The Role And Mechanism Of P2Y₁₃ Receptor In Cervical Sympathetic Ganglionic Neuropathy Induced By HIV-1 Envelope Glycoprotein Gp120

Background and Objective:Human immunodeficiency virus（HIV）infection has become a serious global health problem.The death toll caused by the complications associated with Acquired Immunodeficiency syndrome（AIDS）,such as cardiovascular diseases,is increasing year by year,which become the important threat of death to AIDS patients.HIV-1 envelope glycoprotein gp120 as a pathogenic protein of HIV,can produce neuropathic changes similar to HIV.Studies have shown that cardiovascular disease due to HIV infection is associated with the sympathetic activity,and HIV antigen was found in the sympathetic ganglion of HIV infection patient.However,the effect and its molecular mechanism of gp120 on cervical sympathetic ganglion is not clear.It was found that P2Y₁₃receptor is involved in the occurrence of diabetic neuropathic pain,lipid-induced changes in the number of gastrointestinal neurons and neuronal differentiation,and is involved in ADPβs-induced cardiac sympathetic dysfunction,suggesting that P2Y₁₃receptor may be a potential therapeutic target for neurological diseases.Therefore,this study aims to investigate the role and molecular mechanism of P2Y₁₃receptor in gp120-induced cervical sympathetic ganglion pathological changes,hope to provid experimental basis for the prevention and treatment of HIV infection complicated with cardiac autonomic neuropathy and cardiovascular dysfunction.Methods:Animal experiment method:This study used HIV-1 envelope glycoprotein gp120 to stimulate cervical sympathetic nerve to establish a rat model of sympathetic nerve injury.Experimental animals were randomly divided into:normal group（control）;sham operation group（sham）;gp120 group（gp120）;gp120 and P2Y₁₃sh RNA processing group(gp120+P2Y₁₃sh RNA);gp120 and negative control processing group（gp120+NC sh RNA）,n=10 in each group.Experimental contents:（1）Indirect tail sleeve method was used to detect the heart rate,blood pressure.Heart rate variability（HRV）of rats was measured by ECG;（2）The discharge of cardiac sympathetic nerve was recorded;（3）Real-time PCR was used to detect the m RNA level of P2Y₁₃receptor in SCG.（4）Western blot was used to detect the expression of P2Y₁₃receptor,NLRP3 inflammasome,Caspase-1,GSDMD,inflammatory factors IL-1βand IL-18 in SCG.（5）The co-expression of P2Y₁₃receptor and neuron marker Neu N in SCG was detected by immunofluorescence double label method.Cell experiment:In this study,HIV-1 envelope glycoprotein gp120 was used to establish the cell damage model in PC12.The experimental groups were as follows:normal group（control）,gp120 group（gp120）,gp120 and P2Y₁₃sh RNA processing group(gp120+P2Y₁₃sh RNA),gp120 and negative control group（gp120+NC sh RNA）.Experimental content:（1）Screening of HIV-1 gp120 concentration:The effects of various concentrations of gp120（0,0.2,0.4,0.8,1.6 nmol/L）on cell survival were detected by CCK-8 method,and the expression of P2Y₁₃receptor protein was detected by western blot to select the most appropriate modeling concentration of gp120.（2）Real-time PCR was used to detect the expression of P2Y₁₃receptor m RNA in PC12 cells.（3）The expression levels of P2Y₁₃receptor,NLRP3 inflammasome,Caspase-1,GSDMD,ASC,inflammatory factors IL-1βand IL-18 in PC12 cells were detected by western blotting method.（4）Enzyme linked immunosorbent assay（Elisa）was used to detect the release of IL-1βand IL-18 in the supernatant of PC12 cell culture.（5）Intracellular calcium detection kit（Best Bio,Shanghai,China）was used to observe the changes of intracellular calcium ions concentration in control group and gp120 group after treatment with P2Y₁₃agonist ADP and P2Y₁₃inhibitor MRS2211.