Hmgcs2 And Pank1 Participate In The Regulation Of Fentanyl-induced Hyperalgesia In Rats

Clinically,opioid-induced hyperalgesia(OIH)is a tricky problem.As its mechanism remains unclear,there is no treated method untill now.Dorsal root ganglia(DRG)is the location of primary neurons in sensory transmission,and also is an integral part of the pain signal processing,coding and transmission.It has been suggested that DRG is also involved in OIH,but the specific mechanism is unknown.Based on the animal model,Hmgcs2(Hydroxymethylglutaryl-CoA synthase 2)and Pank1(Pantothenate kinase 1)were charactered in DRG and might be participated in OIH in rats.First,the OIH model was prepared by subcutaneous injection of fentanyl citrate in the rat neck.The significant difference of Hmgcs2 in the DRG between the control group and the OIH group was found by methylation sequencing.Secondly,it was detected by semi-quantitative PCR that the expression level of the Hmgcs2 gene was significantly up-regulated in DRG of OIH rats,which undoubtedly indicated that Hmgcs2 might be involved in the occurrence of hyperalgesia at the DRG level.Then,a knockout vector of Hmgcs2 was constructed using the CRISPR/Cas9 system and the lentivirus was prepared for intrathecal injection.Subsequently,the action potential of DRG cells was recorded by electrophysiological patch clamp experiment.We found that after incubated with fentanyl for 2 hours,the threshold currents and potential threshold amplitudes of action potentials of DRG cells which decreased significantly,which suggested the DRG cells were sensitized induced by fentanyl;while,threshold currents and potential threshold amplitudes of action potential after rat DRG cells injected with knockout Hmgcs2 gene lentivirus group were normal as the same treatment.Finally,the content of substance P in rat DRG cells treated with lentivirus was determined by enzyme-linked immunosorbent assay(ELISA).The substance P in the empty-loaded lentivirus group showed significantly increased after fentanyl incubation.The content of substance P in the Hmgcs2 knockout lentivirus group significantly decreased compared to the control group after fentanyl incubation.Besides,by methylation sequencing,it was also found that Pank1 in the OIH group was significantly different from the control group in the rat DRG.In the semi-quantitative PCR,Pank1 was up-regulated in the OIH group.Subsequently,a knockout vector of Pank1 was constructed,and the corresponding lentivirus was prepared for intrathecal injection.Then,the electrophysiological experiments were used to record the action potential of DRG cells.The threshold current and threshold potential amplitude required to trigger the action potential of DRG cells injected with the vacant lentiviral group were significantly increased after incubated with fentanyl for 2 hours.The corresponding threshold current and threshold potential amplitude of rat DRG cells injected with CRISPR-Pank1 lentivirus group decreased after fentanyl incubation.Finally,the substance P in rat DRG cells was significantly increased in the empty lentivirus group after fentanyl incubation determined by enzyme-linked immunosorbent assay(ELISA).While the content of P substance declined in CRISPR-Hmgcs2 group after incubation by fentanyl.In summary,we conclude that the Hmgcs2 and Pank1 genes in DRG are involved in the regulation of OIH.By decreasing the expression of Hmgcs2 and Pank1,fentanyl-induced hyperalgesia can be alleviated at a certain level.This study focused on the role of Hmgcs2 and Pank1 in the regulation of OIH on DRG,which is helpful to study the mechanism and signaling pathway of OIH and provide new potential therapeutic targets for OIH.