Explore The Effect And Mechanism Of Huayu Xiaozheng Granule In The Treatment Of Tubal Pregnancy Based On Ferroptosis

Tubal pregnancy(TP)refers to the implantation and development of a fertilized ovum in a fallopian tube.Tubal pregnancy rupture is the first cause of blood loss related death during pregnancy.Despite its prevalence and danger for women’s reproductive health,the underlying pathogenesis of TP is poorly understood which hinder the development of its drug treatment scheme.The increase of oxidative stress may reduce tubal ciliary pulsation and smooth muscle contraction,which is an important mechanism of TP.However,only a few studies have discussed the relationship between lipid peroxidation and TP.Ferroptosis,which is closely related to lipid peroxidation,was officially proposed in 2012.Ferrroptosis is characterized by iron dependent free radical generation and excessive accumulation of lipid peroxidation.It was reported that ferroptosis was related to trophoblast injury and placental dysfunction.Recent studies have demonstrated the role of ferroptosis in trophoblastic injury and placental dysfunction(which mainly involves lipid peroxidation in embryo injury)and iron-dependent lipid peroxidation is abundant in trophoblasts.However,the effect of ferroptosis on the occurrence and development of TP is unknown.Previous researches have suggested that blood stasis syndrome was closely linked to lipid peroxidation and iron overload,which are the main characteristics of ferroptosis.The pathogenesis of TP lies in the syndrome of shaoabdominal blood stasis,so the treatment of shaoabdominal blood stasis may be related to the regulation of ferroptosis.Traditional Chinese medicine treats tubal pregnancy with less abdominal blood stasis syndrome by promoting blood circulation and removing blood stasis,eliminating symptoms and killing embryos.Represented by Huayu Xiaozheng compound,which was created by Professor Deng Gaopi on the basis of ectopic pregnancy formula I,and is composed of Salvia miltiorrhiza,red peony,peach kernel,trichosanthin and arnebia,which is specialized in removing blood stasis,eliminating symptoms and killing embryos.Huayu Xiaozheng Granule,a granule of Huayu Xiaozheng compound,is the first hospital preparation in China for adjuvant treatment of tubal pregnancy.The preliminary study showed that the clinical efficacy of Huayu Xiaozheng compound in the treatment of early TP was obvious with an effective rate at 88.9%,but the deep--seated pharmacodynamic mechanism at treating TP is still unclear.Therefore,clarifying its mechanism is of great significance for clinical application.In conclusion,this study puts forward the hypothesis that "Huayu Xiaozheng Granule may regulate ferroptosis in the treatment of tubal pregnancy".Objective:To compare the expression of ferroptosis in the villous tissue between patients with tubal pregnancy and normal early pregnant women,and to explore the relationship betweenferroptosis and tubal pregnancy.The ferroptosis model of HTR-8/SVneo cells was established to explore the underlying mechanism of the medicated serum of Huayu Xiaozheng Granule regulating the ferroptosis of HTR-8/SVneo cells through ACSL4.Methods:1.Clinical studyWith the approval of the ethics committee of the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine(approval No.K[2019]136),the villus tissue from 20 cases of tubal pregnancy patient and 20 cases of normal early pregnancy(NP)women who underwent salpingectomy for TP in the First Affiliated Hospital of Guangzhou University of traditional Chinese medicine were collected.Iron deposition in the villus tissue was detected by Prussian blue staining;Western blotting(WB)assay,immunohistochemistry and immunofluorescence staining were used to analyze the expression of ferroptosis related proteins such as GPx4,SLC7A11,TfR1 and ACSL4 in the villus tissue;MDA(malondialdehyde method)was used to detect the level of lipid peroxidation in in the villus tissue;Glutathione kit and glutathione peroxidase kit were used to detect the content of glutathione(GSH)and the activity of glutathione peroxidase(GPx).Comprehensive analysis to clarify the correlation between tubal pregnancy and ferroptosis from the clinical and organizational level.2.Cell experiment(1)The ferroptosis model of HTR-8/SVneo cells was constructed by using Erastin and RSL3.The cell proliferation activity was detected by CCK8 method,the contents of ROS and unstable iron pool(LIP)in cells were detected by DCFH-DA probe and FeRhoNox-1 respectively,and the changes of ferroptosis related proteins were detected by WB assay.