| Tubal pregnancy（TP）refers to the implantation and development of fertilized eggs in different parts of the fallopian tube,and tubal rupture is one of the main causes of female death in the first trimester.In recent years,with the influence of sexual openness,premature use of oral contraceptives,and unhealthy lifestyles such as smoking and drinking,the incidence of tubal pregnancy has gradually increased,while the age of onset has gradually decreased.These have seriously endangered the reproductive health and life safety of women of childbearing age.The previous research of the research group found that Huayu Xiaozheng granules can promote cell apoptosis through the mitochondrial apoptosis pathway Bcl-2/Bax in trophoblast cells.On the other hand,Huayu Xiaozheng granules can induce trophoblast autophagy and interfere with autophagy homeostasis,thereby achieving the purpose of treating tubal pregnancy.The previous spectrum effect study found that shikonin is one of the main components of Huayu Xiaozheng granules.So there is no research on whether Huayuxiaozheng granule and shikonin can regulate mitochondrial autophagy,which is closely related to mitochondrial apoptosis and autophagy,and play a therapeutic role.Based on the above research basis,this topic mainly starts from the two aspects of mitophagy and mitochondrial function,and discusses the mechanism and mechanism of action of Huayu Xiaozheng granule and shikonin,and further enriches and enriches the foundation of Huayu Xiaozheng granule in the treatment of tubal pregnancy.Objective1.To investigate the effect of Huayu Xiaozheng granules on mitophagy and mitochondrial function of HTR-8/Svneo cells based on PINK 1/Parkin signaling pathway.2.To study the effect of shikonin,the main component of Huayu Xiaozheng Granules,on mitophagy and mitochondrial function of HTR-8/Svneo cells.MethodsClinical study:Study on the expression of mitophagy-related proteins in the villous tissue of patients with tubal pregnancyThe villous tissues of tubal pregnancy patients and 20 normal early-pregnancy women were collected in the clinic.Western blotting and semi-quantitative reverse transcription polymerase chain reaction were used to detect the mitophagy-related proteins PINK1,PARK2,LC3Ⅱ,P62,TOM20 and their genes.Basic research1.The effect of Huayu Xiaozheng granule on mitophagy and mitochondrial function of HTR-8/Svneo cells based on PINK1/Parkin pathway5%blank serum,10%blank serum,15%blank serum,5%medicated serum of Huayu Xiaozheng granules,10%medicated serum of Huayu Xiaozheng granules,and 15%medicated serum of Huayu Xiaozheng granules were used for intervention.After HTR-8/Svneo cells,the cell viability was detected by CCK8 method,and 10%Huayu Xiaozheng granule-containing serum was selected as the best drug concentration for follow-up experiments.Yuxiaozheng granule-containing serum group.（1）Hoechst staining was used to verify the level of apoptosis;（2）ATP detection kit was used to measure the intracellular ATP content;（3）JC-1 fluorescent probe was used to measure the mitochondrial membrane potential;（4）DHE probe was used to measure the intracellular ultra-high temperature.Oxide levels;（5）mitochondrial and lysosomal fluorescent probes to determine the co-localization of the two;（6）Western blotting to determine the expression of mitophagy-related proteins.2.Effects of shikonin on mitophagy and mitochondrial function in HTR-8/Svneo cells by regulating PINK1/Parkin pathwayHTR-8/Svneo cells were intervened with 0.2 μmol/L,0.4μmol/L,0.8 μmol/L and 1.6 μmol/L of shikonin for 6 h,12 h and 24 h,respectively.The drug concentration was 0.8 μmol/L,and the optimal action time was 24 h.The follow-up experiments were carried out under this condition.The experimental groups were divided into control group,Mdivi-1 group（mitochondrial autophagy inhibitor）,shikonin group,and shikonin+Mdivi-Group 1.（1）Flow cytometry to detect cell apoptosis;（2）ATP detection kit to detect intracellular ATP content by chemiluminescence;（3）JC-1 fluorescent probe to determine cell mitochondrial membrane potential;（4）DHE probe The level of intracellular superoxide was determined;（5）mitochondrial and lysosome fluorescent probes were used to determine their co-localization;（6）Western blotting was used to determine the expression of mitophagy-related proteins.ResultsClinical research1.Baseline dataThere was no difference between the TP group and the NP group in terms of BMI,smoking history,drinking history,gestational age,pregnancy times,delivery times,and abortion times（P>0.05）.2.Expression of mitophagy-related proteinsCompared with the NP group,the LC3Ⅱ/Ⅰ,P62,PINK1 and Parlin proteins in the villus tissue of the TP group were up-regulated（P<0.05）,and the TOM20 protein was down-regulated（P<0.01）;3.Expression of mitophagy-related genesCompared with the NP group,the mRNA levels of mitophagy-related genes LC3II,P62,PINK1,and Parkin in the villus tissue of the TP group were increased（P<0.01,P<0.05）,and the mRNA expression level of TOM20 was decreased（P<0.01）.).Basic research1.The effect of Huayu Xiaozheng granule on mitophagy and mitochondrial function of HTR-8/Svneo cells based on PINK1/Parkin pathway（1）Effect of Huayu Xiaozheng Granules on Cell ViabilityHuayuxiaozheng granules can inhibit the cell viability of HTR-8/Svneo cells in a concentration-dependent manner to a certain extent.10%Huayu Xiaozheng granule-containing serum treated HTR-8/Svneo cells for 24 hours,and the cell inhibition rate was about 59%.