Let-7b Regulates Electroacupuncture Tolerance By Targeting Penk1 Gene In Rats

Electroacupuncture(EA)analgesia is one of the most importantly original achievements in Acupuncture Science in the 20 th century.Owing to its features of safety,economy,and less physiological disturbance,EA has been widely used in the remission of surgical pain and the treatment of painful diseases.Researches have confirmed that EA analgesia is a physiological process under the combinative control of neural and humoral regulation by modern neurobiology.Moreover,early studies found that neurotransmitters in the central nervous system are involved in the regulation of analgesia,and the following studies have found that neuromodulators,especially endogenous opioid peptides,have played a more important role in the EA analgesia.EA has a good analgesic effect,but this effect can easily become attenuated after prolonged or repeated EA stimulations,resulting in EA tolerance(EAT),about EAT many studies focused on opiate peptides,anti-opioid peptides and their receptors.MiRNA has been verified participate in morphine tolerance,while chronic EAT and morphine tolerance have a cross tolerance,according to the correlation between chronic EAT and morphine tolerance,recent study from our laboratory has confirmed that miRNAs(including let-7b)are also involved in EAT.Therefore,to further exploring the underlying mechanism of miRNAs involved in EAT in the protein level can help to find an effective way to reducing the EAT,reduce its adverse effects,and promote the popularization and application of EA technology.MiRNAs can be paired with the 3’-UTR of target gene through the base complementary pair,interfering with the translation of target gene,and eventually lead to certain biological effects.Using target gene prediction software,it was found that Penk1,which can translate proenkephalin protein and produce enkephalin after enzymolysis,may be a target gene of let-7b.Furthermore,a large number of researches have shown that enkephalin,the most abundant opioid peptide in the central nervous system,plays an important role in EA analgesia.However,whether let-7b can affect the EA analgesic effect by interfering with the translation expression of Penk1 gene and then cause EAT has not been confirmed.In this research,the hypothalamic neurons were cultured in vitro and the constructions of dual luciferase reporter vectors for co-transfection were performed.The results showed that let-7b could significantly inhibit the fluorescence signal of the WT-psiCHECK2-Penk1-3’ UTR dual luciferase reporter plasmid,indicating that Penk1 is the target gene of let-7b.In vivo experiment,72 female SD rats were randomly divided into 4 groups(n = 18): EA+AgoNC group,EA+AtgNC group,EA+Ago group and EA+Atg group,and established with intracerebroventricular injection models.Four groups of rats were respectively injected with stable negative control(the negative control of agomir),miRNA inhibitor NC(the negative control of antagomir),agomir-let-7b and antagomir-let-7b one day before EA stimulation,and CFA inflammation pathology models were also established in their right hind paw.EA stimulation was performed into bilateral "Zusanli-Sanyinjiao" acupoint once a day,2/15 Hz,for 30 minutes,lasted for 7 days.The pain thresholds of rats were measured by their tails flick latency before and after EA stimulation,and then 6 animals were randomly selected in each groups for the following experiments after 2h EA stimulation at day 1,4 and 7,respectively.Meanwhile,the protein expression of PENK in the thalamus was detected by WB,and the expression of let-7b was detected by qPCR method.The results showed that the change rate of pain threshold in EA+Ago group was significantly lower than that in other experimental groups at day 1 after EA stimulation(p<0.05),while it was significantly higher in EA+Atg group than that in other groups at day 7(p<0.05),indicating that let-7b could affect the EA pain threshold;the Let-7b expression in EA+AgoNC group was significantly increased at day 7 than day 1 after EA stimulation;the PENK protein in EA+Ago group had significant decrease compared with other experimental groups at day 1 after EA stimulation(p<0.05),whereas PENK protein was significantly increased in EA+Atg group compared with other groups at day 7 after EA stimulation(p <0.05),indicating that let-7b can affect the expression of PENK after EA.In this experiment,we demonstrated that Penk1 is a target of let-7b by the dual luciferase reporter experiment.Furthermore,through the change of pain threshold rate and the PENK protein level,we concluded that let-7b regulates EAT via targeting Penk1 gene in the hypothalamus,which further elucidates the molecular mechanism of miRNAs in EAT.