(2)Based on the ferroptosis model of HTR-8/SVneo cells induced by RSL3,the medicated serum of Huayu Xiaozheng Granule was added to explore whether the combination using of Huayu Xiaozheng Granule and RSL3 affected the proliferation,invasion and migration of HTR-8/SVneo cells.HTR-8/SVneo cells were intervened with the medicated serum of Huayu Xiaozheng Granule and/or RSL3.The functions of cell proliferation,invasion and migration were detected by CCK8 test,Transwell assay and wound-healing assay.(3)To explore whether Huayu Xiaozheng Granule could promote the sensitivity of HTR-8/SVneo cells to ferroptosis induced by RSL3.The contents of GSH and MDA were detected by kit,the intracellular ROS level was detected by DCFH-DA fluorescent probe,the content of intracellular LIP was detected by FeRhoNox-1 fluorescent probe,the lipid ROS level of cell membrane was detected by C11-BODIPY581/591 fluorescent probe,and the expression of GPx4,SLC7A11,TfR1 and ACSL4 proteins was detected by WB assay.(4)To explore the mechanism of Huayu Xiaozheng Granule enhancing ferroptosis of HTR-8/SVneo cells.The expression of ACSL4 in HTR-8/SVneo cells was interfered by siRNA.The intracellular ROS level was detected by DCFH-DA fluorescent probe,the lipid ROS level of cell membrane was detected by C11-BODIPY581/591 fluorescent probe,and the protein expressions of GPx4,SLC7A11 and ACSL4 were detected by WB assay.Results:1.Clinical research(1)There was no significant difference in age,gestational weeks,number of pregnancies,number of abortions,number of births and number of previous tubal pregnancies between the normal pregnancies and TP patients.(2)Prussian blue staining showed that iron deposition in the villous of TP group was significantly higher than that of normal early pregnant women.(3)Compared with normal early pregnant women,the content of GSH and GPx activity in the villous of TP patients were decreased,while the level of MDA was increased.The differences were statistically significant.It is suggested that oxidative damage occurred in the villous tissue from TP.(4)Compared with NP normal early pregnant women,the expression of ferroptosis negative regulatory proteins SLC7A11 and GPx4 in the villous of TP patients were significantly lower and the positive regulatory proteins TfR1 and ACSL4 of ferroptosis were notably increased,and there were significant differences.It is suggested that the protein expressions which inhibit ferroptosis were decreased,while the expression of protein which promote ferroptosis were increased in the villous of TP patients.The trend of immunohistochemical manifested the same results as that of WB.(5)The double immunofluorescence labeling method showed that the fluorescence expression of GPx4 in the villous of NP group was significantly higher than that of TP group,while the fluorescence expression of TfR1 was significantly lower than that of TP group.The fluorescent expression of GPx4 and TfR1 was mainly distributed in cytotrophoblast cells.2.Establishment of HTR-8/SVneo cells ferroptosis model by Erastin and RSL3(1)Erastin and RSL3 damaged the viability of HTR-8/SVneo cells and induced cell ferroptosis in a time and dose-dependent manner.Compared with erastin(IC50=2.521)μ M),HTR-8/SVneo cells were more sensitive to cell damage induced by RSL3(IC50=124.3 nm).(2)Both Erastin and RSL3 couuld cause the decrease of negative regulatory protein expression and the increase of positive regulatory protein expression of ferroptosis in HTR8/SVneo cells.(3)Both Erastin and RSL3 could damage the morphology of HTR-8/SVneo cells and promote the accumulation of ROS and LIP in cells.3.Effect of the medicated serum of Huayu Xiaozheng Granule combined wi th RSL3 on the function of HTR-8/SVneo cells(1)Compared with the blank group or the drug alone group,the medicated serum of Huayu Xiaozheng Granule(HYXZ)combined with RSL3 significantly inhibited the proliferation activity of HTR-8/SVneo cells,and could be significantly restored after pretreatment with ferroptosis inhibitor Fer-1.(2)Compared with the blank group or the drug alone group,HYXZ combined with RSL3 significantly inhibited the invasion and migration of HTR-8/SVneo cells.4.Study on the sensitivity of the medicated serum of Huayu Xiaozheng Granule to promote ferroptosis of HTR-8/SVneo cells(1)Compared with RSL3 or HYXZ group alone,decreased GPx activity and evaluated MDA level were observed in combination of HYXZ and RSL3 group.