（2）Effect of Huayu Xiaozheng Granule-containing serum on apoptosisCompared with the control group and 10%blank serum group,10%Huayu Xiaozheng granules-containing serum can reduce the density of HTR-8/Svneo cells and induce cell apoptosis.（3）The effect of Huayu Xiaozheng granule-containing serum on the level of cellular ATPCompared with the control group and the 10%blank serum group,the 10%Huayu Xiaozheng granules-containing serum decreased the ATP content of HTR-8/Svneo cells（P<0.01）.（4）The effect of Huayu Xiaozheng granule-containing serum on the mitochondrial membrane potential level of cellsCompared with the control group and 10%blank serum group,10%Huayu Xiaozheng granules-containing serum significantly reduced the mitochondrial membrane potential of cells（P<0.01）.（5）The effect of Yuxiaozheng granule-containing serum on the level of intracellular superoxideCompared with the control group and 10%blank serum group,10%Huayu Xiaozheng granules-containing serum could increase the level of superoxide in cells（P<0.01）.（6）The effect of Huayu Xiaozheng granule-containing serum on the co-localization of mitochondria and lysosomes in cellsCompared with the control group and the 10%blank serum group,the co-localization area of lysosomes and mitochondria increased after 10%Huayu Xiaozheng granule-containing serum treated cells（P<0.05）.（7）Effects of Huayu Xiaozheng Granule-containing serum on mitochondrial autophagy-related proteinsCompared with the control group and the 10%blank serum group,the expressions of LC3Ⅱ/Ⅰ,P62,PINK1 and Parkin in the 10%Huayu Xiaozheng granules-containing serum group were increased（P<0.05,P<0.01）,and the expression of TOM20 was decreased（P<0.05,P<0.01）.2.Effects of shikonin on mitophagy and mitochondrial function in HTR-8/Svneo cells by regulating PINK1/Parkin pathway（1）The effect of shikonin on cell viabilityShikonin can inhibit the proliferation of HTR-8/Svneo cells in a concentration-and time-dependent manner.Moreover,the inhibitory rate of 0.8 μmol/L and shikonin for 24h on HTR-8/Svneo cells was close to IC50,so this concentration and time were selected as the subsequent experimental conditions.（2）The effect of shikonin on apoptosisCompared with the control group and the Mdivi-1 group,the shikonin group could promote the apoptosis of HTR-8/Svneo cells（P<0.05,P<0.01）;compared with the shikonin group,the shikonin+Mdivi-The apoptosis rate of group 1 decreased（P<0.05）.（3）The effect of shikonin on cellular ATP levelsCompared with the control group and the Mdivi-1 group,the intracellular ATP content of the shikonin group decreased（P<0.01）;compared with the shikonin group,the ATP content of the shikonin+Mdivi-1 group increased（P<0.05）.（4）The effect of shikonin on the level of mitochondrial membrane potential in cellsCompared with the control group and Mdivi-1 group,the mitochondrial membrane potential of the shikonin group decreased（P<0.01）;compared with the shikonin group,the mitochondrial membrane potential of the shikonin+Mdivi-1 group increased（P<0.05）.（5）The effect of shikonin on the level of intracellular superoxideCompared with the control group and Mdivi-1 group,the level of intracellular superoxide in the shikonin group was increased（P<0.01）;Superoxide levels decreased（P<0.01）.（6）The effect of shikonin on the co-localization of mitochondria and lysosomesCompared with the control group and Mdivi-1 group,the co-localization area of lysosome and mitochondria in the shikonin group increased（P<0.01）;The co-localization area of body and mitochondria decreased（P<0.05）.（7）Effects of shikonin on mitochondrial autophagy-related proteinsCompared with the control group and Mdivi-1 group,the expressions of LC3Ⅱ/Ⅰ,P62,PINK1,and Parkin in the shikonin group were increased（P<0.01）,and the expression of TOM20 was decreased（P<0.05,P<0.01）;Compared with the shikonin+Mdivi-1 group,the expressions of LC3Ⅱ/Ⅰ,P62,PINK1,and Parkin in the shikonin+Mdivi-1 group were decreased（P<0.05）,and the expression of TOM20 was increased（P<0.05）.Conclusion1.There is mitophagy in the villous tissue of tubal pregnancy,indicating that mitophagy may be one of the mechanisms of tubal pregnancy,and mitophagy will be a new way to treat tubal pregnancy.2.Huayu Xiaozheng Granules can destroy the mitochondrial function of trophoblasts and promote mitochondrial autophagy in trophoblasts,so as to achieve the effect of treating tubal pregnancy.3.Shikonin can inhibit the proliferation of HTR-8/Svneo cells,induce mitophagy through the PINK1/Parkin pathway,and reduce mitochondrial function. |
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