Neuroprotective Effect Of Salvianolic Acid B On Parkinson’s Disease Model Mice Through Gut-brain Axis Pathway

Objective: The aims of this study is to establish mice model of Parkinson’s disease（PD）induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine（MPTP）,and then investigate the neuroprotective effect and mechanism of salvianolic acid B（salb）on MPTP induced PD model mice through gut-brain axis pathway.Methods: C57 BL / 6 male mice were intraperitoneally injected with MPTP to establish PD mice model and then treated with SalB intraperitoneally.48 healthy adult C57 BL / 6 male mice were randomly divided into four groups: normal（Control）,SalB control（SalB）,PD model（MPTP）,SalB treatment（MPTP + SalB）.The motor function was evaluated by using the pole climbing test and gait test;The intestinal function was assessed by measuring the excretion time of the first black stool,fecal water content and colon length;The number of tyrosine hydroxylase（TH）positive cells in substantia nigra,TH positive cells in colon and the expression level of Toll like receptor 4（TLR4）in colon were analyzed by immunohistochemistry;The pathological changes of colonic mucosa in mice were observed by hematoxylin eosin（HE）staining;The levels of Calprotectin（CP）,Tumor necrosis factor（TNF-α）and Interleukin-1β（IL-1β）in mice colon were measured by enzyme-linked immunosorbent assay（ELISA）;Western blot was used to detect the level of TH in mice midbrain,and also to detect the protein level of TH,tight junction protein（ZO-1）,TLR4,myeloid differentiation factor 88（My D88）and nuclear factor-κB p65（Nuclear NF-κB p65）and phosphorylated p-NF-κB p65 which expressed in colon.Results :1.The results of motor function and gastrointestinal function test showed that compared with the Control group,the climbing time and T-turn time of MPTP group mice were prolonged（P < 0.05）,while the stride of unilateral forepaw was reduced（P < 0.05）,and the distance between hindpaws was widened（P < 0.05）;Compared with MPTP group,the climbing time and T-turn time of MPTP + SalB group were significantly shorter（P < 0.05）,meanwhile the stride of unilateral forepaw increased（P < 0.05）,and the distance between hindpaws decreased（P < 0.05）;Compared with the Control group,the first black stool excretion time of mice in MPTP group was prolonged（P < 0.01）,and the fecal water content decreased（P < 0.01）,the length of colon was shortened（P < 0.01）;Compared with MPTP group,the first black stool excretion time of MPTP + SalB group was shorter（P < 0.01）,fecal water content increased（P <0.01）,the length of colon increased（P < 0.01）.2.Immunohistochemical results demonstrated that compared with the Control group,the number of TH positive cells in substantia nigra in MPTP group decreased（P < 0.05）,and the number of TH positive cells in substantia nigra in MPTP + SalB group increased（P <0.05）;Western blot showed that the levels of TH protein in midbrain and colon in MPTP group were significantly lower than those in control group（P < 0.05）,and the levels of TH protein in midbrain and colon in MPTP + SalB group were higher than those in MPTP group（P < 0.05）.3.The results of HE staining indicated that compared with the Control group,the pathological injury score of colonic mucosa in MPTP group increased（P < 0.05）,Compared with MPTP group,the pathological damage score of colonic mucosa in MPTP + SalB group decreased（P < 0.05）.4.The results of ELISA suggested that compared with the Control group,CP,TNF-α and IL-1β in the colon tissue of MPTP group were increased（P < 0.05）;Compared with MPTP group,the level of CP,TNF-α and IL-1β in colon tissue of MPTP + SalB were decreased（P < 0.05）.5.The results of Western blot demonstrated that the expression level of tight junction protein（ZO-1）in colonic tissue of MPTP group decreased compared with Control group（P <0.05）;Compared with MPTP group,the expression level of tight junction protein（ZO-1）in colon tissue of MPTP + SalB group was significantly restored（P < 0.05）.6.The results of immunohistochemistry and Western blot suggested that compared with the control group,the expression levels of TLR4,My D88,Nuclear NF-κB p65 and p-NF-κB p65 which in the inflammatory signal pathway（TLR4 / My D88 / NF-κB）of colonic tissue in MPTP were increased（P < 0.05）;After SalB treatment,compared with MPTP group,the expression levels of TLR4,My D88,Nuclear NF-κB p65 and p-NF-κB p65 in colon tissue of MPTP + SalB group were decreased（P < 0.05）.Conclusions: 1.SalB can alleviate the degeneration and loss of dopaminergic neurons in substantia nigra and colon of MPTP induced PD model mice,improve the motor dysfunction and gastrointestinal dysfunction of MPTP induced PD model mice,and has dual protective effects on nerves and intestines of MPTP induced PD model mice;2.SalB may improve the neurological dysfunction of MPTP induced PD model mice by alleviating intestinal mucosal pathological injury,maintaining the integrity of intestinal mucosal barrier and alleviating intestinal inflammatory response;3.SalB improves the neurological dysfunction of MPTP induced PD model mice through gut-brain axis pathway,which may be related to the inhibition of colonic TLR4 / My D88 / NF-κB signal pathway.