A Study Of δ-opioid Receptors Inhibiting Ferroptosis By Activating The Nrf2 Pathway To Exert Anti-parkinson’s Disease Effects

Part Ⅰ:δ-opioid receptors have neuroprotective effects in MPTP-induced models of Parkinson’s diseaseObjective:Parkinson’s disease is a common neurodegenerative disease in the elderly characterized by motor dysfunction,and the incidence population is mainly concentrated in the elderly over 60 years old.The current classic treatment options for PD is dopamine replacement therapy and anticholinergic medication,which can relieve symptoms but still have many limitations.Therefore,we urgently need to develop more and more effective therapeutic drugs for treating PD.Studies suggest that δ-opioid receptor may be a potential therapeutic target,but more experimental support is needed to explore the mechanism,so we explored the neuroprotective effects played after δ-opioid receptor activation on models of Parkinson’s disease in this partMethods:Based on PC12 cells and C57BL/6 mice,PD cells and animal models were established,including control group,PD group,PD+DADLE group,PD+DADLE+Naltrindole group,and PD+Naltrindole group.The neuroprotective effect of δ-opioid receptors in MPP+-induced cells was assessed using the CCK-8 assay and the detection of LDH leakage rate while simultaneously observing cell morphology under an inverted microscope.The neuroprotective effect of δ-opioid receptor in MPTP induced mice was assessed by behavioral experiments assessing motor symptoms and immunofluorescence for tyrosine hydroxylase in substantia nigra and striatal regions.Results:1、PC 12 cell model:inverted microscope observation showed that floating cells were observed in MPP+,MPP++DADLE+Naltrindole group,MPP++Naltrindole group,control group and MPP++DADLE group.The CCK-8 test showed decreased cell viability in the MPP+group(52.50%±2.58%)compared with 98.(P<0.01)and the MPP++DADLE group(82.50%±3.08%)compared with the MPP+group(P<0.01).The LDH test showed an increase in LDH leakage rate in the MPP+group(28.72%±1.49%)compared to the control group(4.95%±0.01)and a decrease in LDH leakage rate in the MPP++DADLE group(10.00%±3.08%)compared with the MPP+group(P<0.01).2、C57BL/6 mouse model:Behavioral results showed significantly lower distance(t=86.09,P<0.01)and mean speed(t=28.34,P<0.01)in the MPTP group,and higher distance(t=6.83,P<0.01)(t=21.44,P<0.01)than those in the MPTP group.Immunofluorescence showed a significant loss of tyrosine hydroxylase-positive cells in the substantia nigra(t=28.34,P<0.01)and striatal regions(t=30.50,P<0.01)in MPTP mice compared with the control group.However,compared with the MPTP group,the number of tyrosine hydroxylase-positive cells in nigra(t=33.80,P<0.01)and striatal(t=13.83,P<0.01)of MPTP+DADLE treated mice was significantly higher.Conclusions1、δ-Opioid receptor can improve the cell morphology of MPP+-induced PC12 cells,reduce cytotoxicity,and improve cell viability;2、δ-Opioid receptor ameliorated MPTP-induced motor impairment and alleviated dopaminergic neuronsPart Ⅱ:δ-opioid receptor inhibit Ferroptosis in MPTP-induced C57BL/6 mice by upregulation of Nrf2 pathwayObjective:Although it is currently known that the main pathological alteration in PD is the loss of dopaminergic neurons in the substantia nigra and striatum,the exact mechanism of this alteration is unclear.It has been suggested that the loss of dopaminergic neurons may be closely related to Ferroptosis.So we explored in this part the association of the neuroprotective effect of activated δ-opioid receptor in MPTP-induced C57BL/6 mice with Ferroptosis and its underlying mechanism.Methods:MPTP-induced C57BL/6 mouse model was established,and control group,MPTP group,MPTP+Ferroptosis inhibitor(Ferrostatin-1)group,MPTP+DADLE group,MPTP+DADLE+Naltrindole group,and MPTP+Naltrindole group were established.Motor symptoms were assessed using behavioral experiments,and tyrosine hydroxylase in the substantia nigra and striatal region was measured by immunofluorescence.Lipid peroxidation was assessed by measuring malondialdehyde(MDA)and 4-hydroxynonenal(4-HNE)levels in mouse brain tissue using ELISA-Mitochondrial morphological changes were observed using transmssion electron microscopy.Using WB,the expression levels of ferroptosis proteins including Nrf 2,GPX 4,SLC7a11.Results:1、The results of behavioral experiments indicated that the movement distance(t=32.52,P<0.01)and