| Parkinson’s disease(PD)is the second most common aging neurodegenerative disease.Pathological changes are characterized by the accumulation of α-synuclein in dopamine(DA)neurons in the form of lewy body inclusions.Autophagy is an evolutionarily conserved turnover process in eukaryotic cells,which plays an important role in removing long-lived proteins,aggregation proteins or organelles with dysfunction.NLRP3 inflammasome-mediated neuroinflammation is specifically correlation with neurological diseases.The involvement of central nervous system inflammation and autophagy dysfunction in the pathologic process of PD has been reported by a series of studies.However,the mechanisms driving these phenomena are still unknown.Biochanin A(Bioch A)is a kind of isoflavone estrogen extracted from natural plants,which has many biological functions,such as anti-inflammatory,regulating glucose and lipid metabolism,preventing cancer and neuroprotection.Previous experiments have proved that Bioch A has a good protective effect on neurons,but its mechanism is still unclear.Objective: To clarity the changes of autophagy related proteins and NLRP3 inflammasome signaling in LPS-induced PD model mouse and the neuroprotective effect of Bioch A on LPS-induced PD model mouse.Methods:1.Forty male C57BL/6 mice were randomly divided into 4 groups: sham group,LPS1 d group,LPS 3d and LPS 7d groups.Then,normal saline or LPS solution was injected into the substantia nigra of mice by brain stereotaxic injection instrument.Behavioral detection of the sham group and LPS 7d group began on the seventh days of administration.After completing the behavioral tests,killed the mice and collected its brain tissue were taken for a series of experiments.The damage of DA neurons and the level of Fox O3a、LC3B proteins by immunofluorescence to detect.The level of SIRT1,p-Fox O3a/Fox O3 a,LC3BⅡ/LC3BⅠ,Beclin1,P62 and the expression of CTSB,NLRP3,Caspase-1 and IL-1β by western blot to detect.2.Fifty male C57BL/6 mice were randomly divided into 5 groups: sham group,LPS group,LPS+Bioch A(16 mg/kg),LPS+Bioch A(32 mg/kg),LPS+Bioch A(64 mg/kg).Then,normal saline or LPS solution was injected into the substantia nigra of mice by brain stereotaxic injection instrument.Bioch A was dissolved in distilled water and administered intragastrically(p.o 0.1 m L/10 g/day)to different groups of mice at three different concentrations of 16,32,64 mg/kg.After LPS injection,the mice were continuously treated with Bioch A for 7 days.The sham group and LPS group received equivalent volumes of solvent.Behavioral tests began on the 7th day after surgery.After completing the behavioral tests,killed the mice and collected its brain tissue were taken for a series of experiments.The damage of DA neurons and the level of Fox O3a、LC3B proteins by immunofluorescence to detect.The level of SIRT1,p-Fox O3a/Fox O3 a,LC3BⅡ/LC3BⅠ,Beclin1,P62 and the expression of CTSB,NLRP3,Caspase-1 and IL-1β by Western blot to detect.Result:1.The changes of autophagy-related proteins and NLRP3 inflammasome signaling in LPS-induced PD model mouse(1)Behavioral changes in LPS-induced PD model mouse: the open-field test data showed that compared with the sham group,the moving distance(m),mean speed (m/s),line crossings and the number of stand up showed a significant downward trend in the LPS 7d group.Moreover,the mice grip experiment also showed that compared with the sham group,LPS treatment also reduced the mice grip strength.Behavioral results showed that LPS-induced significantly weakened the spontaneous activity and muscle contraction ability of mice.(2)The damage of DA neurons in the substantia nigra of LPS-induced PD model mouse: the number of DA neurons in the sham group were large and full,while the number of DA neurons in the model group decreased gradually with the time extension of modeling time and the morphology changed from full to dry.(3)Autophagy of microglia and DA neurons in LPS-induced PD model mouse:the co-localization of Iba1 and TH with LC3 B was observed.Compared with the sham group,the number of TH positive neurons decreased and the LC3 B protein increased in the model group,and the co-localization of the LC3 B probe could be observed in the TH positive neurons.Compared with the sham group,although the activation of microglia increased significantly in the model group,the co-localization of Iba1 and LC3 B was difficult to observe in animal models because of the limitations of experimental means.(4)The expression of the Fox O3 a,Sirt1 and p-Fox O3a/Fox O3 a in the substantia nigra of LPS-induced PD model mouse: Immunofluorescence results showed that LPS treatment increased expression of Fox O3 a protein compared with the sham group.Western Blot results showed that compared with sham group,LPS treatment increased SIRT1 protein expression,but significantly reduced p-Fox O3a/Fox O3 a levels.(5)The expression of autophagy-related protein LC3 B,Beclin1 and P62 in the substantia nigra of LPS-induced PD model mouse: Immunofluorescence results showed that LPS treatment increased the expression of LC3 B with the time of modeling compared with the sham group.Meanwhile,Western Blot results showed that compared with the sham group,LPS treatment significantly increased the level of LC3BⅡ/LC3BⅠ,Beclin1 and P62 protein.(6)The expression of CTSB,NLRP3,Caspase-1 and IL-1β in the substantia nigra of LPS-induced PD model mouse: Western Blot results suggested that the expression of CTSB,NLRP3,Caspase-1 and IL-1β were increased to varying degrees in the substantia nigra of LPS model mouse compared with the sham group.2.The protective effect and mechanism of Bioch A on LPS-induced PD model mouse(1)The effect of Bioch A on the behavior of LPS-induced PD model mouse:compared with the LPS group,the moving distance(m),mean speed(m/s),line crossings and the number of stand up were increased in different degrees after Bioch A treatment.Moreover,the mice grip experiment also showed that compared with the LPS group,Bioch A treatment significantly increased the mice grip strength.The above behavioral results suggested that Bioch A treatment significantly improve behavioral dysfunction in LPS-induced PD model mouse.(2)The effects of Bioch A on DA neurons in the substantia nigra of LPS-induced PD model mouse: Immunofluorescence results showed that Bioch A treatment significantly reduced DA neurons damage compared with the LPS group.(3)Bioch A effects on the level of Fox O3 a,Sirt1 and p-Fox O3a/Fox O3 a in substantia nigra of LPS-induced PD model mouse: immunofluorescence results showed that different doses of Bioch A treatment decreased Fox O3 a protein expression compared with the LPS group.Western Blot results showed that compared with the LPS group,Bioch A treatment decreased the expression of SIRT1 protein and the level of p-Fox O3a/Fox O3 a was increased.(4)The effect of Bioch A on the expression of LC3 B,Beclin1 and P62 in substantia nigra of LPS-induced PD model mouse: immunofluorescence results suggested that compared with the LPS group,Bioch A treatment decreased the level of LC3B.Meanwhile,Western Blot results suggested that Bioch A treatment significantly decreased the level of LC3BⅡ/LC3BⅠ,Beclin1 and P62 protein compared with the LPS group.(5)The effect of Bioch A on the expression of CTSB,NLRP3,Caspase-1 and IL-1β in the substantia nigra of LPS-induced PD model mouse: Western Blot results showed that compared with the LPS group,the expression of CTSB,NLRP3,Caspase-1 and IL-1β of Bioch A group were decreased.Conclusion:1.LPS-induced DA neuron damage,which may be related to the activation of NLRP3 inflammasome by CTSB released from autophagy lysosomal dysfunction.2.Bioch A may relieve the dysfunction of autophagy lysosomes,reduce CTSB released by lysosomal damage and the activation of NLRP3 inflammasomes,thereby exerting the protective effect of DA neurons. |
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