| Objective:To explore the mechanism of Gexiazhuyu Decoction by observe the effect of which on tubal fibrosis mediated by TGF-β1/MAPK signaling pathway and in the TGF-β1-induced fibrosis model of fibroblast based on the rat model in theway of the establishment of chronic salpingitis with qi stagnation and blood stasis.Methods:Part Ⅰ:3 2 female SD rats were randomly divided into the Control group,Sham operation group,the Staphylococcus aureus group(SA)and the Complex group.The control group did not receive any surgical operation or stimulation,while the SA group injects the prepared Staphylococcus aureus into the oviduct of the anesthetized rat.In the sham operation group,the fallopian tube of the anesthetized rat is not injected with Staphylococcus aureus but replaced with an equal amount of sterile saline.The Complex group began subcutaneous injection of adrenaline at a concentration of 0.08mL/100g body weight once a day for 7 days after injection of Staphylococcus aureus;after 7 days,the sound,light,electricity and tail stimulation were given for 3 weeks.Day 29 of the experiment,qi stagnation and blood stasis syndrome scores on each group of rats and blood rheology tests on the abdominal aorta in rats were performed;the fallopian tube morphology were scored;the pathological morphology of the fallopian tubes by HE staining and MASSON staining were observed.Part Ⅱ:Mouse embryonic fibroblasts(NIH-3T3)were divided into the Control group,the TGF-β1 group,the Medicated serum group,the SB203580 group and the SB203580+Medicated serum group.Except the Control group,the other 4 groups were induced by 10 ng/mL TGF-β1 for 12 hours to establish the fibrosis model.The Medicated serum group,the SB203580 group and the SB203580+Medicated serum group were given the corresponding drug pretreatment for 6 hours before the model was replicated.The specific methods are as follows:the Medicated serum group was given 1μg/mL of Gexiazhuyu Decoction serum,the SB203580 group was given SB203580 10 ng/mL,and the SB203580+Medicated serum group was given the above two drugs.The TGF-β1 group and the Control group were not given drugs.After that the OD values of the constituent fibroblasts at the 24th hour,48th hour and 72th hour were detected by MTT method,and the proliferation curves were drawn and compared.Cell apoptosis was detected by Flow cytometry.Laser confocal assay for the expression of α-SMA,p38MAPK,p-p38MAPK and TGF-β1.PartⅢ:100 female SD rats were randomly divided into the Control group,the Model group,the GXZY Low group,the GXZY Medium group and the GXZY High group.No operation was performed in the Control group,and the other 4 groups were modeled in the same way as part 1.The GXZY Low group,the GXZY Medium group and the GXZY High group began to administer the model rats every morning on the 29th day of modeling.The concentrated dry powder of Gexiazhuyu Decoction and distilled water were prepared into suspensions of different concentrations,which were 0.38g/mL,0.75g/mL,and 1.50g/mL,respectively,and the intragastric dose was administered according to 2 mL/100g body weight.The Model group was intragastrically administrated with equal amount of distilled water each time for 1 week,total 4 weeks.On the 57th day of the experiment,the whole blood viscosity of 3.0,the plasma viscosity,the syndrome of qi stagnation and blood stasis syndrome were observed.The pathological morphology of the fallopian tubes by HE staining and MASSON staining were observed.Detection of TGF-β1 and IL-1β levels in rat serum by Elisa method.The results of SOD,MDA,COL-1,MMP-9 and TIMP-1 in rat oviduct tissues were detected by Elisa method.The results of MMP-9 and TIMP-1 in rat oviduct tissues were detected by immunohistochemistry.The results of α-SMA,p38MAPK,p-p38MAPK and TGF-β1 in rat oviduct tissues were detected by immunofluorescence and Western blotting.The results of mRNA of α-SMA,p38MAPK,TGF-β1,COL-1,MMP-9 and TIMP-1 in rat oviduct tissues were detected by RT-PCR.Results:Part Ⅰ:1.There were significant differences between the groups in the total score of qi stagnation and blood stasis syndrome,blood viscosity 3.0,and plasma vi sco sity(P<0.01).2.Masson staining showed that the blue collagen fibers were visible in the Complex group,and the color of the Complex group was darker than that of the SA group.Compared with the Sham operation group,the difference with the Complex group was statistically significant(P<0.01).Part Ⅱ:1.The drug-containing serum of Gexiazhuyu Decoction can inhibit the proliferation of fibroblasts.Compared with the Model group,the difference is statistically significant(P<0.01).2.The drug-containing serum of Gexiazhuyu Decoction can promote the apoptosis of fibroblasts.Compared with the Model group,the difference is statistically significant(P<0.05).3.The drug-containing serum of Gexiazhuyu Decoction reduces the fluorescence intensity of α-SMA,p38MAPK,p-p38MAPK and TGF-β1 in fibroblasts.Part Ⅲ:1.The GXZY Medium group and the GXZY High group can reduce the total score of qi stagnation and blood stasis syndrome,the difference has statistical significance(P<0.01).2.Gexiazhuyu Decoction can reduce the whole blood viscosity of 3.0 and plasma viscosity in rats,the difference is statistically significant(P<0.01).3.Gexiazhuyu Decoction can down-regulate the expression of proinflammatory cytokines TGF-β1 and IL-1β in serum,and the difference is statistically significant(P<0.01).4.Gexiazhuyu Decoction can up-regulate SOD and down-regulate the expression of MDA in rat oviduct tissue,and the difference is statistically significant(P<0.01).5.Gexiazhuyu Decoction can down-regulate the expression of COL-1 in rat’s fallopian tubes,and the difference is statistically significant(P<0.01).6.Gexiazhuyu Decoction can up-regulate MMP-9 and MMP-9/TIMP-1,and down-regulate the expression of TIMP-1 in rat’s fallopian tubes.The difference is statistically significant(P<0.05).7.Gexiazhuyu Decoction can down-regulate the expression ofα-SM A,p3 8M APK,p-p3 8MAPK and TGF-β1 protein in rat’s oviduct tissues,and the difference is statistically significant(P<0.01).8.Gexiazhuyu Decoction can down-regulate the mRN A expression of α-SMA,p38MAPK,TGF-β1,COL-1 and TIMP-1 in rat’s oviduct tissues,and the difference is statistically significant(P<0.01);while it can up-regulate the expression of MMP-9 mRNA in rat’s fallopian tubes,and the difference is statistically significant(P<0.05).Conclusions:1.To establish the rat models of chronic salpingitis with qi stagnation and blood stasis by subcutaneous injection of adrenaline after injection of Staphylococcus aureus and the stimulation of sound,light,electricity and tail stimulation.Thro ugh the detection of tissue morphology and pathophysiological indicators,it was confirmed that the model was successfully constructed.2.The drug-containing serum of Gexiazhuyu Decoction can do wn-regulate the expression of TGF-β1/MAPK signaling pathway-related proteins,thereby inhibiting the proliferation of fibroblasts and promoting cell apoptosis.3.Gexiazhuyu Decoction may do wn-regulate the expression of TGF-β1/MAPK signaling pathway-related proteins,reduce collagen deposition and reduce the degree of tubal fibrosis by reducing oxidative stress and inflammatory response in rat tissues. |
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