis. Western-Blot was applied to detect expression of the specific TRP channels;4. Inhibit the expression of TRPM8 channel to detect cell proliferation and oxidative stress.[Recults]1. Zmpste24-/- BMMSCs showed a flat and larger shape than wild-type mice. Positive expression of stem cell antigen Sca-1, CD73 and negative expression of CD34 and CD45 were identified in BMMSCs of both Zmpste24-/- and wild-type mice. CFU-F assay showed that the BMMSCs of Zmpste24-/- mice presented smaller and fewer colony-forming units than wild-type mice.2. The BMMSCs of Zmpste24-/- mice showed reduced proliferation ability(P<0.05) and increased apoptosis(P<0.05) than wild-type mice. Flow cytometry analysis showed that the cell number of Zmpste24-/- in G0/G1 phase of cell cycle was higher than the wild-type, however the G2 and S phase was lower(p<0.05). The senescent cell number of Zmpste24-/- was more than wild-type(p<0.05). The BMMSCs of Zmpste24-/- mice showed a decrease expression of Cyclin D1, CDK1, CDK4, CDK6(p<0.05), while the expression level of P16, P21, P53 was significantly higher than wild-type mice(p<0.05).3. The BMMSCs of Zmpste24-/- mice exhibited an increase of TRPC1、TRPM4、TRPM8、TRPV1、TRPV2、TRPV3、TRPV5 channels expression compared with wild-type mice(P<0.05). Cultured by serum-free and 500μmol/L H2O2 medium, BMMSCs of wild-type mice showed an elevation of TRPM8 expression.4. The BMMSCs of Zmpste24-/- mice had a higher level of MDA expression,however the SOD2, CAT expression was lower(P<0.05). After inhibition TRPM8 with2-APB, the cell proportion of Zmpste24-/- mice in G1 phase was decreased, while G2+M phase cells increased(p<0.05); SOD2, CAT expression level elevated, the expression level of MDA was significantly lower(p<0.05).[Conclusions]1. Zmpste24-/- mice BMMSCs appeared characteristics of senescent cells, including a large, flat shape; poor, decreased proliferative capacity, high level of apoptosis, fewer cell numbers of mitosis and colony-forming units; lower expression of Cyclin D1,CDK1, CDK4, CDK6,SOD2, CAT; higher expression of P16, P21, P53, MDA.2. Zmpste24-/- mice BMMSCs exhibited elevated expression of TRPM8, which might be associated with apoptosis induced by H2O2.3. The inhibition of TRPM8 expression in Zmpste24-/- mice BMMSCs improved cell proliferation; The level of cellular oxidative stress was reduced, while antioxidant capacity enhanced.