Electroacupuncture Alleviates Inflammation-induced Intestional Hypersensitivity Through Regulating The Expression Of PAR2 And PAR4

Visceral hypersensitivity is a common clinical feature in patients with irritable bowel syndrome（IBS）for which there is no good treatment.Electroacupuncture provides good relief for visceral hypersensitivity,but its exact mechanism is not clear.Visceral hypersensitivity is associated with peripheral and central sensitization,with inflammatory substances stimulating nociceptive nerve endings causing peripheral sensitization and repeated injurious stimulation causing central sensitization.In this paper,we induced visceral hypersensitivity by chemical method and detected neurosensitization markers as well as the expression of protein activated receptor 2（PAR2）and protease-activated receptor 4（PAR4）to elucidate the peripheral and central mechanisms of electroacupuncture to alleviate visceral hypersensitivity.1.Electroacupuncture alleviates visceral hypersensitivity by modulating PAR2 and PAR4 expression in the ileum and descending pain modulatory systemIn this experiment,36 goats were randomly divided into control group（CON group）,pure electroacupuncture group（EA group）,model group（TNBS group）and model plus electroacupuncture group（TNBS+EA group）.Ileitis and visceral hypersensitivity were induced by injecting 1.2 m L TNBS solution（30 mg TNBS and 40%ethanol mixture）into the ileum wall of goats in the TNBS and TNBS+EA groups,while equal amounts of saline were injected into the Ileum wall of goats in the CON and EA groups in the same manner,and the changes in body weight of each group were recorded daily.On the 7th day after modeling,the ileum tissue of 6 goats from each of the CON and TNBS groups was taken.The nociception scores and visceral motor response（VMR）values to colorectal dilatation（CRD）in goats were recorded after each electroacupuncture session,which started on day 7 and were administered every three days for a total of seven sessions.The concentrations of MPO,TNF-αand IL-1βin the ileum tissue on days 7 and25,and TNF-α,COX-2 and IL-6 in the T11 spinal cord and brain tissues on day 25 were measured by ELISA at the end of electroacupuncture on day 25 of the experiment.The expression levels of PAR2,PAR4,c-Fos,CGRP and SP in the ileum tissue,T11 spinal cord,periaqueductal gray matter（PAG）of the midbrain and the ventral medial region（RVM）of the cephalic end of the delayed brain were measured by western blotting and real-time fluorescence quantitative PCR,and the distribution of PAR2,PAR4,c-Fos,CGRP and SP were detected by immunofluorescence.The results showed that goats in the TNBS group exhibited diarrhea,poor mental appetite,and significantly higher VMR values and nociception scores in response to colorectal dilatation compared with those in the CON,EA and TNBS+EA groups,and the expression levels of PAR2,c-Fos,CGRP and SP were significantly higher in the ileum tissue,T11 spinal cord,PAG and RVM of goats,while the expression levels of PAR4 did not show significant changes.The expression levels of PAR2,c-Fos,CGRP and SP in the ileum,T11 spinal cord,PAG and RVM of goats in the TNBS+EA group were decreased,but the expression level of PAR4was significantly increased.Compared with goats in the TNBS group,electroacupuncture significantly reduced the VMR values and alleviated the nociceptive responses in the TNBS+EA group.Conclusion:Electroacupuncture may alleviate visceral hypersensitivity by modulating the expression levels of PAR2 and PAR4 in the ileal and descending pain modulatory system of goats.2.Electroacupuncture attenuates visceral hypersensitivity via balancing the expression of PAR2 and PAR4 in the ileum tissue and T11 spinal cord of mouseTo investigate whether electroacupuncture alleviates visceral hypersensitivity by balancing the expression of PAR2 and PAR4,60 mice were randomly divided into control group（CON group）,pure electroacupuncture group（EA group）,model group（TNBS group）and model plus electroacupuncture group（TNBS+EA group）in this experiment.Visceral hypersensitivity was induced by injecting 5 mg of TNBS mixed with 25%ethanol into the ileal intestinal lumen of mice in the TNBS group and TNBS+EA group,mice in the CON and EA groups were injected with equal amounts of saline with the same manner.The mice were treated with electroacupuncture every two days starting from day 7 for a total of seven times,and the mechanical retraction reflex threshold（PWT）and thermal retraction reflex latency threshold（PWL）were recorded on days 3and 5 after modeling and after each electroacupuncture session.At the end of electroacupuncture on day 20,the ileum and T11 spinal cord of mice were taken,and the expression levels of PAR2,PAR4,c-Fos and CGRP in the ileum tissue and T11 spinal cord of mice were detected by western blotting and real-time fluorescence quantitative PCR,and the distribution of PAR2,PAR4,c-Fos and CGRP and their co-localization with neurons,respectively,were detected by immunofluorescence.The results showed that electroacupuncture significantly relieved visceral hypersensitivity,significantly down-regulated the expression levels of PAR2,c-Fos and CGRP,and significantly increased the expression of PAR4 in mouse ileum and T11 spinal cord.The results indicates that PAR2and PAR4 in the ileum and spinal cord may be involved in the regulation of visceral hypersensitivity.To investigate whether electroacupuncture alleviates visceral hypersensitivity in mice by modulating the expression level of PAR2,the mice were divided into four groups,TNBS+saline,TNBS+FSLLRY-NH₂,TNBS+EA+saline and TNBS+EA+SLIGRL-NH₂,FSLLRY-NH₂ and SLIGRL-NH₂ were inhibitor and agonist of PAR2,which were administered by intrathecal injection.The PWT and PWL values of the mice were recorded before and after each electroacupuncture session.After two electroacupuncture injections,the T11 spinal cords of mice were taken and the expression of PAR2,c-Fos and CGRP in the spinal cords of mice were detected by western blotting and real-time fluorescence quantitative PCR.The results showed that intrathecal injection of FSLLRY-NH₂ significantly increased PWT and PWL values in mice,whereas intrathecal injection of SLIGRL-NH₂ significantly decreased PWT and PWL values in mice.No difference was seen in the expression level of PAR2 in the spinal cord of mice before and after injection of PAR2 agonist or inhibitor.The results suggest that electroacupuncture alleviates VH by downregulating PAR2 expression levels in the spinal cord of mice.To investigate whether electroacupuncture alleviates visceral hypersensitivity in mice through regulating the expression level of PAR4,the mice were divided into four groups:TNBS+saline,TNBS+PAR4-AP TFA,TNBS+EA+saline and TNBS+EA+tc Y-NH₂ TFA.PAR4-AP TFA and tc Y-NH₂ TFA were agonist and inhibitor of PAR4,which were administered by intrathecal injection.The PWT and PWL values of the mice were recorded before and after each electroacupuncture session.After two electroacupuncture sessions,the T11 spinal cord of mice were taken and the expression of PAR4,c-Fos and CGRP in the spinal cords of mice were detected by western blotting and real-time fluorescence quantitative PCR.The results showed that intrathecal injection of PAR4-AP TFA significantly increased PWT and PWL values in mice,whereas intrathecal injection of tc Y-NH₂ TFA significantly decreased PWT and PWL values in mice.No difference was seen in the level of PAR4 expression in the spinal cord of mice before and after injection of PAR4 inhibitor or agonist.The results suggest that electroacupuncture alleviates nociceptive hypersensitivity by increasing the expression level of PAR4 in the spinal cord of mice.In summary,electroacupuncture alleviates visceral hypersensitivity through balancing the expression of PAR2 and PAR4 in the spinal cord.