| Purpose1 To establish a rat model of endometriosis(EMs)by autologous transplantation,and then to observe the morphology of the ectopic lesions and their microscopic cytopathological changes to verify the success of modelling and the therapeutic effect of PCJNF.2 The Rat Grimace Scale(RGS)and Pain Threshold(PWT)were measured to verify whether PCJNF could relieve the pain in the EMs model rats.In addition,the levels of Tumour necrosis factor-α(TNF-α)and nerve growth factor(NGF)in the serum of rats were measured by ELISA,and the levels of capsaicin receptors in the in situ endothelium and ectopic lesions in each group were measured by Western Blot(WB).The expression of Transient Receptor Potential Vanilloid1(TRPV1),p-TRPV1,protein kinase C(PKC)and NGF in the endothelium,ectopic lesions of each group was measured by protein immunoblotting(WB)to investigate whether the pain relief mechanism of PCJNF in EMs model rats was based on NGF/TRPV1 pathway,and immunohistochemical The expression of capsaicin receptors TRPV1,p-TRPV1,PKC and NGF in ectopic lesions,dorsal horn and dorsal root ganglion of the spinal cord in each group was detected by staining to verify the results of WB experiments.3 Combined with the experimental findings,the expression of NGF,TRPV1 and PKC in the ectopic lesions,dorsal horn and dorsal root ganglion of the spinal cord in rats was investigated to preliminarily explore the mechanism of PCJNF in relieving pain in EMs.MethodsNinety female SD rats were established as an EMs rat model based on the endometrial autotransplantation method,which was modified from the literature by opening the abdomen medially,ligating the two ends of the uterus,cutting off the middle section of the uterus(1 cm),separating the adipose tissue attached to the plasma surface of the uterus,and dissecting the uterus longitudinally.The two separated endometrial surfaces of the uterine tissue were each sutured to the right and left abdominal walls,gentamicin0.1m L was left in the abdominal cavity to prevent infection and the abdomen was routinely closed.Postoperatively,the EMs lesions were maintained in cages of 4 animals,fasted for one day,housed normally and cleaned,and maintained by subcutaneous injection of estradiol(10mg/kg)every 4 days.Two weeks after surgery,the lesions were observed in the open abdomen.The lesions should be enlarged in size,with a transparent capsule containing yellow fluid,and new blood vessels could be seen on the surface.The rats were divided into 6 groups,including sham-operated group,model group,PCJNF low dose group(3.78g/kg),PCJNF medium dose group(7.56g/kg),PCJNF high dose group(15.12g/kg)and progesterone group(0.26mg/kg),regardless of their body weight,and kept in 4 cages.Each group was administered once a day for 21 days from three weeks post-operatively.The sham-operated and model groups were given distilled water gavage at 0.01ml/g daily,the progesterone group was given progesterone suspension twice a week and equal amounts of distilled water gavage for the other 5 days,and the three groups were given different doses of PCJNF gavage daily.The number of compounds in each batch of PCJNF was measured using high performance liquid chromatography(HPLC)and the most significant compounds were identified.The analgesic effect of PCJNF was also observed by RGS and PWT values preoperatively,3 weeks postoperatively,1 week and3 weeks after administration.At the end of the treatment,the rats in each group were executed,the size of the ectopic lesions in each group was measured,the in situ endothelium and ectopic lesions in each group were extracted,the serum,the dorsal horn of the spinal cord and the dorsal root ganglion were preserved for relevant tests.The expression of TRPV1,p-TRPV1,PKC and NGF in the ectopic lesions was measured by ELISA,and the expression of TRPV1,p-TRPV1,PKC and NGF in the ectopic lesions,dorsal horn and dorsal root ganglion of the spinal cord was measured by WB.Results1 It was demonstrated by HPLC that there was little variability in the number of compounds intersecting each of the three different batches of PCJNF3 samples,and that three samples were qualitatively stable.Also,five compound components were identified that played a greater role,namely linoleic acid,adenosine,oleic acid,D-proline and L-phenylalanine.2 At 3 weeks postoperatively,RGS values were significantly higher and PWT values were significantly lower in the model,PCJNF low-dose,medium-dose,high-dose and progesterone groups than in the sham-operated group,indicating the presence of typical EMs pain symptoms in the modelled rats(p < 0.001).At 3 weeks of administration,in terms of RGS,the rats in the medium-dose group had significantly lower RGS values compared to the model group(P < 0.01),and the differences in RGS values between the low-dose,high-dose and progesterone groups were not statistically significant(P > 0.05);in terms of PWT,the PWT values in the low-dose,medium-dose,high-dose and progesterone groups were all higher compared to the model group(P < 0.05 or P < 0.001).3 After measuring the volume of ectopic lesions in each group,it was found that the size of lesions decreased in the low-dose,medium-dose,high-dose and progesterone groups compared to the size of lesions in the model group(P <0.001).4 At 3 weeks postoperatively,a combination of visual and histopathological observations of the diseased ectopic lesions showed successful modelling in the EMs rats.Also at 3 weeks after administration,pathological damage was reduced in the low,medium and high dose and progesterone groups compared to the ectopic lesions in the model group.5 The ELISA results showed that after 3 weeks of administration,the concentrations of TNF-α and NGF were significantly higher in both the model and dosed groups compared to the sham-operated group;and the concentrations of TNF-α and NGF were significantly lower in the dosed group compared to the model group(p < 0.001).6 The results of WB experiments indicated that the expression levels of NGF,TRPV1,p-TRPV1 and PKC proteins in the ectopic lesions of rats in the medium-dose group were most significantly decreased compared with the model group(P < 0.05 or P < 0.01).And this study was further verified by the immunohistochemical results that the positive expression area of NGF,TRPV1,p-TRPV1 and PKC in the ectopic lesions and dorsal root ganglion of EMs rats in the middle dose group also showed similar results.Conclusion1 PCJNF has a significant analgesic effect and inhibits the growth of ectopic lesions in EMs model rats.2 PCJNF may reduce nociceptive sensitization in EMs rats by downregulating the NGF/TRPV1 pathway,thereby alleviating the symptoms of pain. |
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