Neuroprotective Effect Of GLP-1/GIP Receptor Unimolecular Co-agonist DA-CH5 On 6-OHDA SH-SY5Y Cells Model And Rat Model Of Parkinson’s Disease

Part I:The protective mechanism of DA-CH5 against 6-OHDA induced neurotoxicity in SH-SY5Y cellsObject:1.To investigate whether DA-CH5 can improve 6-OHDA-induced SH-SY5Y cell model with the following toxic effects:（1）decreased cell survival rate,increased reactive oxygen species,and abnormal expression of apoptosis-related proteins;（2）abnormal expression of IRS-1/AKT/CREB proteins;（3）abnormal expression of mitochondrial function-related protein;（4）the effect on the autophagy-related proteins;and reveal its potential neuroprotective mechanism;2.To explore whether the GLP-1/GIP unimolecular co-agonist DA-CH5 is superior to the GLP-1 receptor agonist Exendin-4 in the 6-OHDA induced PD cell model.Methods:1.Experimental grouping:after 4 h of serum shock,the experiment was divided into the following 6 groups by co-incubation of 6-OHDA with Exendin-4 or DA-CH5:（1）CNTRL;（2）Exendin-4;（3）DA-CH5;（4）6-OHDA;（5）6-OHDA+Exendin-4;（6）6-OHDA+DA-CH5.2.Measurement of cell viability:cells were grew in 96-well plates,and after 4 h of serum shock,6-OHDA with gradient concentration（150 M,250 M,350 M,450 M）was added and incubated for 24 h.Then 5 mg/mL MTT solution was added to each well and placed at 37℃for 3 h before the OD value was detected by a microplate.After the optimum concentration of the model was screened,the 6-OHDA of the concentration was added to the medium together with 100 nM Exendin-4 or 100 nM DA-CH5 and incubated for 24 h before OD value was detected.3.The level of reactive oxygen species（ROS）was detected by DCFH-DA probe:the cells that had been treated with drugs were incubated in PBS containing 10M DCFH-DA,and after incubated for 15 min washed by PBS.The fluorescence signal was detected by a flow cytometer.4.Western blot was used to detect the expression levels of the following proteins:（1）apoptosis-related proteins Bax,Bcl-2,p-Bad^ser136and t-Bad;（2）insulin signaling pathway proteins p-IRS-1^ser312,t-IRS-1,p-AKT^ser473,p-CREB^ser133,and t-CREB;（3）mitochondrial function-related proteins PGC-1 and NRF-1;（4）autophagy related proteins SQSTM1 and Beclin-1.The GAPDH orβ-actin were house-keeper proteins.Results:1.MTT cell viability test:（1）after incubation with 350 M 6-OHDA for 24 h,the cell viability decreased significantly compared with the CNTRL group（P<0.001;vs.CNTRL group）.Therefore,the concentration of 6-OHDA solution was used as the mimic the PD condition for the cell model.（2）The cell viability in both 6-OHDA+Exendin-4（P<0.05;vs.6-OHDA group）and 6-OHDA+DA-CH5（P<0.001;vs.6-OHDA group）groups were significantly higher than that in the 6-OHDA group.In addition,the improvement of 6-OHDA+DA-CH5 group was much significant than that of 6-OHDA+Exendin-4 group（P<0.01;vs.6-OHDA+Exendin-4 group）.2.DCFH-DA probe detected the level of ROS:compared with the CNTRL group,the intensity of ROS in the 6-OHDA group increased significantly（P<0.001;vs.CNTRL group）.And the ROS intensity in both 6-OHDA+Exendin-4（P<0.001;vs.6-OHDA group）and 6-OHDA+DA-CH5（P<0.001;Compared with the 6-OHDA group）groups decreased significantly.In addition,compared with the 6-OHDA+Exendin-4 group,the6-OHDA+DA-CH5 group showed a small improvement（P<0.05;vs.6-OHDA+Exendin-4 group）.3.Western blot:（1）Exendin-4（Bax,P<0.01;Bcl-2,P<0.05;vs.6-OHDA group）and DA-CH5（Bax,P<0.001;Bcl-2,P<0.01;vs.6-OHDA group）can significantly improve the expression abnormality of apoptosis related proteins Bax and Bcl-2 induced by6-OHDA.Only DA-CH5（P<0.01;vs.6-OHDA group）can significantly up-regulated the expression level of p-Bad^ser136.