mean speed(t=33.80,P<0.01)were significantly higher than MPTP+Ferrostatin-1 treated mice,and the MPTP+Ferrostatin-1 group and MPTP+DADLE group(t=1.19,P=0.26).Immunofluorescence results indicated that the number of tyrosine hydroxylase-positive cells in t=21.44(P<0.01)and striatum(t=1 P.45,P<0.01)was significantly higher than that in MPTP treated mice.Compared with the MPTP+DADLE group,there was no statistical difference in the number of tyrosine hydroxylase-positive cells in the nigra(t=1.55,P=0.16)and striatum(t=0.12,P=0.90)in the MPTP+Ferrostatin-1 group.2、The levels of MDA(t=25.48,P<0.01)and 4-HNE(t=2.34,P<0.05)in the MPTP group were significantly higher than those in the control group.Compared with mice in the MPTP group,the levels in MDA(t=24.19,P<0.01)and 4-HNE(t=2.34,P<0.05)in the MPTP+DADLE group.The MDA(t=22.70,P<0.01)and 4-HNE(t=2.87,P<0.05)levels of the MPTP+DADLE+Naltrindole group were significantly higher compared with the MPTP+DADLE group.The levels of MDA in the MPTP+Ferrostatin-1 group(t=31.15,P<0.01)and 4-HNE(t=5.87,P<0.01)were significantly lower than those in the MPTP group.Compared with the MPTP+DADLE group,there was no statistical difference in the levels of MDA(t=2.01,P=0.07)and 4-HNE(t=2.51,P=0.06)in the MPTP+Ferrostatin-1 group.3、The intracellular ultrastructural changes of neurons in mouse mesencephalic nigra tissue were examined by TEM,with normal morphological mitochondria,mitochondrial ridge and outer mitochondrial membrane intact in the control group.Mitochondrial ridges disappearance and destruction of the outer mitochondrial membrane were observed in the MPTP group.No inner mitochondrial ridges and no onter mitochondrial membrane and intact mitochondrial morphology from MPTP+Ferrostatin-1 treated mice and MPTP+DADLE treated mice.4、WB results showed that the expression levels of GPX 4(t=178.70,P<0.01),SLC7a11(t=40.36,P<0.01),and Nrf 2(t=16.68,P<0.01)were significantly reduced in the MPTP group compared with the control group.Compared with the mice in the MPTP group,GPX 4 in the mesencephala nigra tissue of the MPTP+DADLE group mice(t=91.31,P<0.01),SLC7a11(t=34.59,P<0.01),Nrf2(t=16.13,P<0.01)with significantly increased expression levels,GPX4 in the MPTP+DADLE+Naltrindole group as compared to the MPTP+DADLE group(t=68.15,P<0.01),SLC7a11(t=30.55,P<0.01),Nrf2(t=8.15,P<0.01)and the expression level again decreased significantly.The expression levels of GPX 4(t=118.0,P<0.01),SLC7a11(t=50.78,P<0.01),and Nrf 2(t=19.84,P<0.01)in the midbrain were significantly higher in the MPTP+Ferrostatin-1 group than those in the MPTP group.At the same time,GPX4(t=2.36,P=0.07),SLC7a11(t=0.64,P=0.55),and Nrf 2(t=0.46,P=0.91)in the MPTP+Ferrostatin-1 group were not significant with those in the MPTP+DADLE group.Conclusions:1、δ-opioid receptor and Ferroptosis inhibitors were neuroprotective in MPTP-induced C57BL/6 mice;2、δ-Opioid receptor and Ferroptosis inhibitor can inhibit lipid peroxidation and mitochondrial destruction in C57BL/6 mice;3、The δ-opioid receptor suppressed the MPTP-induced Ferroptosis in the dopaminergic neurons of C57BL/6 mice,possibly through the upregulation of Nrf 2,SLC7a11,and GPX4 expression.Part Ⅲ:δ-opioid receptors activate Nrf2-GPX4/SLC7a11 and inhibit ferroptosis in MPP+-induced PC12 cellsObjective:Ferroptosis is involved in the pathogenesis of PD.In this study,Ferroptosis by Nrf 2 after δ-opioid receptor(DOR)activation was inhibited in an animal model.Therefore,this study was further validated in a cell model to explore whether the activation of δ-opioid receptor exerts an anti-PD effect by inhibiting Ferroptosis through the Nrf 2-GPX 4/SLC7a11 pathway.Methods:In this study,PC12 cell model induced by MPP+was constructed,and the control group,MPP+group,MPP++Ferrostatin-1 group,MPP++DADLE group,MPP++DADLE+Naltrindole group,and MPP++Naltrindole group were established.Cytotoxicity and viability were assessed by CCK-8 and LDH leakage rate.GPX 4,SLC7a11 and Nrf2 protein expression levels were assessed using Western blot.The level of lipid peroxidation was measured by malondialdehyde(MDA)and 4-hydroxynonenal(4-HNE)levels using ELISA-Mitochondrial membrane potential was measured in MPP+-induced cells,using JC-1 staining Nrf 2 knockdown in PC12 cell model,cytotoxicity and viability were assessed after DOR activation,protein expression levels of GPX 4,SLC7a11 and Nrf 2 were measured,lipid peroxidation