Protective Effects Of Imperatorin On Satellite Glial Cells From Cytotoxicity Induced By High Fat

Background:The increasing number of obese patients has become an increasingly serious worldwide health problem.The neurons in the ganglia of the peripheral nervous system are usually completely surrounded by satellite glial cells（SGCs）In the obese state,activated SGCs release cytokines such as interleukin-1β（IL-1β）and tumor necrosis factor-α（TNF-α）,which can easily spread to neighboring neurons and increase their excitability,thereby transmitting nociceptive signals.P2X4 receptors are widely expressed in SGCs of stellate ganglion and dorsal root ganglion（DRG）and are involved in the transmission of noxious signals.Imperatorin（IMP）is a furanocoumarin that has been shown to have a variety of pharmacological effects,such as anti-inflammatory,cardioprotective effects.Our laboratory previous studies have found that IMP can down-regulate the expression of P2X4 receptors on SGCs in stellate ganglion of obese rats and improve obesity-induced pathologic injury of cardiac sympathetic nerve.However,the protective effect of IMP on SGCs in vitro culture from the cytotoxicity induced by high fat and its possible mechanism remain unclear and require further study.Objective:In this experiment,we investigated the mechanism of P2X4 receptor-mediated high-fat-induced injury in SGCs by co-culturing SGCs of dorsal root ganglia with high-fat（HF）at the cellular level,and observed the effect and possible mechanism of imperatorin on P2X4 receptor-mediated high-fat-induced injury in SGCs to explore new methods for the prevention and treatment of obesity and metabolic syndrome.Methods:The experimental groups were divided:SGCs normal control group（Ctrl group）;imperatorin treatment group（IMP group）;high fat treatment group（HF group）;high fat+imperatorin treatment group（HF+IMP group）;high fat+P2X4sh RNA treatment group（HF+P2X4 sh RNA group）;high fat+NC sh RNA treatment group（HF+NC sh RNA group）.Experiment content:（1）The expression of glial fibrillary acidic protein（GFAP）in cultured SGCs was detected by immunofluorescence and the purity of primary satellite glial cells was identified.（2）Screening of HF concentration:The effects of different HF concentration treatment groups（0.2 m M,0.4 m M,0.6 m M,0.8 m M,1.0 m M）and treatment time of 24 h and 48 h on cell survival were detected by CCK-8 method,and the most suitable modeling concentration was selected.（3）Screening of imperatorin concentration:CCK-8 method was used to detect the effect of imperatorin concentration（1μM,10μM,100μM,1000μM）on cell survival for 24 h,and the concentration range of imperatorin was screened out.Then,the concentration gradient of imperatorin（0μM,10μM,20μM,40μM,80μM,160μM）was set up with the selected HF concentration as the model,and the concentration of imperatorin was screened by CCK-8 method.（4）The effects of imperatorin on the survival rate of SGCs induced by HF were detected by CCK-8 method（Ctrl group,high fat group,high fat+imperatorin group）.（5）Real-time PCR and protein blotting（Western Blot,WB）were used to detect changes in the expression of P2X4 m RNA and protein in SGCs.The co-expression of P2X4 and Glial fibrillary acidic protein（GFAP）was detected by immunofluorescence double-labeling method in SGCs of each group.（6）Flow cytometry was used to detect the apoptosis of SGCs in each group.（7）BBCell Probe（?）F03 fluorescent probe method was used to detect the changes of Ca²⁺concentration in SGCs of each group.（8）The changes of ROS content in SGCs were detected by fluorescence method.（9）Enzyme linked immunosorbent assay（ELISA）was performed to measure the changes of interleukin 1β（IL-1β）,tumor necrosis factorα（TNF-α）,and interleukin18（IL-18）release in the supernatant of SGCs in each group and in the supernatant of neuronal cells in the lower chamber of the Transwell co-culture system.（10）WB detection of IL-1β,TNF-α,NOD-like receptor protein 3（NLRP3）,cysteine aspartate protein hydrolase-1（Caspase-1）,GSDMD（Gasdermin-D）,IL-18,p38 MAPK and p-p38 MAPK protein expression changes in SGCs.Results:（1）Purity identification of SGCs:The purity of primary SGCs was identified by immunofluorescence.The results showed that the purity of satellite glial cells was more than 95%,which met the requirements of later experiments.