(2)Compared with RSL3 or HYXZ group alone,the expression of ROS and LIP in HYXZ combined with RSL3 group was significantly increased,which revealed that the content of ROS and Fe2+ decreased with statistical difference.After the intervention of iron death inhibitor Fer-1 in advance,the increased levels of ROS and LIP induced by RSL3+HYXZ group were reversed,and there were significant differences.(3)RSL3 or HYXZ alone could promote the production of lipid ROS in HTR-8/SVneo cell membrane.HYXZ combined with RSL3 further amplified the production of lipid ROS in cell membrane.In addition,the oxidative form of cells caused by HYXZ combined with RSL3 can be inhibited by Fer-1.(4)Compared with the normal group,after HYXZ intervention,GPx4 protein decreased significantly,ACSL4 and TfR1 protein expression increased significantly,and SLC7A11 protein had no significant difference.In addition,compared with RSL3 alone,HYXZ combined with RSL3 further inhibited the expression of GPx4 and SLC7A11 proteins and promoted the expression of ACSL4 and TfR1 proteins.(5)Immunofluorescence staining showed that HYXZ combined with RSL3 inhibited the expression of GPx4 compared with the normal group and promoted the expression of ACSL4.In addition,GPx4 protein was mainly expressed in the cytoplasm of HTR-8/SVneo cells,while ACSL4 was mainly expressed in the cell membrane,which further supported the argument that RSL3 combined with Huayu Xiaozheng Granule could promote the production of intracellular ROS and cell membrane lipid ROS.(6)Under the pre-intervention of ferroptosis inhibitor Fer-1 and DFO,the decrease of GPx4 and SLC7A11 protein expressions and the increase of ACSL4 and TfR1 proteins caused by HYXZ combined with RSL3 were reversed,with statistical difference.However,the administration of apoptosis inhibitor Z-VAD-FMK and autophagy inhibitor chloroquine could not save the above protein expressions induced by the combination of the medicated serum of Huayu Xiaozheng Granule and RSL3.5.The medicated serum of Huayu Xiaozheng Granule regulated ACSL4 mediated ferroptosis sensitivity of HTR-8/SVneo cells(1)The expression of ACSL4 in HTR-8/SVneo cells transfected with siRNA-was verified by WB assay.The results showed that the expression levels of ACSL4 protein in the three sequences of si-ACSL4 group were significantly lower than that of si-Control group,and there were statistical differences,which suggested that the siRNA transfection of ACSL4 in HTR-8/SVneo cells was successful.Besides,si-ACSL4-1 had the best transfection efficiency among the three sequences of si-ACSL4.(2)The results of intracellular ROS detected by DCFH-DA probe showed that compared with HYXZ+RSL3 group,the level of ROS in si-ACSL4 group and HYXZ+RSL3+siACSL4 group decreased significantly.The results showed that excessive ROS produced by Huayu Xiaozheng Granule combined with RSL3 could be reversed after interfering with the expression of ACSL4.(3)The results of C11-BODIPY581/591 fluorescent probe showed that compared with HYXZ+RSL3 group,the lipid ROS of cell membrane in si-ACSL4 group and HYXZ+RSL3+si-ACSL4 group decreased significantly.The results showed that the increase of cell membrane lipid peroxide induced by HYXZ combined with RSL3 could be reversed after interfering with the expression of ACSL4.(4)Results of WB assay showed that compared with HYXZ+RSL3 group,the expression levels of GPx4 and SLC7A11 protein in si-ACSL4 group and HYXZ+RSL3+siACSL4 group increased significantly,and the expression level of ACSL4 protein decreased significantly,and there were significant differences.The results showed that after siRNA interference with the expression of ACSL4,the expression of ACSL4 was down-regulated and GPx4 and SLC7A11 were increased,that is,the expression of ferroptosis related protein was reversed and ferroptosis was inhibited.Conclusions:1.There are differences in the expression of ferroptosis in the villous tissue between normal early pregnant women and TP patients.Lipid peroxidation,rich iron and PUFA substrate were jointly involved in the process of ferroptosis during TP.2.Single use of RSL3 or Huayu Xiaozheng Granule inhibited the proliferation,invasion and migration of HTR-8/SVneo cells,which could help to clarify that targeting ferroptosis of villous trophoblast may be a potential method for the treatment of TP,and clarify that Huayu Xiaozheng Granule could inhibit the function of HTR-8/SVneo cells in treating TP.3.Knocking down the expression of ACS14 in HTR-8/SVneo cells significantly inhibited ferroptosis induced by Huayu Xiaozheng Granule combined with RSL3,suggesting that regulating the expression of ACS14 might be the molecular mechanism of Huayu Xiaozheng Granule enhancing the sensitivity of HTR-8/SVneo cells to ferroptosis.