Study On Pharmacodynamic Substances And Mechanisms Of Prevention And Treatment Of With Mouse Parkinson's Disease By Inhibiting Microglial Activation

Objective:Parkinson's disease(PD)is the second largest neurodegenerative disease in the world.Many clinical symptoms such as resting tremor,muscle stiffness,and dyskinesia appear in the clinic,which reduces the quality of life of patients.Current studies have found that the pathogenesis is mainly due to the loss of dopaminergic neurons in the midbrain,resulting in reduced dopamine secretion in the striatum and exercise Obstructive disease.Western medicine treatment of PD is mostly dopamine supplementation and prolonged dopamine action time,but there are problems such as large side effects.From the perspective of traditional Chinese medicine,PD is mostly due to liver and kidney deficiency,so it is believed that the purpose of treatment can be achieved by supplementing liver and kidney.Du Zhong formula,including Eucommia ulmoides,Dendrobium,Dihuang,Dried Ginger,can nourish liver and kidney,strengthen bones and muscles,and also have effects of nourishing yin and dispersing cold.This study intends to further clarify the pharmacodynamic effect of Du Zhong formula on PD on the basis of the previous period,and analyze its pharmacodynamic material basis.In view of the important role of microglia activation in the pathogenesis of PD,this study focuses on its intracerebral components,from Inhibit the activation of microglia to explore the protective mechanism of Duzhongfang's brain,and provide experimental basis for the clinical treatment of PD and the development of new drugs.Method:In this experiment,a mouse model of PD was established by the combination of probenecid intraperitoneal injection and MPTP subcutaneous injection.The experimental animals were randomly divided into normal group,model group,low dose group(10g/kg),high dose group(20g/kg),high dose group(40g/kg).The extracts of Du Zhong formula were given by intragastric administration.MPTP was injected once every 3.5 days,10 consecutive injections.After the model was finished,the drug was continued for 1 week.1.Pharmacodynamic study of Du Zhong formula in preventing and treating experimental mouse with PDFirst,behavioral experiments such as climbing rods,rotating rods,and suspension were used to test the mice's motor coordination ability at the middle and end stages of administration respectively.The HPLC-electrochemical detection method was used to detect striatum dopamine and its levels in mice Metabolites homovanillic acid and 3,4-dihydroxyphenylacetic acid;immunohistochemical staining was used to detect the number of tyrosine hydroxylase positive cells in each group of mice,and to evaluate the therapeutic effect of Du Zhong formula on Parkinson's disease.2.Study on the transitional components in Du Zhong formulaBased on the pharmacodynamic effects of Du Zhong formula,UPLC-Q-MS-TOF was used to analyze the cerebrospinal fluid,blood,urine,and feces of rats administered orally with Du Zhong formula.3.Study on the mechanism of Du Zhong formula's brain component in preventing and treating PDOn the basis of clarifying the eucommia components in the body,this study focuses on the in vitro pharmacological experiments on the intracerebral components.The analyzed intracerebral components act on neurons and microglia in vitro to explore their neuroprotective effects and mechanisms.The nerve cells SH-SY5Y were stimulated with MPP~+,and 6-Gingerol and 6-Shogaol were intervened at different concentrations to detect their cytotoxicity and cell viability to evaluate the efficacy.Culture BV2 microglia in vitro.LPS was used to stimulate microglial BV2,and different concentrations of and 6-Gingerol and 6-Shogaol were administered at the same time to detect cell viability and NO release;RT-PCR was used to detect IL-6,IL-1?,TNF-?,TGF-?m RNA expression levels;ELISA method was used to detect the contents of IL-6 and TNF-?in the supernatant of BV2 cells to evaluate the inhibition of LPS-induced BV2 inflammation by 6-Gingerol and 6-Shogaol effect.Use protein interaction technology to discover the key targets of inflammatory activation of microglia and investigate the potential role of active ingredients.Result:1.Effect of Du Zhong formula on MPTP-induced PD miceAt the middle stage of modeling and after the modeling was finished,the behavioral indexes of the mice in each group were measured.The results showed that:compared with the normal group,the model group