levels were assessed,and mitochondrial membrane potential was determined.Results:1.The cytotoxicity and viability results showed that The PC12 cell survival rate(t=17.60,P<0.01)and the LDH leakage rate(t=18.62,P<0.01)were significantly increased in the MPP++DADLE group as compared to the MPP+group.PC12 cells survival was significantly increased in the MPP++Ferrostatin-1 group compared with the MPP+group(32.80%±1.79%,P<0.05)and significantly lower LDH leakage(22.70%±0.66%,P<0.05).The LDH leakage rates in the MPP++DADLE and MPP++Ferrostatin-1 groups were not significant(t=10.56,P=0.26).Cell survival was insignificant in the MPP++DADLE+SiRNA(-)group(t=0.19,P=0.85),but was significantly lower in the MPP++DADLE+SiRNA(Nrf 2)group(t=15.80,P<0.01).No significant LDH leakage rate was compared with the MPP++DADLE+SiRNA(-)group(t=0.44,P=0.66).However,the LDH leakage rate increased significantly in the MPP++DADLE+SIRNA(Nrf 2)group(t=14.30,P<0.01)2,WB results showed that the expression levels of GPX 4(t=9.10,P<0.01),SLC7a11(t=8.64,P<0.01),and Nrf 2(t=3.92,P<0.01)were significantly reduced in the MPP+group compared with the control group.Compared with the MPP+group,GPX 4 in the MPP++DADLE group(t=5.35,P<0.01),SLC7a11(t=6.80,P<0.01),Nrf2(t=3.91,P<0.01)with significantly increased expression levels,However,the GPX 4 in the MPP++DADLE+Naltrindole group versus the MPP++DADLE group(t=5.14,P<0.01),SLC7a11(t=5.50,P<0.01),Nrf2(t=4.00,P<0.01)and the expression level again decreased significantly.The expression levels of GPX 4(t=4.87,P<0.01),SLC7a11(t=4.72,P<0.01),and Nrf 2(t=3.38,P<0.01)were significantly higher than those in the MPP+group in the MPP++Ferrostatin-1 group.At the same time,the expression levels of GPX 4(t=2.73,P=0.052),SLC7a11(t=0.58,P=0.59),and Nrf 2(t=0.78,P=0.44)were not significant between MPP++Ferrostatin-1 and MPP++DADLE groups.For MPP++DADLE+SiRNA(-)group,Nrf 2,GPX 4(t=0.17,P=0.86),P=0.48),and SLC7a11(t=1.71,P=0.16)were not significantly compared with MPP++DADLE group.However,the expression levels of the MPP++DADLE+SiRNA(Nrf 2)group of Nrf 2(t=5.49,P<0.01),GPX 4(t=4.13,P<0.01),and SLC7a11(t=7.27,P<0.01)were significantly lower than those of the MPP++DADLE group.3,ELISA results showed that the MDA(t=19.50,P<0.01)and 4-HNE(t=5.27,P<0.01)levels were significantly higher in PC12 cells induced by MPP+than in the control group.Compared with the MPP+group,the MDA levels decreased significantly in the MPP++DADLE group(t=16.92,P<0.01)and the MPP++Ferrostatin-1 group(t=15.53,P<0.01).Compared with the MPP+group,the 4-HNE level were significantly lower in the MPP++DADLE group(t=3.35,P<0.01)and the MPP++Ferrostatin-1 group(t=4.12,P<0.01).There were no statistical differences in MDA(t=0.36,P=0.72)and 4-HNE(t=0.26,P=0.79)levels between the MPP++DADLE and MPP++Ferrostatin-1 groups.MDA(t=0.11,P=0.91)and 4-HNE(t=0.28,P=0.78)levels for MPP++DADLE+SiRNA(-)group compared with MPP++DADLE group.However,the MDA(t=17.70,P=<0.01)and 4-HNE(t=1.94,P=<0.05)levels increased significantly in the MPP++DADLE+SiRNA(Nrf 2)group.4,The mitochondrial membrane potential results showed a significantly decreased membrane potential level in the MPP+group compared with the control group(t=86.09,P<0.01).Compared with the MPP+group in the MPP++DADLE group(t=35.52,P<0.01).However,the MPP++DADLE+Naltrindole group was significantly lower compared with the MPP++DADLE group(t=33.51,P<0.01).The level of membrane potential was significantly higher in the MPP++Ferrostatin-1 group than in the MPP+group(t=24.83,P<0.01).No significant membrane potential levels were found in the MPP++Ferrostatin-1 group or the MPP++DADLE group(t=1.07,P=0.31).No significant mitochondrial membrane potential was compared with the MPP+ +DADLE+DADLE+SiRNA(-)group(t=0.10,P=0.92),but the MPP++DADLE+SiRNA(Nrf 2)group(t=33.21,P<0.01).Conclusions:1,δ-opioid receptor alleviated the cytotoxicity of MPP+-induced PC 12 cells,increased cell viability,inhibited lipid peroxidation and improved mitochondrial membrane potential,and the above effects of δ-opioid receptor were reversed after Nrf 2 knockdown;2,δ-opioid receptor and Ferroptosis inhibitor inhibited MPP+-induced Ferroptosis in PC12 cells by upregulating Nrf 2,SLC7a11,and GPX 4 expression,and the anti-Ferroptosis effect of δ-opioid receptor was reversed after Nrf 2 knockdown;3,δ-opioid receptor may induce Ferroptosis in PC12 cells through Nrf 2-GPX 2/SLC7a11 pathway.