The Mechanism Of Zuogui Pill Ameliorative Proliferation Of Aging Rats Mesenchymal Stem Cells

ObjectiveTo especially explore the mechanism of Zuogui pill on the proliferation of aging rats mesenchymal stem cells. According to the hypothesis of "Kidney essence deficiency leads to aging theory" and "stem cells have the same properties as congenital essence and is a existence form in the cellular level", combining with previous studies of Zuogui pill on the proliferation of aging rats mesenchymal stem cells, deeply exploring molecular mechanism of Zuogui pill which regulars and controls the proliferation of aging rats mesenchymal stem cells, further defining scientificity of hypothesis, furnishing more adequate scientific basis for the "essence" of the stem cell theory, this study will get improvement to the level of molecular, and provide strong experimental evidence for assisting clinical stem cell transplantation for treatment of diseases by Chinese medicine.Methods1. Isolation, culture, purification and identification of MSCMSC was isolated and purified by whole bone marrow adherent method:MSC was identified by morphologie charaeteristies, surface antigens detected by immunofluorescence.2. The mechanism of Zuogui pill on proliferation of aging rats MSC2.1 The whole rats except the normal group were intraperitoneally injected with D-galactosidase to make the model of aging rats. MSC was isolated and purified by whole bone marrow adherent method. Then the morphology of mesenchymal stem cells was observed by inverted microscope.2.2 Cell cultured to P3 generation, different mRNA expressive of cytokines, which are BMP-2、G-CSF、GM-CSF、IL-6、IL-7、LIF、OSF、SCF、SDF-1、p21、p16、 p53、c-Myc were detected by RT-PCR. Discrepant expression of genes were detected by Real time-PCR. Discrepant expressive of proteins were detected by Western blot.2.3 Affymetrix analysis showed about 89 mRNAs in Wnt/β-Catenin signaling pathway significantly altered in expression arrays. Wnt3a and Kremen2 protein expression detected by Western blot.3. A preliminary research on effect and mechanism of Zuogui pill on aging mesenchymal stem cells proliferation in vitro.3.1 MSC were isolated and cultured by method of whole bone marrow adherent. P3 MSC induced to senescent by D-galactose. Zuogui pill groups were treated with a certain concentration culture of a decoction of zuogui pill intervention training.3.2 We observed cellular form, growth curve, growth cycle and β-galactosidase staining senescent cells. And compared the MSC proliferation capacity of different groups3.3 Genes expression of different cell, such as BMP-2、G-CSF、GM-CSF、 IL-6、IL-7、LIF、OSF、SCF、SDF-1、p21、p16、p53、c-Myc, were detected by RT-PCR and Real-time PCR.Results1. Isolation, culture, purification and identification of MSCThe results of the surface antigens of MSC detected by immunofluorescence showed the positive expression of CD44 and the negative expression of CD45.2. The mechanism of Zuogui pill ameliorating the proliferation of MSC from aging rats.2.1 The growth curve of P3 MSC showed that:normal cells grew well fusiform cells swirling growth nucleus was round the rules; The model group grew slowly and had, large cells, irregular nucleus, cytoplasm visible particulate matter, and could see different levels of cell membrane rupture; The MSC of middle-dose Zuogui pill group is better that most of cells had regularly distributed nucleus with a small amount of particulate matter.2.2 RT-PCR results showed that SCFmRNA expression of the aging group was significantly lower than those of the normal group and the Zuogui pill group while p21 and SDF-1mRNA expression were significantly higher than those of the normal group and the Zuogui pill group. (P<0.01)2.3 Real time-PCR results showed that SCFmRNA expression of the aging group was obviously lower than those of the normal group and aging group (P <0.05) while p21mRNA expression of aging group was obviously higher than the normal group and aging group (P<0.05)2.4 Western blot results showed that SCF proteins expression of the aging group was significantly lower than the normal group (P<0.01) while p21 protein was higher (P<0.01);SCF proteins expression of the Zuogui pill group was obviously higher than aging group (P<0.05) while p21 protein was lower (P <0.05).2.5 About 89 genes in the Wnt/β-Catenin were screened. Compared with normal group Kremen1、Kremen2、Wnt4 and other 18 genes expression of aging group were up-regulated and Wnt2、Wnt3a and Wnt7a genes expression were down-regulated. Compared with the aging group, Wnt2、Wnt3a、Jun gene expression of the Zuogui pill were up-regulated and Kremen2、sFRP5、Wnt9b gene expression of the Zuogui pill group were down-regulated.2.6 Western blot results showed that Wnt3a proteins expression of the aging group was significantly lower than the normal group (P<0.01), Kremen2 protein expression of aging group was significantly higher than the normal group (P <0.01);Wnt3a proteins expression of the Zuogui pill group was obviously higher than aging group (P<0.05),Kremen2 protein expression of the Zuogui pill group was obviously lower than aging group (P<0.05).3. A preliminary research involved effect and mechanism of the Zuogui pill ameliorative proliferation of aging mesenchymal stem cells in vitro.3.1 The morphology of P3 MSC presented features of senescent cells in aging group, after Zuogui pill intervened, the morphology of MSC are similar with the normal MSC.3.2 The results of β-galactosidase staining showed that model group of aging cells had the highest rate while the middle group significantly reduced the positive rate of senescent cells (P<0.05)3.3 After aging cell model was established. the proliferation capacity of MSC in aging group was significantly lower than the normal and the Zuogui pill group (P<0.01)3.4 Flow cytometry showed that the cycle of cell proliferation. The most MSC of model group were stagnated in the early period of the DNA replication the almost whole number of cells of the Zuogui pill group ahead inter into the period of DNA rapid replication.3.5 Real-time PCR results showed that SCF mRNA expression of the aging group were obviously lower than the normal group and the middle group (P< 0.05) while p21 mRNA expression of aging group was obviously higher than the normal group and the middle group (P<0.05).Conclusions1. The stable and uniformal MSC could be obtained by method of whole bone marrow adherent.2. Zuogui pill can be significant delaying senescence of mesenchymal stem cells, Wnt/β-Catenin signaling pathway can be activated by inhibiting Kremen2 and promoting Wnt3a pathway, promoting the expression of SCF and inhibit the expression of p21, regulating the cycle of cell, so as to promote MSC proliferation of aging rats.3. The decoction of Zuogui pill can promote the proliferation of MSC of aging model in vitro, which may be related to expression of p21 and SCF.4. This study further on the level of the molecular and protein proved that the "stem cells have the same properties as congenital essence and is a existence form in the cellular level" hypothesis of science to rich "kidney essence" new substance of modern theory.