（2）Exendin-4（P<0.05;vs.6-OHDA group）and DA-CH5（P<0.05;vs.6-OHDA group）to down-regulated the expression of p-IRS-1^ser312.Exendin-4(p-AKT^ser473/t-AKT,P<0.001;p-CREB^ser133/t-CREB,p<0.01;vs.6-OHDA group)and DA-CH5(p-AKT^ser473/t-AKT,P<0.001;p-CREB^ser133/t-CREB,P<0.01;vs.6-OHDA group)can increase the expression of p-AKT^ser473and p-CREB^ser133.In addition,DA-CH5(p-AKT^ser473/t-AKT,P<0.05;vs.6-OHDA+Exendin-4 group)significantly up-regulated the expression of p-AKT^ser473.（3）Only DA-CH5（PCG-1α,P<0.05;NRF-1,P<0.01;vs.6-OHDA group）can improve the expression of 6-OHDA induced mitochondrial function related protein PCG-1 and NRF-1 decreased.And compared with Exendin-4,the expression level of NRF-1 was in the 6-OHDA+DA-CH5 group（P<0.05;vs.6-OHDA+Exendin-4 group）was statistically significant up-regulated.（4）Compared with the normal control group,Exendin-4（SQSTM1,P<0.01;Beclin-1,P<0.05;vs.CNTRL）and DA-CH5（SQSTM1,P<0.01;Beclin-1,P<0.01;vs.CNTRL）can increase the expression of autophagy-related proteins SQSTM1 and Beclin-1.Conclusion:Exendin-4 and DA-CH5 can effectively inhibit the decrease of cell viability and the increase of ROS induced by 6-OHDA.In addition,by regulating IRS-1/AKT/CREB pathway,the abnormal expressions of apoptosis-related proteins were improved and the autophagy related protein expressions were promoted.In addition,DA-CH5 can partially restore the expression levels of proteins related to mitochondrial function,such as PCG-1 and NRF-1.DA-CH5 is more advantageous than Exendin-4 in improving cell viability,reducing the level of intracellular ROS,and increasing the expression levels of p-Bad^ser136,p-AKT^ser473er473 and NRF-1.This study will provide a new basis for clinical treatment of PD.Part II:The protective mechanism of DA-CH5 against 6-OHDA induced neurotoxicity in SD ratsObject:1.To investigate whether DA-CH5 can improve PD-like behavioral disorders,the reduction of dopamine level in the nigro-striatal system,elevation inflammatory factors levels,and abnormal expression ofα-synuclein oligomers,mitochondrial function-related proteins,apoptosis-related proteins,and autophagy-related proteins in the substantia nigra region induced by 6-OHDA in SD rat PD model.2.To investigate whether the GLP-1/GIP unimolecular co-agonist DA-CH5 is more advantageous than the GLP-1 receptor agonist Exendin-4 in SD rat models induced by6-OHDA.Methods:1.Stereotaxic surgery and experimental grouping:the rats were anesthetized and fixed on the stereotaxic locator,and the skull drill was used to drill holes in the corresponding position of the rat skull according to the stereotaxic map of the rat brain.6-OHDA solution（2 mg/mL）was injected into the right lateral frontal tract（MBF）through a micro syringe.The rats were intraperitoneally injected with Exendin-4,DA-CH5 and saline once a day for 30 days after surgery,and were divided into the following four groups:（1）Sham+Saline group;（2）6-OHDA+Saline group;（3）6-OHDA+Exendin-4 group;（4）6-OHDA+DA-CH5 group.2.Behavioral test:the following behavioral experiments were conducted on the 31st day after surgery:（1）Open-field experiment:rats were placed in a square open-field surrounding a wall of 75 cm long and 35cm high and moved autonomously for 10min.A tracking capture system recorded the rat movement and calculated the movement distances.（2）Apomorphine-induced rotation experiment:after intraperitoneal injection of 0.05 mg/kg apomorphine for 10min,the rats were placed in a square open field for 30min,and the movement track of the rats was recorded by the animal motion tracking system.After that,the number of turns to the contralateral side of stereotactic surgery was recorded by a video system.3.Materials:the animals which had completed the behavioral experiment were sacrificed on the 32nd day.