Protection Of Pioglitazone Against LPS-induced Damage To The Substantia Nigra

Parkinson’s disease(PD)is the second largest neurodegenerative disease,second only to Alzheimer’s disease.Degenerative death of dopaminergic neurons in the midbrain substantia nigra is the main pathological feature.The clinical symptoms include motor symptoms and non-motor symptoms.The motor symptoms are resting tremor,muscle rigidity and bradykinesia and other behavioral disorders,non-motor symptoms are depression,cognitive disorders,anxiety,insomnia and other non-motor symptoms.In recent years,a growing number of studies have found that the main causes of PD are mitochondrial dysfunction,oxidative stress,excitotoxicity,apoptosis and immune inflammation.Currently,a lot of research still needs to provide a new direction and found new therapeutic targets for effective treatment methods and drugs.Peroxisome proliferator-activated receptors(PPARs)are ligand-activated transcription factors of the nuclear receptor superfamily,and PPAR-γ is the most widely studied subtype.PPAR-γ plays an important role in adipocyte differentiation and lipid storage.However,in recent years studies have found that activation of PPAR-γ will play a neuroprotective effect.Thiazolidinediones are one of the agonists of PPAR-γ,which can selectively activate PPAR-γ.Pioglitazone(Pioglitazone,Pio)is such a drug,which can also selectively activate PPAR-γ and is clinically used to treat type 2 diabetes.Whether Pio can also have neuroprotective effect in the PD model induced by lipopolysaccharide(LPS),and what are the mechanisms? Further research is needed.Kunming mice were used in this study,and the mice were stereotactic injection of LPS in bilateral striatum induced PD model.Pio was administered by gastric administration to intervene.Behavioral test,immunofluorescence experiments,ELISA and Western Blot were used to explore the protective effect of Pio on LPS-induced damage of substanti nigra(SN).The experiment results are as follows:1.The blood glucose and body weight of the experimental mice were measured and recorded.We found that Pio had no effect on the blood glucose and body weight of the mice during this experiment period.2.The number of activities and times of standing-rear,the rest time in swimming were recorded.The results showed that there was no statistical difference in the behavior among the five groups before stereotactic injection of LPS(P>0.05).After stereotaxic injection of LPS behavioral results showed that the times of standing-rear and locomotor activities were significantly reduced,and the time of resting in swimming was significantly increased in LPS group compared with the control group.While after Pio treatment,which improves behavioral disorders,but no statistical difference compared with LPS group(P > 0.05).3.Immunofluorescence experiments was used to detected the expression of TH,GFAP and Iba1 in SN.The results showed that after LPS treatment,the number of TH-positive neurons significantly decreased,the expression of Iba1 significantly increased,and the fluorescence intensity of GFAP increased significantly(P<0.05).However,the number of TH positive neurons in the other three Pio groups increased significantly,the expression of Iba1 decreased significantly,and the fluorescence intensity of GFAP decreased significantly after Pio intervention(P<0.05)compared with LPS group.4.The expression levels of several inflammatory factors in the SN,including interleukin-6(IL-6),tumor necrosis factor α(TNF-α)and interleukin-1β mice(IL-1β),were determined by ELISA.The results showed that LPS increased the expression of these three inflammatory factors in the LPS group,while the Pio decreased the expression level.Only IL-6 expression level had significantly difference,the expression level in LPS+10 mg/kg Pio group and LPS+20 mg/kg Pio group were significantly lower than that of the LPS group(P<0.05).5.The protein expression levels of TH,GFAP,PPAR-γ,NF-κB and Caspase 3 in SN were detected by Western Blot.The results showed that TH protein expression in the substantia nigra decreased significantly after injection of LPS(P<0.05),and after Pio intervention TH expression level increased,but it had no difference compared with LPS group(P>0.05).The GFAP protein expression level in the LPS group was significantly higher than that of Control group.Three pioglitazone interventions at different concentrations resulted in significantly lower levels of GFAP protein expression than the LPS group.The expression level of PPAR-γ in the substantia nigra significantly decreased(P <0.05)in the LPS group compared with Control group,and after treatment with different concentrations of Pio PPAR-γexpression increased.But there was no significant difference(P> 0.05)among the three groups.The expression levels of NF-κB and Caspase 3 in the substantia nigra are consistent.The expression levels in the LPS group were significantly higher than those in the Control group,and the expression levels of these two proteins in the three different concentrations of Pio group were significantly lower than those in LPS Group(P <0.05).6.Immunofluorescence,ELISA and western results showed that in the pioglitazone intervention group at three concentrations,the intervention effect of 20 mg/kg pioglitazone was better.We can draw the conclusions through analyze the above results,the conclusions are as follows:1.Pio had no effect on the blood glucose and body weight during the experiment period.2.Pio can improve the behavioral disorder of mice by increasing the times of activity and standing,and reducing the resting time of swimming.3.Pio activates PPAR-γ and inhibits the expression of NF-κB and thereby inhibit the activation of microglia and astrocytes,reducing the expression of inflammatory factors and immune inflammation reduce the damage and increase the survival of dopaminergic neurons.At the same time,Pio may play a role in the expression of anti-apoptosis by reducing the Caspase 3.In conclusion,Pio can reduce the inflammatory response by activating PPAR-γ and inhibiting NF-κB pathway.Moreover,Pio can also play a neuroprotective role in LPS-induced nigra injury by reducing the expression of Caspase 3.