Mechanism Of Purine 2 Receptor Involved In Diabetic Cardiac Autonomic Neuropathy In Stellate Ganglion

Background:Diabetes mellitus?DM?as a global chronic disease,seriously affects people's health and quality of life.Long-term hyperglycemia in diabetes can lead to various organ system damage and serious complications.Diabetic cardiac autonomic neuropathy?DCAN?is a common complication.It is often ignored and misdiagnosed.Its pathogenesis is not clear,may be caused by many factors.Autonomic ganglia include sympathetic ganglia and parasympathetic ganglia.Nerve fibers from stellate sympathetic ganglion can distribute to the heart plexus and influence heart activity.Neurons from peripheral sympathetic ganglion are tightly surrounded by surrounding satellite glial cells?SGCs?.SGC is an important part of the noxious signaling pathway.The abnormal interaction of neuron-SGC is involved the pathological changes of peripheral sympathetic nerve.Noxious stimuli can cause the increase of ATP released by neurons.As a neurotransmitter,ATP acts on purine 2?P2?receptors on neurons and glial cells.P2 receptors contain P2X and P2Y receptors.It has been reported that P2X receptor-associated subtypes are involved in autonomic neuropathy.Our previous study found that cervical sympathetic ganglia were associated with diabetic autonomic neuropathy,but the mechanism by which sympathetic ganglia function in autonomic neuropathy of diabetic hearts was unclear.Objective:The purpose of this study was to explore the role of P2 purinergic receptors in stellate ganglia in DCAN and its mechanism via type 2 diabetic rats.Methods:Type 2 diabetic rat models were established.The rats were randomly divided into five groups:control group?Ctrl?,type 2 diabetes mellitus group?DM?,DM combined with P2X₃ short hairpin RNA?shRNA?treatment group?DM+P2X₃shRNA?,DM combined with P2Y₁₂2 shRNA treatment group(DM+P2Y₁₂ shRNA)and DM combined with plasmid vector treatment group?DM negative control group:DM+NC shRNA?.Experimental contents:?1?Measure the heart rate,blood pressure and heart rate variability;?2?Quanlitify the serum epinephrine by enzyme linked immunosorbent assay?ELISA?;?3?Observe the changes of mRNA and protein expression levels of P2Y₁₂ in stellate ganglion by Real-time PCR and Western blotting;?4?Immunofluorescence double-labeled method was used to detect the co-expression of P2Y₁₂ receptor and glial cell marker?glutamine synthetase,GS?;?5?Observe the changes of expression of P2X₃ in stellate ganglion by Real-time PCR,Western blotting and Immunohistochemistry;?6?Observe the changes of mRNA and protein expression levels of IL-1?and TNF-?in stellate ganglion by Real-time PCR and Western blotting;?7?Observe the changes of mRNA and protein expression of Cx43 in stellate ganglion by Real-time PCR and Western blotting;?8?Electron microscopy was used to observe the gap junctions?GJs?,and determine whether diabetic damage leads to gap junctions mediated coupling increased;?9?The phosphorylation of p38MAPK in SG was detected by Western blotting.Result:?1?Blood pressure and heart rate in the DM group were significantly higher than those in the Ctrl group?p<0.01?,P2X₃ shRNA or P2Y₁₂2 shRNA treatment decreased the elevated blood pressure and heart rate induced by DM?p<0.01?.There was no significant difference in blood pressure and heart rate between the DM+NC shRNA and DM group?p>0.05?;The heart rate variability in the DM group was significantly lower than that in the Ctrl group?p<0.01?,P2X₃ shRNA or P2Y₁₂2 shRNA treatment increased the reduced heart rate variability induced by DM?p<0.01?.There was no significant difference in heart rate variability between the DM+NC shRNA and DM group?p>0.05?.?2?ELISA result showed that compared with the Ctrl group,the concentration of serum epinephrine increased in the DM group.The concentration of serum epinephrine was lower in the DM+P2X₃ shRNA or DM+P2Y₁₂2 shRNA group than that in the DM group?p<0.01?.There was no significant difference between the DM+NC shRNA and DM group?p>0.05?.?3?Real-Time PCR and Western blotting results indicated that compared with the Ctrl group,the expression levels of P2Y₁₂ mRNA and protein in the DM rats were significantly higher than those in the Ctrl group?p<0.01?.After P2X₃ shRNA or P2Y₁₂2 shRNA treatment,the expression levels of P2Y₁₂2 receptor mRNA and protein were significant decreased?p<0.05?.There was no significant difference between the DM+NC shRNA and DM group?p>0.05?.?4?Double immunofluorescence staining results showed that P2Y₁₂ receptor in SG was co-expressed with GS.The co-expression fluorescence intensity of P2Y₁₂ receptor and GS in the DM and DM+NC shRNA groups was higher than that in the Ctrl group.The co-expression fluorescence intensity of P2Y₁₂ receptor and GS in the DM+P2Y₁₂2 shRNA and DM+P2X₃ shRNA group was lower than that in the DM group.?5?Real-time PCR,Western blotting and immunohistochemical results showed that the expression of P2X₃ receptor in the DM group was higher than that in the Ctrl group?p<0.01?,after treatment with P2X₃ shRNA or P2Y₁₂2 shRNA,the expression of P2X₃ receptor in SG was decreased?p<0.01?.There was no significant difference between the DM and DM+NC shRNA group?p>0.05?.?6?The expression levels of IL-1?and TNF-?mRNA and protein in SG were detected by Real-time PCR and Western blotting.Compared with the Ctrl group,the expression levels of IL-1?and TNF-?mRNA and protein in the DM group were significantly increased?p<0.05?.After P2X₃ shRNA or P2Y₁₂2 shRNA treatment,the expression levels of IL-1?and TNF-?mRNA and protein were significantly lower than those in the DM group?p<0.01?,there was no significant difference between the DM+NC shRNA and DM group?p>0.05?.?7?The results of Real-time PCR and Western blotting showed that the expression levels of Cx43 mRNA and protein in the DM rats were significantly higher than those in the Ctrl group?p<0.05?,after treatment with P2X₃ shRNA or P2Y₁₂2 shRNA,the expression levels of Cx43 mRNA and protein were significantly lower than those in the DM group?p<0.01?.There was no significant difference between the DM+NC shRNA and DM group?p>0.05?.?8?Electron microscopy results showed that compared with the Ctrl group,the number of gap junctions?GJs?in the DM group increased and the intercellular coupling enhanced.?9?Compared with the Ctrl group,the phosphorylation of p38 protein in the DM group increased?p<0.01?.After P2X₃shRNA or P2Y₁₂2 shRNA treatment,the phosphorylation of p38 decreased?p<0.01?.There was no significant difference in the phosphorylation of p38 between the DM+NC shRNA and DM group?p>0.05?.Conclusion:P2 receptors in the stellate ganglion participated in the pathological process of cardiac autonomic neuropathy in type 2 diabetic rats,and its mechanism may be related to the interaction of neuron-SGC and the activation of p38 MAPK pathway.P2Y₁₂ shRNA and P2X₃ shRNA attenuated cardiac autonomic neuropathy in type 2 diabetic rats.