（1）4 rats in each group were infused with saline and 4%paraformaldehyde,then their brains were extracted,dehydrated in 30%sucrose solution,and then frozen for immunofluorescence staining.（2）6 rats in each group were perfused with saline,and the striatum（for Elisa）and substantia nigra（for Western blot）on the affected side were extracted,respectively.4.The concentration of dopamine and TNF-in striatum was detected by Elisa:the striatum was grinded in a homogenizer containing RIPA lysate（1 mg/10μL）and protease inhibitor PMSF（1:100 RIPA lysate）,and the supernatant was taken after centrifugation.After the protein concentration was measured by BCA method,RIPA lysate was used to adjust the protein concentration of each sample.The specific steps in the instructions for the dopamine Elisa kit and TNF-αkit were then followed to incubate the samples.Finally,OD value was measured by microplate and the concentrations of dopamine and TNF-αwere calculated by the standard curve.5.Evaluation of the number of dopamine neurons in the substancia nigra and striatal astrocytes by immunofluorescence staining:droping 10%goat serum on slides which incubated for 30min at room temperature,and then incubated overnight at 4℃with 1:100diluted rabbit anti-TH antibody or rabbit anti-GFAP antibody.Finally,these slides incubated with Goat anti-rabbit IgG FITC conjugated secondary antibody labeled with luciferin at 37℃for 2h,then were photographed by fluorescence microscopy.6.Western blot was used to detect the expression of the following proteins in the substantia nigra:（1）tyrosine hydroxylase TH;（2）synaptic ribosomal protein oligomers;（3）inflammatory cytokines TNF-αand IL-1β;（4）insulin signaling pathway proteins and mitochondrial function-related proteins p-IRS-1^ser312,t-IRS-1,PGC-1 and NRF-1;（5）apoptosis-related proteins active-caspase-3,caspase-3,Bax and Bcl-2;（6）autophagy related proteins SQSTM1 and Beclin-1.The internal reference was GAPDH orβ-actin.Results:1.Behavioral test:（1）Open-field experiment:the movement distance of rats in the 6-OHDA Saline group decreased significantly compared with that in the Sham Saline group（P<0.001;vs.Sham Saline group）.Compared with the rats in the 6-OHDA Saline group,the rats in the 6-OHDA+DA-CH5 group significantly recovered their motor ability,while the rats in the 6-OHDA+Exendin-4 group（P<0.05;vs.6-OHDA Saline group）only slight improvement was observed.（2）Apomorphine-induced rotation experiment:the number of turns to the injured rats in the 6-OHDA Saline group（P<0.001;vs.Sham Saline group）showed a significant increase compared with Sham Saline group.The6-OHDA+Exendin-4 group（P<0.001;vs.6-OHDA Saline group）and 6-OHDA+DA-CH5 group（P<0.001;vs.6-OHDA Saline group）showed significant improvement.Rats in the 6-OHDA+DA-CH5 group（P<0.05;vs.6-OHDA+exeindin-4 group）also slightly improved compared to 6-OHDA+Exendin-4 group.2.Elisa assay:（1）dopamine content in striatum of rats with 6-OHDA Saline group（P<0.01;vs.Sham Saline group）showed a significant decrease compared with Sham Saline group,while only the rats treated with DA-CH5（P<0.05;vs.6-OHDA Saline group）can restore dopamine level.（2）TNF-αcontent in rat striatum of 6-OHDA Saline group(P<0.05;vs.Sham Saline group,Exendin-4（P<0.05;vs.6-OHDA Saline group）and DA-CH5（P<0.05;vs.6-OHDA Saline group）could reverse this phenomenon.3.Immunofluorescence staining:（1）TH labeled dopamine neurons in the substancia nigra of rats with 6-OHDA Saline group（P<0.001;vs.Sham Saline group）showed a significant increase compared with Sham Saline group.Exendin-4（P<0.01;vs.6-OHDA Saline group）and DA-CH5（P<0.001;vs.6-OHDA Saline group）could significantly reverse this phenomenon.