The Effect And Mechanism Of Glaucocalyxin B On Parkinson' Disease Model Induced By LPS

Objective: Parkinson's disease(PD)is a serious neurological degenerative disease that is closely related to the patient's age.The main clinical features of PD are severe dyskinesia and other non-motor-related symptoms such as cognitive impairment.The loss of dopaminergic(DA)neurons in the brain and the formation of Lewy bodies are major changes and important features of PD histopathology.At present,the cause and pathogenesis of PD induction is still not fully proved,there are a variety of mechanisms can lead to neuronal degeneration is believed to be induced Parkinson's disease.Studies have shown that oxidative stress and neurological damage are the main pathologic mechanisms of Parkinson's disease and play a key role in the loss of DA neurons.The presence of microglia in the nervous system is activated,the release of a large number of oxygen free radicals and cell inflammatory factors caused by the brain nerve inflammation and peroxidation damage,causing the brain in the substantia nigra DA neuronal degeneration and apoptosis,and ultimately lead to PD happened.Therefore,finding drugs with anti-neuroinflammatory and oxidative stress has become an important direction for the alleviation and treatment of Parkinson's disease.Glaucocalyxin B(GLB)is a kind of diterpene compound extracted and purified from Rabdosia japonica(R.japonica)(Burm.f.)var.glaucocalyx(Maxim.)Hara.It has been reported that have anti-inflammatory and anti-oxidative stress and neuroprotective effects on microglia cells.Therefore,it may be a potential therapeutic drug for Parkinson's disease.This study focused on elucidating the therapeutic effect of GLB on Parkinson's disease in vivo and in vitro and to explore the relevant molecular mechanisms to provide a theoretical basis for application of GLB in clinical treatment of Parkinson 's disease.Methods : 1.Established the rat model of PD induced by injection of LPS.2.Behavioral test of Parkinson 's disease model rats.3.Immunohistochemical staining was used to detect the expressions of ?-Syn and cell-specific markers TH,GFAP and CD11b in PD model rats brain tissue.4.The expression of TNF-?,IL-1?,IL-6 m RNA in rats brain was detected by Real-time PCR.5.Commercial kits was used to detect the effect of GLB on NO,ROS,MPO and SOD in PD model rats brain.6.The expression of i NOS,Bax,Bcl-2,cleaved caspase-3,NF-?B,Nrf2/HO-1 signaling pathway protein in the rat brain was detected by western blot.7.Cultured BV2,PC12 cells.8.MTT assay was used to detect the effects of LPS and GLB on the proliferation of BV2 cells.9.Real-time PCR was used to detect the effect of GLB on the expression of TNF-?,IL-1?,IL-6 m RNA in LPS-induced BV2 cells.10.Commercial kits was used to detect the effect of GLB on LPS-induced NO,ROS,MPO and SOD in BV2 cells.11.Western blot was used to detect the expression of NF-?B and Nrf2/HO-1 signaling pathway proteins in LPS-induced BV2 cells.12.MTT assay was used to detect the effect of LPS-induced BV2 cell supernatant on PC12 cell viability.13.Annexin V-FITC cell apoptosis detection kit was used to detect the apoptosis of PC12 cells.Results:1.Animal behavior test to detect the effect of GLB on PD rats.The results showed that after GLB treatment,the Stepping Test,Whisker Test and Cylinder Test data of rats were significantly higher than those of PD group.These results suggest that GLB can alleviate the disturbance of walking,climbing and sensory in PD model rats.2.Expression of ?-Syn and cell-specific markers TH,GFAP and CD11 b in brain tissues were detected by immunohistochemistry.The results showed that after GLB treatment,Compared with PD group,expression of ?-Syn decreased,the number of TH positive cells increased,the number of GFAP and CD11 b positive cells decreased significantly.These results suggested that GLB could decrease expression of ?