P2Y₁₂ Receptor Mediates The Effect Of Natural Drugs On Primary Sensory Nerve Injury In Rats With Impaired Glucose And Lipid Metabolism

Background and Objective:Diabetic peripheral neuropathy results in diabetic neuropathic pain?DNP?.Satellite glial cells?SGCs?enwrap the neuronal soma in the dorsal root ganglia?DRG?.SGCs have multiple receptors for neurotransmitters and other bioactive molecules.The purinergic 2?P2?Y₁₂ receptor is expressed on SGCs in the DRG.Both neurons and glial cells in the DRG release ATP.Peripheral inflammation increases SGC sensitivity to ATP in the primary sensory ganglia.ATP signals of sensory inputs can activate ionotropic P2X receptors and metabotropic P2Y receptors in the DRG.The P2Y₁₂ receptor is activated by ATP,and ADP plays an important role in the transmission of painful signals.ATP is the major transmitter mediating neuronal-SGC communication.Inflammatory stimulation can activate SGCs,SGC activation plays an important role in the pathogenesis of DNP.Curcumin has anti-inflammatory and antioxidant properties.Because curcumin has poor metabolic stability in vivo and low bioavailability,nanoparticle-encapsulated curcumin was used to improve its targeting and bioavailability.In this study,a diabetic neuropathic animal model was established.However,the effects of the P2Y₁₂receptor on DNP in rats are unclear.This study will provide a new way for prevention and treatment of DNP.In the present study,our aim was to investigate the effects of nanoparticle-encapsulated curcumin on DNP mediated by the P2Y₁₂ receptor on SGCs in the rat DRG.Quercetin,an effective ingredient of the natural drug monomer,has anti-inflammatory,antibacterial,anti-viral,anti-oxidative,immunomodulatory effects?Roslan et al.,2017?.Laboratory experiments showed that quercetin can effectively relieve DNP.This study explored the effect of quercetin on P2Y₁₂ receptor-mediated hyperlipidemia?HLP?and hyperlipid-induced neuropathological injury in rat SGCs of dorsal root ganglion?DRG?,which provided a new basis for the prevention and treatment of hyperlipidemia.The first partMethods:1.Nanomaterial carrier?Polymer–Poly?ethylene glycol?methyl ether methacrylate–2-?Diethylamino?ethyl methacrylate–macromolecule,PDAEMA?was prepared through reversible addition-fracture chain transfer polymerization?RAFT?polymerization.2.The experiments for the effects of curcumin nanoparticles on DM neuropathic pain model rats:Rats were randomly divided into control group,type II diabetic model group?DM?,diabetic model combined curcumin nanoparticles treatment group?DM+nano curcumin?,diabetic model combined nano carrier treatment group?DM+nano carrier?.The effects of curcumin nanoparticle treatment on the pathological changes of DM rats were observed by blood glucose and body weight measurements.The behavioral test was used to mesure mechanical withdrawal threshold?MTW?and thermal withdrawal latency?TWL?in the rats.The co-localization of the P2Y₁₂receptor and glutamine synthetase?GS??a marker of satellite glial cells?was measured using double-label immunofluorescence staining.The effects of curcumin nanoparticles on the expression of P2Y₁₂ receptor,IL-1?and Cx43 mRNA and protein can be detected by Q-PCR and Western Blot.The effect of curcumin nanoparticles on PKB/AKT and its phosphorylation level was detected by Western blotting.The score of docking of curcumin and P2Y₁₂ protein molecules was used to simulate the matching and interaction of curcumin and P2Y₁₂ receptors.Results:The synthesized PDAEMA was uniform in size and spherical in shape,and the curcumin nanoparticles PDAEMA-CUR obtained after encapsulating curcumin had good dispersibility in water.The experiments for the effect of Curcumin Nanoparticles on DM Rats were as following:At the 5^th,7^th,and 10^th week,the blood glucose level of the DM group was significantly higher than that of the Ctrl group?p<0.001?.At 10^th weeks,the blood glucose level of DM+nano curcumin group was significantly lower than that of DM group?p<0.05?.There was no significant difference between the DM group and the DM+nano carrier group?p>0.05?.At 5 ^th week,the body weight of the DM group was significantly higher than that of the Ctrl group?p<0.05?.At 7^th week,compared with the Ctrl group,the body weight of the DM group was significantly decreased?p<0.01?.At 10^th weeks,the body weight of DM rats was significantly lower than that of the Ctrl group?p<0.01?.There was no significant difference in body weight between the DM+nano carrier group and