G9a In The Anterior Cingulate Cortex Is Involved In Postoperative Incision Pain By Regulating Voltage-gated Potassium Ion Channel Kv1.4

BackgroundPain is a type of unpleasant feeling and emotional sensation.Because of its complex mechanism,it has always been a difficult problem in the medical community.Whether it is a peripheral sensitization mechanism or a central sensitization mechanism,the ultimate nociative stimulus needs to be transmitted from the sensory cortex to a higher neocortex to integrate into unpleasant emotions to perceive pain.Previous studies have shown that the anterior cingulate cortex（ACC）is a key brain region for handling pain and its associated emotions,however the molecular regulatory mechanisms that contribute to the enhancement of ACC excitability are unclear.The voltage gated potassium channels（Kv）family is the cornerstone of the formation and maintenance of cell membrane potentials,and Kv1.4,as an important member of Kv,is involved in regulating the excitability of neurons.G9a is a histone dimethyltransferase that often causes chromatin condensation and inhibits gene expression.In this study,we intend to explore whether G9a in glutamatergic neurons in ACC in pain state participates in the occurrence and development of pain by inhibiting the expression of Kv1.4,to provide new ideas for the study of pain mechanism and clinical treatment.PurposesTo explore whether G9a regulates the excitability of glutamatergic neurons in ACC by acting on Kv1.4,thereby participating in the occurrence and development of pain.Methods1.Establish a rat model of plantar incision pain and observe the excitation of glutamatergic neurons in rat ACC.2.Stereotactic injection of glutamatergic neurons in rat ACC inhibits the activity of glutamatergic neurons and detects its effect on rat pain behavior and related molecules.3.Stereotactic injection of glutamatergic neuron excitatory chemical genetic virus in rat ACC,excitatory activity of glutamatergic neurons,and detect its effect on rat pain behavior and related molecules.4.The expression changes of Kv1.2 protein,Kv1.4 protein and Kcna4 m RNA in rat ACC of plantar incision pain model were detected.5.By cytology experiments,the Kv1.4-si RNA that can effectively knock down Kv1.4 is screened out in PC12 cells.The expression of Kv1.4 protein and Kcna4 m RNA and the pain behavior of rats after localization injection of Kv1.4-si RNA in rat ACC were detected.6.After high-throughput RNA sequencing of rat ACC tissue,the expression of G9a protein and Ehmt2 m RNA in rat ACC after plantar incision was detected and cell typing was detected.7.By cytology experiments,it is verified in PC12 cells that G9a-si RNA can be effectively knocked down.The expression changes of G9a and Kv1.4 proteins and Ehmt2 m RNA and Kcna4 m RNA in ACC were detected in ACC,and the effect on pain behavior in rats was detected.8.Detection of expression changes of G9a and Kv1.4 proteins and Ehmt2m RNA and Kcna4 m RNA in ACC after localization injection of G9a-LV,lentivirus G9a-LV overexpressed with G9a,and effect on pain behavior.9.Detection of the histone methylation product H3K9me2 of G9a on the targeting of Kv1.4 and the co-localization distribution of G9a and Kv1.4 in ACC.Results1.Successfully established a model of plantar incision pain induced by plantar incision injury,and the excitation of glutamatergic neurons in the model group rat ACC increasedThe thermal paw withdrawal latencies（PWL）and the mechanical paw withdrawal threshold（PWT）of rats in the Incision group were significantly reduced at 6 h postoperatively,gradually permissioned at the 1st,3rd,5th day after surgery,and returned to normal at postoperative 7th day,when it was compared to the Control group,indicating successful molding.Immunofluorescence technology detected the activation of neurons in the right ACC of rats and found that the stained localization of glutamatergic neurons and c-Fos in the right ACC of rats in the model group was significantly increased compared with the control group,displaying that contralateral ACC glutamatergic neurons may be involved in the development of incisional pain.2.Inhibition of