| Objective: To investigate the inhibitory mechanism of Dendrobium huoshanense polysaccharide(DHP)on the inflammatory damage of neurons in Parkinson’s disease(PD)model.Methods: 1-methyl-4-phenylpyridinium(MPP+)was induced in SH-SY5 Y cells and microglia(BV-2)cells to establish PD cell models,respectively.SH-SY5 Y cells were divided into blank group,model group and DHP group,and the survival rate of cells was measured by MTT colorimetric assay.The levels of lactate dehydrogenase(LDH),reactive oxygen species(ROS),malondialdehyde(MDA)and superoxide dismutase(SOD)were measured by colorimetric analysis.Microglia(BV-2)were divided into blank group,model group,DHP group and MCC950 group(positive control group),and enzyme-linked immunosorbent assay(ELISA)was applied to detect tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-18(IL-18).The expression of NLRP3,ASC,Caspase-1 and IL-1β was measured by Western blot.50 C57BL/6 mice were randomly divided into blank group,model group,DHP low dose(100 mg-kg-1)group,DHP equivalent dose(350 mg-kg-1)group and MCC950 group(positive control group),10 mice in each group.The motor balance and coordination ability of C57BL/6mice were observed by balance beam test,suspension test and baton-turning test;the expression levels of Iba-1 and Tyrosine hydroxylase(TH)were detected by immunofluorescence staining;the damage of dopamine neurons in the substantia nigra was detected by FJB staining.The expression levels of inflammatory factors such as IL-1β,IL-18 and TNF-α in mouse midbrain tissues were detected by ELISA;the expression levels of NLRP3,ASC,Caspase-1 and IL-1β protein were detected by Western blot.Results: Compared with the blank group,the SH-SY5 Y model group showed decreased cell survival,increased levels of LDH,ROS and MDA(P<0.05)and decreased levels of SOD(P<0.05);compared with the model group,the DHP group showed increased cell survival,decreased levels of LDH,ROS and MDA(P<0.01)and increased levels of SOD(P<0.01).Compared with the blank group,the levels of inflammatory factors IL-1β,IL-18 and TNF-α were increased in the BV-2 cell model group(P<0.05),and the expression of NLRP3,Caspase-1,IL-1β and ASC protein was increased(P<0.05);compared with the model group,the levels of inflammatory factors IL-1β,IL-18 and TNF-α were decreased in the DHP and MCC950 groups(P<0.01).The levels of inflammatory factors IL-1β,IL-18 and TNF-α were reduced in the DHP and MCC950groups(P<0.01),and the expression of NLRP3,Caspase-1,IL-1β and ASC were reduced(P<0.01).Compared with the blank group,the C57BL/6 mouse model group had increased time to pass the balance wood(P<0.05),decreased time at the stick in the stick in the stick-turning experiment(P<0.05),increased levels of midbrain inflammatory factors IL-1β,IL-18 and TNF-α(P < 0.05),increased expression of Iba-1in the midbrain substantia nigra(P<0.05)and decreased levels of TH expression(P<0.05)The number of neuron-positive cells in the midbrain substantia nigra was increased by FJB staining(P<0.05),and the expression of NLRP3,Caspase-1,ASC and IL-1β proteins was increased(P < 0.05);compared with the model group,the mice in the DHP and MCC950 groups had reduced time to pass the balance beam(P<0.01)and increased time on the stick in the stick-turning experiment(P<0.01),and the midbrain inflammatory factors IL-1β,IL-18 and TNF-α levels were decreased(P<0.01),midbrain substantia nigra Iba-1 expression was decreased(P<0.01),TH expression levels were increased(P<0.01),the number of neuron-positive cells in the midbrain substantia nigra was decreased in FJB staining(P<0.01),NLRP3,ASC and IL-1β proteins were decreased in the MCC950 group(P< 0.01),and NLRP3,ASC,Caspase-1 and IL-1βproteins were decreased in the DHP equivalent dose group(P<0.01).Conclusion:DHP has anti-oxidative stress effects;DHP may exert neuroprotective effects by regulating NLRP3 inflammatory vesicle expression and inhibiting microglia over-activation,thereby reducing neuroinflammatory damage in PD models. |
