Research Of Let-7b-5p Targeting Dusp1 Gene On The Electroacupuncture Tolerance Rats

Intracellular miRNA can repress the expression of a target mRNA with partially complementary binding sites in its 3‘UTR which plays a key role in regulating gene expression and these researches which related to the pain and these phenomenon attracted the people‘s attention.The researchers found that miRNA let-7b influencd the pain regulation by TLR7/TRPA1 in nociception neuron which proved that let-7b was important in pain regulation.The electroacupuncture(EA)tolerance was defined as prolonged or repeating EA stimulation resulted in a gradual fading away of its analgesics effect.Our laboratory has proved that let-7b-5p participates in the EA tolerance by deep sequencing technique and intracerebroventricular injection technique.The present study found that the let-7b-5p target gene is the Dusp1 gene by miRNA-target prediction software and it has been proved that Dusp1 gene playing an important role in MAPK signal pathway.The Dusp1 gene can be translated into MKP-1 and it is a kind of bidirectional specificity Sue/tyrosine phosphatase,not only can make the phosphorylation of serine/threonine phosphortlation,but also can make the phosphorylation of tyrosline phosphorylation.MKP-1 could act specifically upon p38 and study showed that interfer the activity of the MKP-1 would regulate the MAPKs signal pathway and it could reduce the pain as well as delay the progress of pain development.Our laboratory found that the EA caused the inflammatory pain rat‘s brain p38 MAPK reducing in nuclei periaqueductal gray matter,rostral ventromedial medulla and spinal cord horn and this proved that MAPK signals pathway plays a significant role in EA analgesia.The present study was designed to explore whether the let-7b-5p influencing the MAPK signal pathway(or influencing the p38MAPK)by Dusp1 gene to disturb the development of EA tolerance.Thus,the present study tried to explore the relationships between the let-7b-5p with Dusp1 gene and(or)MKP-1 protein in the EA tolerance rats.Using dissociated neural cell culture of rat in vitro and transferring the dual-Luciferase reporter expression vector,the present study found that the let-7b-5p inhibited the psi CHECK2-Dusp1-3‘UTR-WT luciferase activity comparing with the control group.Later,54 SD female rats(weighting 210±20g)with 6 week aged were selected.The rats were randomly divided into three groups: the Ago+EA group,the Atg+EAgroupand the CT+EA group.Each group contained 18 rats and was operated with intracerebroventricular injection.The Ago+EA group was injected with agomir-let-7b-5p;the Atg+EA group was injected with antagomir-let-7b-5p;the CT+EA group were injected with Stable Negative Control(random base sequence).After the intracerebroventricular injection,all the rats were injected 0.1m L CFA in to left hind paw immediately.After 24 h,the EA was applied to all rats at bilateral ?Zusanli‘ and ?Sanyinjiao‘ acupoints for 30 min and the EA would last for 7days,once a day.Before and after the EA,the paw pain withdrawl threshold and the tail flick latency were detected by the touch pain measurement instrument and tail threshold detector.Randomly selecting 6 rats sacrificed and separated the hypothalamus in each group at day 1,day 4 and day 7 to detect the MKP-1 protein level.The change rate of pain threshold results showed that the rats pain threshold in Atg+EA group was significant increased(p<0.05)than the Ago+EA group in day 1 to day 4;in the day 1 and day 3,the rats pain threshold in Ago+EA group was lower than the CT+EA group.The rats pain threshold in Atg+EA group were significant higher than rats in Ago+EA group by dectecting the TFL from day 1 to day 7(p<0.05).Comparing CT+EA group with Atg+EA group,the pain threshold were sighnificantly different in these two groups(p<0.05);in the day 1,day 3 and day 4,the rats‘ TFL in Ago+EA group was lower than the CT+EA group.The results of the MKP-1 expression in hypothalamus showed that the expression level of MKP-1 in Ago+EA group were decreased significantly(p<0.05)decreased after the first EA.However,the expression level of MKP-1 showed no difference between Atg+EA group and CT+EA groups(p>0.05),and the expression level of MKP-1 in Ago+EA group were lower than the other two groups(p<0.05)after the 4 days EA.In the last day,the expression level of MKP-1 in Atg+EA was higher than the other groups(p<0.05)and there were no differences between the CT+EA groups and Ago+EA groups(p>0.05).These results indicated the involvement of the let-7b-5p interference with the Dusp1 gene and influenced the MAPK signal pathway in EA analgesia and EA tolerance regulation in rats CNS by in vitro and in vivo biotechnology.