The Role And Mechanism Of Hmgcs2 And Pank1 In Opioid-induced Hyperalgesia

Pain is one of the biggest problems facing human beings.Opioids have been used as analgesics for thousands of years.In addition to their strong analgesic effects,opioids can also increase the body’s sensitivity to nociceptive pain,leading to hyperalgesia,a phenomenon known as opioid-induced hyperalgesia(OIH).OIH is an urgent clinical problem to be solved.Although the mechanism of OIH has been studied to some extent,its complete mechanism cannot be explained.Dorsal root ganglia(DRG)is the site of the primary afferent neuron of pain,an important part of the body’s transmission and regulation of pain signals,and a large number of studies have demonstrated that DRG is involved in the process of hyperalgesia.In the early stage,we found that Hmgcs2(Hydroxymethylglutaryl-Co A synthase2)and Pank1(Pantothhenate kinase 1)were significantly up-regulated in the DRG of the OIH rat model by methylation sequencing.We speculate that Hmgcs2 and Pank1 may be involved in the regulation of OIH process.While there are no reports that Hmgcs2 and Pank1 involve in the OIH process.In this study,we preliminarily explored the role and mechanism of these two genes in the rat OIH process.First,the rats were repeatedly injected with fentanyl,and we determined whether the rats developed hyperalgesia by measuring the changes in the mechanical pain threshold before and after the injection.We acutely isolated rat DRG cells and recorded their action potentials by electrophysiological patch-clamp technique,and we found that DRG cells in OIH rats were sensitized.Second,previous laboratory experiments demonstrated that Hmgcs2 and Pank1 were significantly up-regulated in DRG cells of OIH rats by semi-quantitative RT-PCR experiments.Therefore,we constructed gene knock-down plasmids of CRISPR-Hmgcs2 and CRISPR-Pank1 based on the CRISPR/Cas9 system.In this project,lentiviruses were prepared using CRISPR-Hmgcs2 and CRISPR-Pank1 plasmids,and the rats were injected intrathecally,and then repeatedly injected with fentanyl.Measuring changes in mechanical pain thresholds before and after injection,we found that intrathecal injection of CRISPR-Hmgcs2 and CRISPR-Pank1 lentiviruses could effectively inhibit fentanyl-induced hyperalgesia.After acute isolation of DRG cells,semi-quantitative RT-PCR experiments showed that intrathecal injection of CRISPR-Hmgcs2 and CRISPR-Pank1 lentiviruses could down-regulate the expressions of Hmgcs2 and Pank1 in rat DRG cells,and could inhibit the up-regulation of Hmgcs2 and Pank1 induced by fentanyl.Then we recorded the action potentials of DRG cells by electrophysiological patch-clamp experiments and analyzed their characteristic values,and found that intrathecal injection of CRISPR-Hmgcs2 and CRISPR-Pank1 lentiviruses could i n h i b i t t h e s e n s i t i z a t i o n o f D R G c e l l s i n d u c e d b y f e n t a n y l.In summary,we infer that Hmgcs2 and Pank1 in DRG may affect the sensitization of DRG cells by altering the occurrence of action potentials,thereby participating in the regulation of OIH in rats.This study aims to study the mechanism of Hmgcs2 and Pank1 involved in the process of OIH in rat DRG cells,expand the mechanism of OIH,and provide a potential targe for the treatment and diagnosis of OIH.