-Syn,inhibit the decrease of dopamine,and the increase of astrocytes and microglia astrocytes and microglia.3.Real-time PCR was used to detect the expression of inflammatory factors TNF-?,IL-1 and IL-6 in brain tissue of PD model rats.The results showed that GLB could significantly inhibit the expression of TNF-?,IL-1? and IL-6 in brain tissue of PD model rats.These results suggest that GLB can inhibit the inflammatory reaction in the brain of PD model rats.4.The results of NO and i NOS in brain tissue of rats showed that GLB could significantly reduce the content of NO,and significantly inhibited the expression of i NOS protein in brain tissue of PD model rats.5.Westernblot was used to detect the expression of cytosolic NF-?B,nuclear NF-?B,nuclear Nrf2 and whole-cells HO-1 level in the brain tissue of PD model rats.The experimental results show that compared with the Sham group,the cytosolic NF-?B in the brain tissue of PD model rats was significantly reduced,the nuclear NF-?B and Nrf2 were significantly increased,and the expression of HO-1 in whole-cells was significantly increased.GLB could significantly increased the expression of NF-?B in the cytoplasm of PD model rats,reduced the expression of NF-?B in the nucleus,and further promote the expression of Nrf2 and HO-1.These results suggest that GLB can inhibit the activation of NF-?B,activate the Nrf2/HO-1 signaling pathway,and alleviate the occurrence of dyskinesia,inflammatory response,oxidative stress and apoptosis in PD rats.6.Expression of inflammatory factors in BV2 cells after LPS induction by Real-time PCR.The results showed that the expression of TNF-?,IL-1? and IL-6 were significantly up-regulated in the cells after LPS induction,and after GLB treatment the levels of inflammatory factors were significantly decreased.These results suggest that GLB can inhibit the inflammatory reaction induced by LPS in BV2 cells.7.Commercial kits was used to detect the effect of GLB on on LPS-induced NO,ROS,SOD and MPO in BV2 cells.The results showed that the NO and ROS contents in the cells of BV2 cells were significantly increased,MPO activity was significantly increased and SOD activity was significantly decreased after LPS induction.After treatment with GLB,the content of NO and ROS decreased significantly,the activity of MPO was decreased and the activity of SOD was significantly increased.These results suggest that GLB can inhibit the oxidative stress induced by LPS in BV2 cells.8.Western blot was used to detect of the expression of NF-?B and Nrf2/HO-1 pathway related factors in BV2 cells.The data showed that the expression of cytosolic NF-?B was significantly decreased,the expression of nucleus NF-?B and Nrf2 was significantly increased and the expression of HO-1 in whole-cells was significantly increased after BV2 cells were induced by LPS.After treatment with GLB,the expression of cytosolic NF-?B was significantly increased,the expression of nuclear NF-?B was significantly reduced,and the expression of Nrf2 and HO-1 increased.These results suggest that GLB can alleviate LPS induced inflammatory and oxidative stress in BV2 cells by inhibiting the activation of NF-?B and activating the Nrf2/HO-1 pathway.9.The cell viability of PC12 cells was significantly decreased and the apoptotic rate of PC12 cells was significantly increased after 48 hours of incubation with LPS-induced BV2 cell supernatant.After treatment with GLB,the activity of PC12 cells was significantly increased and the apoptosis was significantly reduced.These results suggest that GLB can decrease the cytotoxicity of microglia in vitro.Conclusion:1.GLB could alleviate the disturbance of walking,climbing and sensory in PD model rats.2.GLB could reduce the expression of ?-syn,inhibit the reduction of TH-positive cells and the increase of GFAP and CD11b-positive cells in PD model rats.3.GLB can inhibit the expression of inflammatory cytokines and oxidative stress in PD rat models.4.GLB can reduce the apoptosis of brain tissue in PD model rats.5.GLB could inhibit the activation of NF-?B and activate the Nrf2/HO-1 pathway to alleviate the dyskinesia,inflammatory response,oxidative stress and apoptosis in PD rats.6.LPS can induce inflammation and oxidative stress in BV2 cells.7.GLB could down-regulate the expression of inflammatory cytokines in LPS-induced BV2 cells.8.GLB can inhibit the oxidative stress in LPS-induced BV2 cells.9.GLB attenuates LPS-induced inflammatory and oxidative stress in BV2 cells by inhibiting NF-?B activation and activating the Nrf2/HO-1 pathway.10.GLB can reduce the cytotoxicity of LPS-induced microglia in vitro.