the DM group?p>0.05?.After the rats were modeled?weeks 6-10?,the MWT and TWL in the DM rats were significantly lower than those in the Ctrl group?p<0.01?,and after two injections of curcumin nanoparticles?weeks 9-10?MWT and TWL were significantly higher in DM+nano curcumin group than in DM group?p<0.01?.At week 6-10,there was no significant difference between DM group and DM+nano carrier group?p>0.05?.Q-PCR and Western blot results showed that the expression levels of both P2Y₁₂mRNA and protein were increased compared with Ctrl group rats?p<0.01?.Compared with DM rats,P2Y₁₂ mRNA and protein expression levels in DM rats treated with curcumin nanoparticles were significantly lower?p<0.01?.The co-expression of the P2Y₁₂ receptor and GS in the DM+nanoparticle-encapsulated curcumin group was significantly decreased compared to that in the DM group.A difference in the co-expression of the P2Y₁₂ receptor and GS was not observed between the DM and DM+nanoparticle-encapsulated carrier groups.The Q-PCR results of IL-1?and Cx43 mRNA showed that curcumin nanoparticles-treated DM rats were significantly lower than those in DM rats?p<0.01?.There was no significant difference between DM group and DM+nano carrier group?p>0.05?.Western blotting results showed that the expression of IL-1?,Cx43,and p-AKT protein in the DM+nano curcumin group was significantly lower than that in the DM group?p<0.01?.There was no significant difference between the DM group and the DM+nano carrier group?p>0.05?.Molecular docking scores of rat P2Y₁₂ protein and curcumin?-7.3,Kcal/mol?indicate that curcumin is fully suitable for interaction with rat P2Y₁₂ receptor.The second partMethod:The experiments for the effects of quercetin on high fat diet-induced obesity?DIO?rats:The rats were randomly divided into normal control group?Ctrl?,obesity model group?DIO?,obesity model combined with quercetin group?DIO+Quercetin?,obesity model combined with DMSO treatment group?DIO+DMSO?,normal control combined with quercetin group?Ctrl+Quercetin?.The body weight,tail sensory nerve conduction velocity,serum total cholesterol?TC?,triglyceride level,high density lipoprotein cholesterol?HDL?and low density lipoprotein cholesterol?LDL?were measured in each group.After intraperitoneal injection of quercetin,the mRNA and protein expression levels of P2Y₁₂ receptor in dorsal root ganglion?DRG?were detected by real-time quantitative PCR?Q-PCR?and Western Blot.Immunofluorescence double labeling was used to detect the co-expression of P2Y₁₂ receptor and glial fibrillary acidic protein?GFAP?in DRG satellite glial cells?SGC?.The effects of quercetin on ERK1/2 and its phosphorylation level were detected by Western blot.In order to simulate the interaction between quercetin and P2Y₁₂ receptor,molecular docking method was used to detect the molecular docking between quercetin and P2Y₁₂ protein.The experiments for the effects of quercetin on SGCs treated with high fat?HF?:Different concentrations of high fat?0.25 m M,0.5 m M,0.75 m M,1 m M,1.25 m M,1.5 m M?and different treatement time of 24h,48h and 72h were used to select the appropriate high lipid concentration and suitable treatment time in the primary culture of SGCs.The cultured cells were divided into:normal control group?Ctrl?,high fat model group?HF?,high fat model with quercetin treatment group?HF+Quercetin?,high fat model combined with DMSO treatment group?DM+DMSO?,normal control combined with quercetin treatment group?Ctrl+Quercetin?.The expression changes of P2Y₁₂ mRNA and protein treated by different concentrations of high fat were observed.Real time quantitative PCR?Q-PCR?and Western blot?Western Blot?were used to detect the expression changes of P2Y₁₂ mRNA and protein induced by high fat in SGCs treated with quercetin.In SGCs,the effect of quercetin on ERK1/2 and its phosphorylation level induced by high fat was detected by Western blot.Enzyme linked immunosorbent assay?ELISA?was used to detect the effects of quercetin on the changes of inflammatory factors?IL-1 beta,TNF-alpha?in high fat treated cell supernatant.Results:The animal experiments showed that the weight,the total cholesterol?TC?,triglyceride,and low density lipoprotein cholesterol?LDL?were increased and the level of high-density lipoprotein cholesterol?HLD?was reduced in the obese?DIO?model rats.The conduction velocity of the sensory nerve in the tail of the rat was reduced.Quercetin can improve abnormal blood lipid levels and improve the tail sensory nerve conduction velocity in