Experimental Study On Treatment Of Parkinson's Disease Using TBA Combined With Bone Marrow Mesenchymal Stem Cells

Parkinson's disease is a common degenerative disease of central nervous system in mid-aged and aged people, PD pathogenesis has not yet fully understood, but a large number of studies confirmed that inflammation reaction in neurodege- nerative diseases played an important role. The most commonly drugs for treatment of Parkinson's disease are dopamine receptor agonists such as L-dopa, but these drugs can not cure the disease, and a series of serious complications will appear with the length of medication time. Therefore, it becomes a hotspot to search for minimum side effects, safe and effective drug ingredients. As a common component of traditional Chinese medicine, 3,4,5 - trihydroxy benzoic acid ( TBA ) extensively exists in Humifusae, gall, grapes, tea and lemon plants, with stopping bleeding, anti-inflammatory, antioxidant, anti-mutation, and other biological activity [5-8]. It's a polyphenolic compound which is widespread in nature [9-11]. But its effect on the treatment of PD has not been reported.Cell transplantation therapy is one of the hotspots of the treatment of PD. Because bone marrow mesenchymal stem cells have some unique biological characteristics, they have become an ideal seed cells for the treatment of neurological diseases. Currently a large number of studies demonstrated that after bone marrow mesenchymal stem cells were transplanted into the brain of PD model animals, abnormal behavior of the animals was improved, and it was determined that bone marrow mesenchymal stem cells could protect, repair and promote the growth of DA neurons by pathologic examination. But the only cell transplantation treatment or drug therapy has a certain shortcoming, in order to achieve better effects, this is the first time to use TBA combined with bone marrow mesenchymal stem cells to treat PD.In this study, the purpose is to discuss therapy of PD using TBA combined with bone marrow mesenchymal stem cells, provide experimental evidence and theoretical basis for searching for high efficient, low toxicity new drug and develop a new drug therapy for PD. First, the anti-inflammatory mechanisms of TBA were discussed, with classical macrophage cells, inflammatory cells, as an entry point. Macrophages were activated by classic cell activator lipopolysaccharide (LPS), the regulation of TBA on inflammatory cytokine production was analyzed. The effect of TBA on the expression of activation NF-κB, and IkB-а. were performed using the methods of MTT, double antibody sandwich enzyme linked immunosorbent assay, EMSA, Western blot and the others. The results showed that TBA inhibited the NF-κB activation through inducing the expression of IkB-а., blocked the synthesis of inflammatory cytokines regulated by NF-κB, further eliminated the accumulation of inflammatory factors in the body, and finally blocked the inflammatory response.In order to determine the protective effect of TBA on the DA neurons, in vitro PC12 cells as study objects, the protective effect of TBA on neurons against inflammatory effect was discussed by determining the contents of LDH, ON, NOS, apoptosis rate and expression of inflammatory cytokines. The results showed that TBA can antagonized PC12 cell injury cause by LPS, 100.0-25μg/mL TBA could improve the cytotoxicity of LPS at different degree, reduce the release of inflammatory cytokines, decreased cell apoptosis rate. The behavior changes of rats and tyrosine hydroxylase expression were performed using the behavioral test, RT-PCR technique in PD model rats induced by LPS after TBA was administered to PD model rats. The results showed that the abnormal behavior of PD rats could be improved at different degree by TBA (0.50g/kg, 0.25 g / kg and 0.13 g / kg ) oral administration, and the treatment of Parkinson's disease by TBA was dose-dependent relationship in a certain dose range. TH mRNA transcription level increased induced by TBA by real-time RT-PCR in brain.The characteristics of the neurobiology of BMSCs were detected by mmunocy- tochemistry and RT-PCR in vitro. The results showed that neural-like cells appeared after BMSCs were cultured 8th generation. Immunocytochemistry staining showed that Nestin, Vimentin and PSA-NCAM positive cells existed in BMSCs. The results of RT-PCR showed target band of GFAP and Nestin. Immunocytochemistry results of Nestin protein expression were consistent with PCR results, which confirmed BMSCs possessed nerve cells characteristics. In order to further analyze BMSCs how to protect dopaminergic neurons under specific micro-environment and the secretion quantities of GDNF under different micro-environmental conditions, ELISA and immunocytochemistry methods were performed. The results showed that DA neurons avoid the damage from neurotoxin, DA neurons delayed death was inhibited by upregulating the expression of GDNF. Our Long-term research confirmed that BMSCs could move to injured brain tissue, donor's BMSCs homed in the recipient's brain tissue, proliferated and differentiated into neuron, further to treat the disease. Based on the previous research results, this subject emphasized on the effect of uninduced BMSCs transplantation on the TH mRNA expression in brain. At 6 week after BMSCs were transplanted into PD model rats, total RNA was extracted, TH mRNA transcript level were detected using real-time PCR. The results showed that TH mRNA transcript level significantly increased in transplantation group compared with PD model group(P<0.05), suggesting that BMSCs improved the abnormal behavior of PD model rats by increasing the TH mRNA level in brain and relieving the PD syndrome.Currently a large number of studies confirmed BMSCs transplantation has an treatment effect on PD disease, but there are still some shortcomings. Therefore, this study will apply TBA combined with BMSCs to treat rat model of PD disease using the methods of behavior change detection, RT-PCR, immunohistochemistry. The results showed that the treatment effect of TBA combined with BMSCs was better than each alone, TH mRNA transcript level was highest in all the groups, with regular striaturn, the formation of distinct network structure, in which TH–positive cells was dense with strong immune activation and dark color by pathological observation. Conclusion:1 It may be a main pathway that TBA upregulated the inhibitory protein (IκB-а.) expression, antagonized nuclear factor (NF-κB) activation, thereby reduced the expression of inflammatory cytokines.2 Culture research in vitro confirmed that TBA antagonized injury of PC 12 cells caused by inflammatory factors by decreasing cell apoptosis rate, reducing the leakage of LDH, NO release and NOS formation of dopaminergic neurons in PD model prepared by PC 12 cells.3. BMSCs cultured in vitro have a vigorous proliferation and passage potency containing some neural stem / progenitor cells. BMSCs may protect DA neuron by upregulate GDNF expression.4. After uninduced undifferentiated BMSCs in vitro were transplanted into the brains of PD model rats, the abnormal behaviors of PD rats were improved, TH mRNA level of brain tissue were increased, and PD syndrome was relieved.5 Both TBA and BMSCs transplantation could therapy the PD model rats, but the combination effect of both was significantly better than that of each alone.