The Function Of TRPV1/TRPA1 Heteromers In Endometriosis Pain

Part I: The effect of TRPV1/TRPA1 heteromeric on pain in endometriosis at clinical levelBackgroundEndometriosis is a benign chronic disease in women.The exact mechanism of its occurrence is not clear.The most common symptom of endometriosis is pain.The involvement of TRPV1/TRPA1 in pain mechanism has been confirmed by studies,but the effect of TRPV1/TRPA1 heteropolymer on pain in endometriosis remains unclear.Research purposeTo investigate the correlation between TRPV1/TRPA1 heteropolymer and pain level in patients with endometriosis.MethodsThe pathological tissues of 28 patients diagnosed with endometriosis were collected and divided into different pain grades according to the Numerical rating scale of pain(0 no pain,1 ? 3 mild,4 ? 6 moderate,7 ? 10 severe).Normal endometrial tissue was obtained from patients with adenomyosis who underwent hysterectomy.The expression level of TRPV1/TRPA1 m RNA was detected by fluorescence quantitative PCR.The expression of TRPV1/TRPA1 protein was detected by immunohistochemistry.The expression and localization of TRPV1/TRPA1 heteromer were detected by immunofluorescence.ResultsCompared with normal tissue,the expression of TRPV1/TRPA1 m RNA,protein and TRPV1/TRPA1 heteromeric were increased with the increase of endometriosis pain score.Was positively correlated with pain scores.ConclusionThe results of clinical samples showed that the formation of TRPV1/TRPA1 heteropolymer may be positively correlated with endometriosis pain.Part II: Studies the effect of TRPV1/TRPA1 heteropolymer on LPS-induced human uterine ectopic endometrial epithelial cellsBackgroundPain in endometriosis is associated with the inflammatory microenvironment of endometrium stroma and endothelial cells,as well as inflammation-induced peripheral and central nervous sensitization.TRPV1/TRPA1 expression affects nerve sensitization.The exact mechanism of pain in endometriosis remains to be further studied.The role of TRPV1/TRPA1 heteromers in the inflammatory microenvironment of endometrial cells is unclear.Research purposeLPS induced human endometrial cells to establish an inflammatory model,to investigate the changes of TRPV1/TRPA1 m RNA,protein expression and TRPV1/TRPA1 heteromer under inflammatory conditions.MethodsLPS induced human endometriosis cells,combined with TRPA1 agonist cinnamaldehyde,TRPA1 inhibitor HC030031,TGF?1 intervention cells were compared.Cell proliferation was detected by EDU.The contents of IL-1?,IL-6,TNF-? and PGE2 in cell supernatant were detected by ELISA.q PCR was used to measure TRPV1/TRPA1 m RNA expression and using Western blot for TRPV1/TRPA1 protein expression.Immunofluorescence was used to detect the co-localization of TRPV1/TRPA1 heteromers.ResultsCompared with solvent-treated cells(Control),cinnamaldehyde,HC030031 and TGF?1 had little effect on the proliferation of HEM15 A cells,while LPS had a tendency to induce a decrease in cell proliferation.Notably,neither cinnamaldehyde nor HC030031 played a more prominent role in LPS-induced HEM15 A cell proliferation.ELISA results showed that LPS-induced increased IL-1?,IL-6,TNF-? and PGE2 concentrations in the supernatant of HEM15 A cells,while cinnamaldehyde,HC030031 and TGF?1 had little effect on HEM15 A cell secretion or LPS-induced inflammation-related factors.Western blotting results showed that cinnamaldehyde,TGF?1 and LPS all induced the expression of TRPV1 and TRPA1 proteins,and LPS had the most significant effect on the expression of TRPV1/TRPA1.Cinnialdehyde and HC030031 attenuated rather than reversed the effect of LPS on the expression of TRPV1/TRPA1 protein.Immunofluorescence staining of HEM15 A cells showed that LPS induced the upregulation of TRPV1/TRPA1 isomer,cinnamaldehyde rather than TGF?1 could enhance the LPS induction of TRPV1/TRPA1 isomer in HEM15 A cells,and HC030031 could reduce the effect of LPS.There is a feedback effect between the TRPV1/TRPA1 heteromer and the inflammatory microenvironment.ConclusionLPS induced the increase of TRPV1/TRPA1 heteromeric in human endometriosis cells.TRPV1/TRPA1 heteropolymer increases the inflammatory microenvironment of human endometriosis cells.Part III : Study on the effect of animal TRPV1/TRPA1 heteromeric on pain in mice with endometriosisBackgroundPain in endometriosis is associated with inflammation and hormones.The role of TRPV1/TRPA1 associated ion channels in steroid-induced endometriosis pain needs further investigation.Research purposeTo investigate the effect of TRPV1/TRPA1 heteromer on pain in mice with endometriosis.MethodsMice endometriosis model was established by implantation method.Combined with TRPA1 agonist cinnamaldehyde and TRPA1 inhibitor HC030031 intervention.Pain behavior experiment was carried out by oxytocin injection.The contents of IL-1?,IL-6,TNF-? and PGE2 in serum and abdominal cavity cleaning fluid were determined by ELISA.q PCR was used to measure TRPV1/TRPA1 m RNA expression and using Western blot for TRPV1/TRPA1 protein expression.Immunofluorescence was used to detect the co-localization of TRPV1/TRPA1 heteromers.ResultsThe writhing frequency of oxytocin induced mice was significantly increased and cinnamaldehyde increased the writhing frequency of mice.HC030031 reduced the wriggling frequency of the mice.The contents of TRPV1 and TRPA1 protein in the endometriosis mice were the highest in the cinnamaldehyde treatment group,corresponding to the trend of the torsion test results in each group.Compared with solvent control mice with endometriosis,the expression of TRPV1/TRPA1 protein was the lowest in HC030031 treatment group and sham operation group.Similar to the results of TRPV1/TRPA1 expression detection,the contents of IL-1?,IL-6,TNF-? and PGE2 in serum and abdominal cavity washing fluid of oxytocin induced model mice were significantly increased.Furthermore,cinnamaldehyde increased the contents of IL-1?,IL-6,TNF-? and PGE2 in serum and abdominal cavity cleaning fluid of mice.HC030031 reduced the contents of IL-1?,IL-6,TNF-? and PGE2 in serum and peritoneal cleaning fluid of mice.ConclusionThe results of animal level study indicated that the TRPV1/TRPA1 heteromeric was positively correlated with pain in mice with endometriosis pain in vivo.It was positively correlated with IL-1?,IL-6,TNF-? and PGE2 in serum and peritoneal cleaning fluid of mice.