obese?DIO?rats.Q-PCR and Western blot analysis showed that the expression levels of P2Y₁₂mRNA and protein in DIO group were increased compared with those in Ctrl group?p<0.01?.Compared with the untreated DIO rats,the expression levels of P2Y₁₂mRNA and protein in DIO rats treated with quercetin were significantly decreased?p<0.01?.Immunofluorescence double labeling results showed that P2Y₁₂ receptor was coexpressed with glial fibrillary acidic protein?GFAP,a SGC marker?.The co-expressed immunofluorescence intensity of DRG P2Y₁₂ receptor and GFAP in DIO group was significantly increased compared with that in Ctrl group.The co-expression immunofluorescence intensity of DRG P2Y₁₂ receptor and GFAP in DIO+Quercetin group was significantly decreased compared with that in the untreated DIO group.There was no significant difference in the co-expression of P2Y₁₂ receptor and GFAP between DIO group and DIO+DMSO group.The results in Western blot showed that the expression level of p-ERK1/2protein in the DIO+Quercetin group was significantly lower than that in the untreated DIO group?p<0.01?,and there was no significant difference between the DIO group and the DIO+DMSO group?p>0.05?.The molecular docking score?-7.7,Kcal/mol?of rat P2Y₁₂ receptor protein and quercetin showed that quercetin interacted with P2Y₁₂ receptor.The experiments in the culture cells showed that the effects of high fat?HF??0.25 m M,0.5 m M,0.75 m M,1 m M,1.25 m M,1.5 m M?on cell activity were detected.The obvious damage to cell activity was induced by the concentrations of0.75 m M,1 m M,1.25 m M,1.5 m M HF.After SGCs cultured with 0.5 m M HF for24h,48h and 72h,the cell survival rates were 85%,78.8%,50.5%,respectively.Compared with Ctrl group,the cell survival rates were significantly decreased?p<0.01?.According to the results of cell viability test,the HF inducing cell injury model was established by using the concentration of 0.5 m M HF for 48 hours.The experiments in real time quantitative PCR showed that there was no significant change for P2Y₁₂ mRNA expression in 0.25 m M HF group compared with the Ctrl group?p>0.05?.P2Y₁₂ mRNA expression was significantly increased in 0.5m M HF,0.75 m M HF,and 1 m M HF groups?p<0.001?.Compared with the Ctrl group,the expression of P2Y₁₂ protein in the 0.25 m M HF group was not significantly changed?p>0.05?.The expression of P2Y₁₂ protein in the 0.5 m M HF group and the 0.75 m M HF group was significantly increased?p<0.001?.The expression level of P2Y₁₂ protein in 1 m M HF group was significantly lower than that in Ctrl group?p<0.001?.The results in real time quantitative PCR and Western blotting showed that the expression of P2Y₁₂ mRNA and protein was significantly increased in the 0.5 m M concentration HF group compared with Ctrl group?p<0.01?.There was no significant difference between HF group and HF+DMSO group?p>0.05?.Compared with the untreated HF group,the expression of P2Y₁₂ mRNA and protein in HF+Quercetin group was significantly decreased?p<0.01?.Western blot analysis showed that the expression of p-ERK protein was significantly increased in HF group compared with Ctrl group?p<0.01?.There was no significant difference in the expression of p-ERK between HF+DMSO group and HF group?p>0.05?.Compared with the untreated HF group,the expression of p-ERK protein in HF+Quercetin group was significantly decreased?p<0.01?.ELISA results showed that compared with Ctrl group,the IL-1 beta and TNF-alpha levels in HF group were significantly increased?p<0.01?.The IL-1 beta and TNF-alpha levels in HF+Quercetin group was significantly lower than that in HF group?p<0.01?.There was no significant difference between HF+DMSO group and HF group?p>0.05?.Conclusion:Successful preparation of curcumin nanoparticles with good dispersibility in water and slow release.The prepared curcumin nanoparticle reduced the P2Y₁₂ receptor up-regulated in the DRG of DM rats,decreased the expression of Cx43 and IL-1?in DRG of DM rats,decreased the phosphorylation of AKT,and inhibited the P2Y₁₂ receptor-mediated the transmission of nociceptive signaling in DM rats.Therefore,curcumin nanoparticles can effectively relieve the mechanical and thermal hyperalgesia in diabetic rats.Quercetin decreased the up-regulated P2Y₁₂ receptor in SGC of DRG,decreased the phosphorylation of ERK and improved the sensory nerve conduction velocity in DIO rats.In the high-fat cell injury model,quercetin reduced the up-regulated expression of P2Y₁₂ in SGCs,reduced the inflammatory cytokines IL-1?and TNF-?.