（2）The number of GFAP-labeled astrocytes in the striatum of rats in the 6-OHDA Saline group（P<0.001;vs.Sham Saline group）showed a significant increase compared with Sham Saline group.Exendin-4 group was significantly higher than Sham Saline group（P<0.05;vs.6-OHDA Saline group）and DA-CH5（P<0.01;vs.6-OHDA Saline group）could partially improve the abnormal increase of astrocytes in the6-OHDA treated rats.4.Western blot:（1）TH level in the substancia nigra was significantly decreased by6-OHDA injury（P<0.001;vs.6-OHDA Saline group）.After Exendin-4(P<0.001;vs.Sham Saline group and DA-CH5（P<0.001;vs.6-OHDA Saline group）can improve the toxic injury to a certain extent.（2）the level ofα-synuclein oligomers in the substantia nigra region was abnormally increased by 6-OHDA induction（P<0.001;vs.Sham Saline group）.Exendin-4（P<0.05;vs.6-OHDA Saline group）and DA-CH5（P<0.01;vs.6-OHDA Saline group）treatments could effectively inhibit the up-regulated expression ofα-synuclein oligomers.（3）6-OHDA induced significantly increased the release of inflammatory cytokines in the substantia nigra（TNF-α,P<0.001;IL-1β,P<0.001;vs.Sham Saline group）.Exendin-4 treatment（TNF-α,P<0.05;IL-1β,P<0.05;vs.6-OHDA Saline group）only slightly improved the inflammatory response,while DA-CH5（TNF-α,P<0.001;IL-1β,P<0.001;vs.6-OHDA Saline group）can significantly inhibit the release of inflammatory cytokines.The inhibitory effect of DA-CH5（P<0.05;vs.6-OHDA+Exendin-4 group）on IL-1βexpression was statistically significant compared with that of the Exendin-4 group.（4）6-OHDA induction significantly up-regulated the expression of insulin substrate protein p-IRS-1^ser129in the substantia nigra（P<0.001;vs.Sham Saline group）,and Exendin-4（P<0.05;vs.6-OHDA Saline group）and DA-CH5（P<0.01;vs.6-OHDA Saline group）treatments showed slight improvement on this phenomenon.In addition,Exendin-4（PGC-1,P<0.05;NRF-1,P<0.001;vs.6-OHDA Saline group）and DA-CH5（PGC-1,P<0.05;NRF-1,P<0.01;vs.6-OHDA Saline group）could effectively alleviate the low expressions of mitochondrial function-related proteins PGC-1 and NRF-1induced by 6-OHDA（PGC-1 expression,P<0.001;NRF-1,P<0.001;vs.Sham Saline group）.（5）Expression of active-Casepase-3 in substantia nigra was significantly up-regulated by 6-OHDA lesion（P<0.001;vs.Sham Saline group）,and Exendin-4（P<0.01;vs.6-OHDA Saline group）and DA-CH5（P<0.001;vs.6-OHDA Saline group）treatments were significantly improved this phenomenon.Meanwhile,Exendin-4（Bax,P<0.05;Bcl-2,P<0.01;vs.6-OHDA Saline group）and DA-CH5（Bax,P<0.001;Bcl-2,P<0.001;vs.6-OHDA Saline group）could effectively alleviate the over expression of apoptosis-related proteins Bax and Bcl-2 induced by 6-OHDA lesion（Bax,P<0.001;Bcl-2,P<0.001;vs.Sham Saline group）.In addition,Bcl-2 level in DA-CH5 group（P<0.05;6-OHDA+Exendin-4 group）was significantly improved compare with Exendin-4group.（6）Beclin-1 level was slightly increased after treatment with Exendin-4（P<0.05;vs.Sham Saline group）or DA-CH5（P<0.01;vs.Sham Saline group）.Only DA-CH5（P<0.001;vs.Sham Saline group）significantly up-regulated the expression level of SQSTM1,and the difference between DA-CH5（P<0.01;vs.6-OHDA+Exendin-4group）and Exendin-4 was statistically significant.Conclusion:Exendin-4 and DA-CH5 can alleviate the behavior disorders,decreased dopamine in the substantia nigra striatum and release of inflammatory cytokines in the rat model lesioned by 6-OHDA.Moreover,the abnormal expressions ofα-synuclein oligomers,mitochondrial function-related protein and apoptotic protein were modulated,and the expression of autophagy related protein was up-regulated.In addition,DA-CH5has slight advantages over Exendin-4 in improving PD-like behaviors in animals,inhibiting the release of inflammatory cytokines and regulating the expression of apoptosis-related proteins.This study will provide a new basis for clinical treatment of PD.