（2）Screening of HF concentration:CCK-8 test results showed that after 24hours of action,compared with the Ctrl group,the survival rate of HF treated cells at a concentration of 0.4 m M was about 90%,while the survival rate of HF treated cells at a concentration of 0.6 m M decreased to about 75%（p<0.01）,and the survival rate of HF treated cells at a concentration of 0.8 m M decreased to about 65%（p<0.01）.After 48h,compared with Ctrl group,the survival rate of cells treated with 0.4m M HF was about 85%,while the survival rate of cells treated with 0.6m M HF was about65%（p<0.01）,and the survival rate of cells treated with 0.8m M HF was about 60%（p<0.01）.Therefore,we used 0.6m M HF to treat SGCs for 24 h to establish a high-fat cell injury model.（3）Screening of imperatorin concentration:The results of CCK-8 showed that there was no significant difference in cell viability between 1μM,10μM and 100μM imperatorin treatment groups and Ctrl group（p>0.05）.Compared with Ctrl group,the cell survival rate of 1000μM imperatorin treatment group decreased to about 60%（p<0.01）.Therefore,we used 0.6m M HF as a model to set a concentration gradient（0μM,10μM,20μM,40μM,80μM,160μM）between 0μM and 160μM to determine the concentration of imperatorin.The results showed that 20μM imperatorin began to affect cell activity,which was statistically different from the Ctrl group（p<0.05）,and40μM imperatorin treatment group was significantly different from the Ctrl group（p<0.01）.The 80μM imperatorin treatment group began to show cytotoxicity.Therefore,we selected the concentration of 40μM as the concentration of imperatorin.（4）Changes in P2X4 receptor expression:P2X4 m RNA and P2X4 protein expression levels were significantly higher in the HF group than in the Ctrl group（p<0.001）;while IMP treatment and P2X4 sh RNA treatment both significantly reduced P2X4 expression（p<0.001）;there was no significant difference between the HF+NC sh RNA group and the HF group（p>0.05）.P2X4 was co-expressed with GFAP in SGCs;the co-expression level of P2X4 and GFAP was significantly increased in HF group compared with Ctrl group（p<0.001）;while IMP treatment and P2X4 sh RNA treatment both significantly decreased the co-expression of P2X4 and GFAP（p<0.001）;there was no significant difference in HF+NC sh RNA group compared with HF group（p>0.05）.（5）Changes in inflammatory factor levels:both protein blotting and ELISA results showed that the expression levels of IL-1β,TNF-α,and IL-18 were significantly increased in the high-fat-treated group compared with the normal control group（p<0.01）,while IMP treatment and P2X4 sh RNA treatment both inhibited the expression of IL-1β,TNF-α,and IL-18 in SGCs（p<0.01）.There was no significant difference in the HF+NC sh RNA group compared with the HF group（p>0.05）.（6）The results of flow cytometry showed that high lipid significantly increased the apoptosis rate of SGCs compared with the control group（p<0.01）;while IMP treatment and P2X4 sh RNA treatment both decreased the apoptosis rate of SGCs（p<0.01）;there was no significant difference between HF+NC sh RNA group and HF group（p>0.05）.（7）The results of intracellular Ca²⁺concentration showed that the intracellular Ca²⁺concentration was significantly increased in the HF group compared with the Ctrl group（p<0.01）,while IMP treatment and P2X4 sh RNA treatment both reduced intracellular Ca²⁺concentration（p<0.01）;there was no significant difference between the HF+NC sh RNA group and the HF group（p>0.05）.（8）The results of intracellular ROS content showed that the intracellular ROS content was significantly increased in HF group compared with Ctrl group（p<0.01）;while IMP treatment and P2X4 sh RNA treatment reduced the intracellular ROS content（p<0.01）;there was no significant difference between HF+NC sh RNA group and HF group（p>0.05）.（9）Cellular pathway results showed that the expression levels of NLRP3,Caspase-1,GSDMD,and p38 MAPK proteins were significantly increased in the HF group compared with the Ctrl group（p<0.01）,while IMP treatment and P2X4 sh RNA treatment both inhibited the expression of the above proteins（p<0.01）,and there was no significant difference between the HF+NC sh RNA group and the HF group（p>0.05）.Conclusion:P2X4 receptor can mediate the cell injury of SGCs induced by high fat,cause the release of inflammatory factors,increase ROS content,increase intracellular Ca²⁺concentration,increase apoptosis,activate of NLRP3/Caspase-1 pathway to promote cell pyroptosis,and enhance phosphorylation of p38 MAPK protein,and ultimately lead to cell injury;IMP intervention can reverse the above changes and ameliorate the cell injury of SGCs induced by high fat,probably by downregulating P2X4 receptor expression,alleviating the level of inflammatory factors,and inhibiting NLRP3/Caspase-1 pathway to reduce cell pyroptosis.Therefore,P2X4 receptor is expected to be a therapeutic target for the treatment of obesity and metabolic syndrome,and IMP is also expected to be a potential therapeutic agent for obesity.