glutamatergic neuronal excitation in ACC relieves painCompared with the threshold of basal pain,the changes in PWT and PWL in rats28 day after the injection of inhibitory chemogenetic virus in the right ACC stereotaxic injection（no intraperitoneal injection of CNO）was not significant.Compared with the intraperitoneal injection of normal saline（+Saline）group,the PWT on the operative side of rats after intraperitoneal injection of CNO（+CNO）activation of inhibitory chemical genetic virus was elevated,PWL was elevated,compared with the pre-CNO injection of intraperitoneal injection（-CNO）,the PWT on the operative side of rats after injection of CNO was elevated,and PWL was elevated,indicating a relief of pain symptoms.The results of the immunofluorescence test displayed that when compared to the intraperitoneal injection of saline（+Saline）group,the expression of c-Fos in the right ACC of rats with intraperitoneal injection of CNO（+CNO）was reduced,and the Stained reduction with m Cherry was reduced,indicating a decrease in glutamate neuron activation in the ACC of rats in the intraperitoneal injection CNO（+CNO）group.3.Excitement of glutamate neurons in ACC can lead to hyperalgesiaThe changes were not significant that in PWT and PWL in rats 28 d after the stereotaxic injection of excitatory chemogenetic virus on the right side of the ACC no intraperitoneal injection of（CNO）when we compared them with the threshold of basal pain.Compared with the intraperitoneal injection of normal saline（+Saline）group,the PWT on the operative side of rats after intraperitoneal injection of CNO（+CNO）activated excitatory chemical hereditary virus was elevated,PWL was elevated,compared with before intraperitoneal injection of CNO（-CNO）,the PWT on the operative side of rats after injection of CNO was elevated,and PWL was elevated,indicating a relief of pain symptoms.The results of the immunofluorescence test revealed that compared with the intraperitoneal injection of saline（+Saline）group,the expression of c-Fos in the right ACC of rats with intraperitoneal injection of CNO（+CNO）was increased,and the co-stained with m Cherry was increased,indicating that the activation of glutamate neurons in the ACC of rats in the intraperitoneal injection CNO（+CNO）group was increased.4.Plantar incision injury caused a decrease in Kv1.4 expression in the right ACC of ratsThe Western Blot results revealed a statistically different decrease in Kv1.2proteins in the postoperative 7 d compared with 0 d.Kv1.4 proteins in the postoperative6 h,1 d and 3 d after the operation Kv1.4 protein expression level gradually decreased,in the postoperative 3 d to the minimum,in the postoperative 5 d tends to recover,the postoperative 7d basically returned to normal,similar to the rat behavior trend,so the follow-up experiments selected Kv1.4 proteins for further study.The results Immunofluorescence staining revealed that the relative intensities of Kv1.4 proteins in the right ACC in the Incision group was weakened compared to the Control group.We can tell that the expression of Kcna4 m RNA in the right ACC of rats in the Incision group was reduced from the q RT-PCR experiment when compared to the Control group.5.Inhibition of expression of Kv1.4 in rat ACC induces hyperalgesiaIn PC12 cells,the Western Blot test results revealed that the Kv1.4-si RNA-1 group had the most significant decrease in Kv1.4 protein compared with the Naive group,so it was used in subsequent rats in vitro experiments and directly labeled with Kv1.4-si RNA.In rats,compared with the Naive group,the PWT of rats in the Kv1.4-si RNA group decreased at the 1st,3rd and 5th days after si RNA injection all decreased at the time,most significantly decreased at the 3rd,and basically returned to normal at the 7^thday,and the PWL and PWT trends were similar,that is,compared with the Naive group,the PWL of rats in the Kv1.4-si RNA group was reduced at the 1 d,3 d and 5 d after si RNA injection,and the decrease was most significant at the 3rd,At 7 d,the results of the Western Blot test showed that the Kv1.4 protein expression level in the Kv1.4-si RNA group decreased when it was compared to the