| Background and Objective：Diabetes is a chronic diseases characterized byhyperglycemia, sustained hyperglycemia leads to metabolic disorders of the bodytissue. Diabetic cardiac autonomic neuropathy (DCAN) is a common chroniccomplications of diabetes, which can cause life-threatening serious consequencessuch as myocardial infarction, sudden cardiac death and so on. Extracellularnucleotides such as adenosine triphosphate (ATP), uridine triphosphate (UTP) can acton P2receptors, which have an important role in the transmission of nervousinformation. P2receptors are divided into ligand-gated ion channel receptors (P2Xreceptors) and G protein-coupled receptors (P2Y receptors). ATP and P2X receptors(acted by ATP) related to the transmission of cardiac sympathetic information insuperior cervical ganglia. This study aims to understand the effects of sinomenine onthe pathophysiological changes mediated by P2X2and P2X3receptors in superiorcervical ganglia under conditions of hyperglycemia and hyperlipid.Methods:1. Homology modeling and molecular docking experiments: Thethree-dimensional structure model of P2X2, P2X3(P2X2/3) receptors was constructedby homology modeling. The small molecule drugs of docking P2X2, P2X3receptorswith lower freedom were screened by molecular docking method.2. Animalexperiments: Type2diabetic rat model was used in the experiment. The rats wererandomly divided into control group (Ctrl), diabetic model group (DM), then thediabetic model group (DM) were randomly divided into diabetic model+salinegroup (DM+NS) and diabetic model+sinomenine treatment group (DM+SIN)(40mg/kg) after type2diabetic rat model was successfully constructed at the end of7thweek, control group (Ctrl) treated with saline, named saline control group (Ctrl+NS).Experimental content:(1) The changes of fasting plasma glucose and postprandialblood glucose in the rats were measured during the experiment.(2) The heart rate,blood pressure and heart rate variability in each group of rats were measured.(3) Theexpressions of P2X2and P2X3receptors in rat superior cervical ganglia were detected by immunohistochemistry and western blot.(4) The expression of P2X2and P2X3receptors mRNA in rat superior cervical ganglia were detected by reversetranscription-polymerase chain reaction (RT-PCR).(5) The effects of sinomenine onATP-activated currents in the superior cervical neurons were detected by using wholecell patch-clamp method.Results:1. The results of homology modeling and molecular docking:(1) P2X2,P2X3models were obtained by homology modeling. More than90%of the aminoacids was located in the permitted area tested by PROCHECK, and the amino acidresidues of ATP binding pocket did not appear in the not permitted region.(2) Bymolecular docking of sinomenine and P2X2, P2X3receptors, the free energy in thedocking system was greatly reduced and its absolute value was the same to thechange value of binding free energy between P2X2, P2X3receptors and knownagonist, it may show to have strong interactions.2. The results of animal experiments:(1) The values of fasting plasma glucose and postprandial glucose in diabetic modelrats had been increased to reach the standard of diabetes diagnosis, indicating thesuccessfully modeling of type2diabetic rats. The values of fasting plasma glucoseand postprandial glucose in Ctrl rats were within normal range. After being treatedwith sinomenine in diabetic rats, the values of fasting plasma glucose andpostprandial glucose in DM+SIN group were significantly reduced in comparisonwith those in DM+NS group (p <0.01). There was no significantly differencebetween DM+SIN group and Ctrl+NS group (p>0.05).(2) The values of heart rate,blood pressure in diabetic rats were increased in comparison with those in control rats.The values of heart rate, blood pressure in DM+SIN group were decreased incomparison with those in DM+NS group (p <0.05). There was no significantlydifference between DM+SIN group and Ctrl+NS group (p>0.05). LF/HF indiabetic model group was significantly higher than that in control group (P <0.01),VLF, LF, HF and TP in diabetic group were significantly lower than those in controlgroup (p <0.01). After being treated with sinomenine in diabetic rats, above abnormalchanges had been significantly improved in comparison with those in diabetic rats (p<0.01).(3) The results of immunohistochemistry and Western blot experimentsshowed that the expression levels of P2X2and P2X3receptors in the superior cervical ganglia of diabetic rats were increased in comparison with those in control rats (p<0.01). The expression levels of P2X2and P2X3receptors in DM+SIN group weredecreased in comparison with those in DM+NS group (p <0.01).(4) The results ofRT-PCR experiments showed that the expression levels of P2X2and P2X3mRNA inthe superior cervical ganglia of diabetic rats were increased in comparison with thosein control rats (p <0.01). The expression levels of P2X2and P2X3mRNA in DM+SIN group were decreased in comparison with those in DM+NS group (p <0.01).(5)The results of whole-cell patch-clamp experiments showed that sinomenine inhibitedthe ATP-activated currents in the superior cervical ganglion neurons.Conclusion: Sinomenine has an inhibitive effect on the signal transmission ofdiabetic cardiovascular autonomic neuropathy which mediated by P2X2/3receptor insuperior cervical ganglia. |
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