had longer rod climbing time,increased stick rotation time,decreased hanging score,and had statistical significance.The results suggest that the PD model was successful.Compared with the model group,the rod climbing time of the high-dose(40 g/kg)group of Du Zhong formula in the middle period of the model was significantly reduced(P<0.05).The behavioral climbing rods,rotating rods,and suspensions of the other groups had an improvement trend compared with the model group,and the results suggest that Du Zhong formula has a certain effect on improving PD behaviors.High-performance liquid chromatography-electrochemical detection results showed that:Compared with the normal group,the content of DA,HVA and DOPAC in the striatum of the model group were significantly reduced(P<0.001).Compared with the model group,the DA and HVA of the Du Zhong formula group There is a tendency to increase(P>0.05),the content of DOPAC increases significantly(P<0.001),the results suggest that Du Zhong formula has a certain effect of increasing the level of DA and its metabolites in PD mice.The results of immunohistochemical staining showed that compared with the normal group,the number of TH positive cells in the model group was significantly reduced(P<0.05),compared with the model group,the number of TH positive cells in the middle dose group of Du Zhong formula was significantly increased(P<0.05).It is suggested that Du Zhong formula can protect DA neurons.2.Identify the ingredients that Du Zhong formula enters the bodyDu Zhong formula was administered orally to normal rats,and plasma,cerebrospinal fluid,urine,and feces of rats were collected.Analysis by liquid chromatography-mass spectrometry revealed 52 prototype products,of which 5components were found in cerebrospinal fluid:6-Gingerol and 6-Shogaol,Dendrobone,Dendrobiumane A and Dendrobiumane A.3.Into the brain component inhibits LPS-induced microglial inflammation and its mechanismMPP~+was used to stimulate nerve cells SH-SY5Y,at the same time,the components of 6-Gingerol and 6-Shogaol that entering the brain were used to intervene with the cells.The results showed that 6-Gingerol and 6-Shogaol could not improve SH-SY5Y cytotoxicity caused by MPP~+And damage to cell viability.LPS was used to activate microglial BV2,and 6-Gingerol and 6-Shogaol were intervened.The results showed that the NO content in the model group was significantly higher than that in the normal group(P<0.001).Compared with 5?M,10?M 6-Gingerol and 2.5?M,5?M,10?M 6-Shogaol NO production was significantly reduced,and statistically significant,the 2.5?M 6-Gingerol group had lower NO content than the model group but not statistically significant.The RT-PCR results showed that compared with the normal group,the expression levels of the pro-inflammatory factors IL-6,IL-1?,and TNF-?m RNA in the model group were increased,while the expression levels of the anti-inflammatory factor TGF-?m RNA were significantly reduced.Compared with the model group,2.5?M,5?M,and 10?M 6-Gingerol and 6-Shogaol significantly reduced the expression levels of IL-6 and IL-1?m RNA after intervention.;The expression levels of TNF-?m RNA in 2.5?M,5?M,10?M 6-Shogaol and 5?M and 10?M 6-Gingerol groups were significantly reduced;the expression level of TGF-?m RNA in 10?M 6-Shogaol group was significantly increased,and there was statistical significance.ELISA results showed that the levels of IL-6 and TNF-?in the supernatant of the model group were significantly increased(P<0.001).Compared with the model group,2.5?M,5?M,10?M 6-Shogaol and 10?M 6-Gingero The content of IL-6 in the cell supernatant of the group was significantly reduced,and the content of TNF-?in the cell supernatant of the 6-Gingero group at 5?M and 10?M was significantly reduced.Compared with the model group,the content of IL-6 in the supernatant of the cells of the 2.5?M,5?M,10?M 6-Shogaol and 10?M 6-Gingero groups were significantly reduced,and the TNF-?content was significantly reduced.The results of protein interaction showed that Pomc played an intermediate role in the process of inflammatory activation of microglia.It was further verified that 6-Shogaol could inhibit the expression of Pomc m RNA in microglia.Conclusion:It is further confirmed that Du Zhong formula can prevent and cure PD in mice.Among the active ingredients in the brain,6-Gingerol and 6-Shogaol may inhibit the inflammatory reaction in the brain by inhibiting the Pomc signal of microglia,which may be one of the ways to prevent and treat PD.