group of Naive,besides,according to the q RT-PCR test results we can learn the Kv1.4-si RNA group Kcna4 m RNA expression level decreased compared with the Naive group.6.G9a（Ehmt2）expression increased in rat ACC after plantar incisionHigh-throughput RNA sequencing techniques were found to have increased expression of histone lysine methyltransferase 2（Ehmt2）m RNA in postoperative ACC in rats in the Incision group in the 3rd day postoperative ACC compared with the Control group.The results of q RT-PCR experiments indicated that the level of Ehmt2m RNA in the 3rd postoperative ACC of rats in the Incision group was elevated when compared to the Control group.The Western Blot test results indicated that the contrast with 0 d,the G9a protein expression level gradually increased at the 6 h,1 d and 3 d after surgery,rose to the highest in the 3rd day after surgery,tended to recover in the5th day after surgery,and basically returned to normal at the 7th day after surgery.The results of immunofluorescence technology and fluorescence in situ hybridization that the relative intensity of Kv1.4 proteins and the relative intensity of Ehmt2 m RNA in the right ACC of rats in the Incision group were enhanced when compared to the Control group.Immunofluorescence staining results revealed that G9a was Stained with Neu N,not with GFAP（astrocytes）and IBA1（microglia）.7.Inhibition of G9a expression can restore the expression of Kv1.4 and alleviate pain-related symptomsIn PC12 cells,the Western Blot assay results revealed that the G9a protein expression in the G9a-si RNA group was reduced and the expression of the Kv1.4 proteins was elevated compared with the Naive group.In rats,contrasted with the Incision group,the PWT of rats in the Incision+G9a-si RNA group was increased in the 1^st day,3^rd day and 5^th day after si RNA injection,and basically returned to normal at 7^th day,and the PWL was similar to the change trend of PWT,that is,PWL was increased in the1^st day,3^rd day and 5^th day after si RNA injection,and basically returned to normal at the 7^th day.The Western Blot test results displayed that at the 3rd day after plantar incision,the G9a expression of the Incision+G9a-si RNA group decreased and the expression of Kv1.4proteins was elevated compared with the Incision group.The q RT-PCR test results indicated that in the 3rd day after si RNA microinjection,the Expression of Ehmt2m RNA in the Incision+G9a-si RNA group decreased and the expression of Kcna4m RNA was elevated compared with the Incision group.8.Overexpression of G9a in ACC inhibits Kv1.4 expression and induces hyperalgesiaContrasted with the Naive group,when the G9a-Lv group mice were mechanically stimulated with 0.07 g and 0.4 g of fibrous filaments on the left posterior foot,the mechanical foot withdrawal frequencies（PWF）gradually increased in the 3^rd day,the5^th day and the 7^th day after G9a-Lv injection,and the PWL was similar to the change trend of PWF,that is,compared with the Naive group,the PWL of the naive+G9a-Lv group mice decreased in the 3^rd day,the 5^th day and the 7^th day after G9a-Lv injection.It was shown that ACC injection of G9a-Lv caused hyperalgesia in mice.The Western Blot detection results revealed that when compared to the Naive group,G9a protein expression was elevated and Kv1.4 protein expression decreased in the 7th day after G9a-Lv microinjection,in the right ACC of mice.The q RT-PCR detection results revealed that the expression of Ehmt2 m RNA in the right ACC in the 7^th day after G9a-Lv microinjection was elevated and the expression of Kcna4 m RNA was decreased when contrasted to the Naive group.9.G9a in rat ACC affects the expression of Kcna4 by H3K9me2CHIP experimental results revealed that H3K9me2 is bound to the Kcna4promoter region.After the ACC of rats was stained by Ehmt2 m RNA fluorescence in situ hybridization,we retained it with Kv1.4 proteins by immunofluorescence technology,the results indicated that Ehmt2 was Stained with Kv1.4.ConclusionBy inhibiting the expression of Kv1.4,G9a leads to an increase in the excitability of glutamatergic neurons in ACC,involved in the occurrence and development of pain.