（6）The expression of P2Y₁₃receptor in PC12 cells was detected by immunofluorescence method.Results:Animal experiment results:（1）Blood pressure and heart rate results showed:The blood pressure and heart rate of gp120 group were higher than those of control group（P<0.01）,while the elevated blood pressure and heart rate in gp120group were inhibited by P2Y₁₃sh RNA（P<0.01）but not by NC sh RNA（P>0.05）.HRV results showed that the LF/HF ratio of gp120 group was significantly higher than that of control group（P<0.01）.P2Y₁₃sh RNA can decrease LF/HF of gp120group（P<0.01）.There was no significant difference in LF/HF between gp120+NC sh RNA group and gp120 group（P>0.05）.（2）Cardiac sympathetic nerve discharge results showed:Compared with the control group,the discharge amplitude in gp120group was significantly increased（P<0.01）,while it was significantly decreased in gp120+P2Y₁₃sh RNA group（P<0.01）.There was no significant difference between gp120+NC sh RNA group and gp120 group in sympathetic nerve discharge（P>0.05）.（3）Real-time PCR results showed that the P2Y₁₃m RNA level in SCG of gp120 group was significantly higher than that of control group（P<0.001）,while it can be reduced by application of P2Y₁₃sh RNA on gp120 group（P<0.001）.But NC sh RNA had no the same effect as P2Y₁₃sh RNA（P>0.05）.（4）Western blot results showed that the protein expression levels of P2Y₁₃receptor,NLRP3 inflammassome,Caspase-1,GSDMD,IL-1βand IL-18 in SCG of gp120 group were significantly increased compared with control group（P<0.01）.P2Y₁₃sh RNA could decrease the elevated expression levels of above proteins in gp120 group（P<0.01）,while NC sh RNA had no the same effect as P2Y₁₃sh RNA（P>0.05）.（5）Immunofluorescence results showed that the co-expression of P2Y₁₃receptor and Neu N in SCG in gp120 group was significantly higher than that in control group（P<0.01）.Compared with gp120group,the co-expression level of gp120+P2Y₁₃sh RNA group was decreased（P<0.01）.gp120+NC sh RNA group had no significant difference compared with gp120 group（P>0.05）.Cell experiment results:（1）CCK-8 results showed that 0.2 nmol/L gp120had no significant effect on cell survival compared with control group（P>0.05）;The cell viability of 0.4,0.8,1.6 nmol/L gp120 group was about 90%,80%,50%,respectively.Western blot results showed that the expression of P2Y₁₃receptor protein increased most obviously in 0.8 nmol/L gp120 group.Therefore,0.8 nmol/L gp120 was selected as the optimal concentration inducing cell injury model.（2）Real-time PCR results showed that the m RNA level of P2Y₁₃in gp120 group was significantly higher than that in control group（P<0.01）.P2Y₁₃sh RNA could decrease significantly the P2Y₁₃m RNA of gp120 group（P<0.01）,while NC sh RNA had not the same effect（P>0.05）.（3）Western blot results showed that the protein expression levels of P2Y₁₃receptor,NLRP3 inflammassome,Caspase-1,GSDMD,ASC,IL-1βand IL-18 in gp120 group were significantly increased compared with control group（P<0.01）.The expression levels of above proteins in gp120+P2Y₁₃sh RNA group were significantly lower than those in gp120 group（P<0.01）.gp120+NC sh RNA group had no significant difference compared with gp120 group（P>0.05）.（4）Elisa results showed that the contents of IL-1βand IL-18 in supernatant of PC12 cells in gp120 group were increased compared with control group,and the difference was statistically significant（P<0.01）,while decreased significantly after application of P2Y₁₃sh RNA（P<0.01）in gp120 group.（5）Intracellular calcium concentration results showed that the intracellular calcium concentration in gp120 group was increased after adding P2Y₁₃agonist ADP compared with control group.It can be blocked by P2Y₁₃inhibitor MRS2211（P<0.01）.（6）Immunofluorescence results showed that the expression level of P2Y₁₃receptor in gp120 group was significantly higher than that in control group（P<0.01）.Compared with gp120 group,the expression level of gp120+P2Y₁₃sh RNA group was decreased（P<0.01）.gp120+NC sh RNA group had no significant difference compared with gp120 group（P>0.05）.Conclusion:P2Y₁₃receptor is involved in gp120-induced sympathetic neuropathy,and its molecular mechanism is implicated in the activation of NLRP3inflammasome followed by Caspase-1 activation,GSDMD formation,and the release of inflammatory factors IL-1βand IL-18,which is a classical pyroptosis pathway.