The Mechanism Of KCNN4-mediated Activation Of Spinal Dorsal Horn Neurons Involved In Inflammatory Pain

Objective: Chronic pain,the consequence of the dynamic interaction among psychological,physiological and social factors,can lead to tremendous personal and socioeconomic burden.Therefore,elucidating the internal mechanism of chronic pain is essential for the exploration of effective clinical treatment strategies.The development of pain is the result of the interaction of many factors.Peripheral sensory neuron sensitization induced by nociceptive stimulation and central sensitization caused by the changes of synaptic plasticity are the main mechanisms of chronic pain.Because the spinal cord is the primary center of pain signal processing,then the excitability of its neurons is an important basis for pain development.Potassium channel KCNN4 can regulate neuronal excitability by affecting cell membrane potential.While,whether KCNN4 is involved in inflammatory pain and its possible mechanism remain unclear,this project intends to explore the role and possible molecular mechanism of KCNN4 in the model of inflammatory pain induced by Complete Freund’s Adjuvant（CFA）.Methods: Male C57BL/6 mice（6-8 weeks old）were selected and randomly divided into four groups: Sham group（Sham group）,inflammatory pain group（CFA group）,inflammatory pain + negative control virus group（CFA + NC-LV group）and inflammatory pain + KCNN4 interference lentivirus group（CFA + KCNN4-LV group）.The mice in the Sham group were injected with 20 μL of normal saline into the sole of the right hind limb,while the mice in the CFA group were injected with 20 μL CFA.The CFA + KCNN4-LV group and the CFA + NC-LV group were injected with KCNN4 interfering lentivirus and negative control virus in the dorsal horn of the spinal cord,respectively.The CFA model was established on the 7th day after virus injection.The Paw withdrawal threshold（PWT）of the four groups was measured on days 0,1,3,5,and 7 after CFA injection.Then the mice were sacrificed after the pain threshold measurement.The co-localization of spinal KCNN4 and neuronal dendrite markers MAP2,c-fos were determined by using the immunofluorescence double standard staining,and expression of KCNN4 protein and m RNA in spinal cord was detected by Western blot and qRT-PCR,respectively.Result:1.Pain threshold measurement showed that there was no significant difference in the basic mechanical foot withdrawal threshold before operation（P > 0.05）.The PWT of CFA group began to decrease from the 1st day and the tendency was the most notable on the 3rd day,and gradually recovered from the 5th day,but there was still a statistical difference when compared with Sham group（P < 0.05）;while,the PWT in the CFA + KCNN4-LV group was lower than that in the Sham group,higher than that in the CFA + NC-LV group,and the difference was statistically significant（P < 0.05）.2.The results of qRT-PCR and Western blot demonstrated that in the CFA group the KCNN4 m RNA and protein expression were increased when compared with the Sham group（P < 0.05）;when compared with the CFA + NC-LV group,the KCNN4 m RNA and protein expression of CFA + KCNN4-LV group were decreased（P < 0.05）.3.The immunohistochemical staining showed that KCN4 and MAP2 were colocated in the dorsal horn of the spinal cord,and KCNN4 was located in the membrane of spinal dorsal horn neurons.Besides,there was a co-localization relationship between KCNN4 and c-fos in the dorsal horn of the spinal cord.The expression of c-fos in the dorsal horn of the spinal cord in inflammatory pain mice was increased.After lentivirus interferes with the expression of KCNN4,the expression of c-fos was down-regulated,indicating that the downregulation of KCNN4 reduces neural activationConclusion:1.The expression of KCNN4 in the spinal dorsal horn of mice with inflammatory pain was increased.After knockdown of KCNN4 by virus transfection,the paw withdrawal threshold increased with the downregulation of KCNN4.These results indicated that KCNN4 is involved in the development of inflammatory pain.2.The expression of c-fos in the spinal dorsal horn of mice with inflammatory pain decreased with the downregulation of KCNN4,indicating that KCNN4 is involved in the development of inflammatory pain by promoting the activation of spinal dorsal horn neurons.