To investigate the effects of Gongqing granules on cell apoptosis and signal transduction in human early pregnancy chorionic villi and meconium tissue, and to provide biological basis for the clinical prevention and treatment of abnormal uterine bleeding after medication abortion (referred to as post-medication abortion hemorrhage). Methods: 120 subjects were randomly divided into 30 cases each in Gongqing drug-abortion group, Xizhi drug-abortion group, simple drug-abortion group and negative-pressure suction group, and the chorionic villi and meconium tissues of each group were collected to observe the ultrastructural changes of the cells; apoptosis rate (AR) was detected by the TUNEL method, the intracellular concentration of calcium ions ([Ca2+]i)（Ca2+］ i） was detected by the fluorescence spectrophotometer, and the protein kinase C (PKC) protein expression was detected by immunohistochemical assay. protein expression. Results:The ultrastructure of chorionic villi and meconium cells in the Miyagin abortion group showed typical apoptotic changes, with increased AR, [Ca2+]i, and decreased PKC protein expression, which were statistically significant compared with the other three groups (P<0.05 or P<0.01). Conclusion:Gongqing granules can affect intracellular Ca2+ and PKC-mediated cell signal transduction, induce apoptosis of human early pregnancy chorionic villi and moult tissue cells, promote the complete discharge of chorionic villi and moult tissue, and then prevent and control bleeding after medication abortion. Drug abortion has the advantages of safety and less pain, but there are problems such as long bleeding time, heavy bleeding, etc. In some cases, the bleeding time is as long as 1~4 months, and there is 6%~10% incomplete abortion rate and potential risk of hemorrhage. The formula of Chinese medicine Gongqing granules is an effective formula that has been used in clinic for a long time, and has been approved by the State Drug Administration for clinical research of new drugs, and has successfully completed the phase II clinical study. In this study, we investigated the mechanism of Gongqing granules in preventing and controlling post-drug abortion hemorrhage from the perspectives of cell apoptosis and signal transduction, so as to provide a biological basis for clinical application and to enrich the scientific connotation of the theory of "many deficiencies and stagnation in the postpartum period" in Chinese medicine. Data and Methods I. Subjects 120 cases, all of whom were seen in the gynecology outpatient clinic of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine from May 2006 to October 2007, were included and excluded according to the contents of Chinese Obstetrics and Gynecology, Obstetrics and Gynecology Diagnostic and Treatment Routine and Guidelines for Clinical Research of New Traditional Chinese Medicines. According to the randomized numerical table, the subjects were divided into Gongqing drug abortion group (Group A), Xi Zhi drug abortion group (Group B), simple drug abortion group (Group C) and negative pressure suction abortion group (Group D) by double-blind randomized grouping method, each group consisted of 30 cases. Drugs and reagents: Mifepristone (China National Drug Administration (CNDA) H20000628, batch no. 050501) and misoprostol (CNDA H10960144, batch no. 050309) were produced by Shanghai Hualian Pharmaceutical Co. Ltd. (State Drug Administration Clinical Research Approval for New Drugs 2003L01386). Group A took mifepristone and misoprostol according to the routine method of drug abortion. Group B: Mifepristone and misoprostol were administered on the 1st day of abortion, 48g/d in 3 doses for 3 days. Group B: Mifepristone and misoprostol were administered on the 1st day of abortion, 15 capsules/d in 3 doses for 3 days. Group C: Mifepristone and misoprostol were administered in the usual way of drug abortion. 4. Group D: Negative pressure suction abortion. Histological analysis 1, material collection 30 cases of fresh chorionic villi and 20 cases of meconium tissues discharged from Groups A, B and C after taking medication, and 30 cases of chorionic villi and meconium tissues obtained from Group D at the time of surgery, which were immediately washed with saline and fixed routinely. 2.Transmission electron microscope observation of cell ultrastructure Epoxy resin (812) embedding, ultra-thin sectioning machine, thickness of about 60 nm, double staining of dioxiranyl acetate and lead citrate, JEM-1200EX transmission electron microscope observation, and photographs. 3, TUNEL method to detect cellular AR specimen sections after HE staining, dewaxing, proteinase K incubation at room temperature for 20 min; drop 50 μl of TUNEL reaction mixture, incubated in a wet box at 37 ℃ for 60 min; add 50 μl of the transforming agent POD, incubated at 37 ℃ for 30 min; after the DAB color, hematoxylin nuclear staining, dehydration, transparency, sealing the film. All the above steps were rinsed with PBS, and the negative control was incubated with TUNEL reaction solution without terminal deoxynucleotidyl transferase. Quantitative image analysis: TUNEL-positive reactants were localized in the nucleus and appeared in brownish-yellow color, and the color-positive cells were apoptotic cells [6]. Ten non-overlapping fields of view (×100) were randomly selected, and the percentage of positive cells was AR. 4. Detection of [Ca2+] by fluorescence spectrophotometer i The excitation grating was set at 5 nm, the emission grating at 10 nm, and the temperature of measurement was 37°C in a Hitachi F-4500 fluorescence spectrophotometer. The excitation spectra of Fura-2/AM standard solution and loading solution were measured spectroscopically, and their maximum wavelengths decreased from 380 nm to 340 nm, indicating that the Fura-2/AM loading entered into the cell. The fluorescence intensity (F value) of Fu-ra-2 loading solution was determined in a temporal manner (excitation wavelength of 340 nm), and the maximum fluorescence intensity Fmax was detected after 30 min with the addition of 10% TritonX-100 at a final concentration of 0. 1%, and the minimum fluorescence intensity Fmin was measured after 30 min with the addition of EDTA at a final concentration of 5 mmol/,l. The maximum fluorescence intensity Fmin was measured after 30 min with the addition of EDTA at a final concentration of 5 mmol/,l. The maximum fluorescence intensity Fmax was measured after 30 min with the addition of EDTA at a final concentration of 5 mmol/,l. The minimum fluorescence intensity Fmin was measured after 30 min with the addition of EDTA at a final concentration of 0. 1%. nmol/L)=Kd(F-Fmin/Fmas-F),Kd is the dissociation constant for the reaction between Fura-2 and Ca2+,224nmol/L at 37℃. 5,Immunohistochemistry to detect the changes of cellular PKC protein expression Specimen sections were dewaxed, incubated with 3% H O at room temperature for 10min;rinsed and repaired by microwave oven antigens; 10% normal goat serum was closed and added with primary antibody. After rinsing, add horseradish enzyme labeled secondary antibody dropwise, incubate at 37℃ for 30min; after color development with DAB colorant, rinse well with tap water, re-stain and seal the slices. The results were analyzed by Leica QW in V3 image analysis software, and there was a negative correlation between the strength of protein expression and the size of grayscale value, i.e., the stronger the protein expression, the lower the grayscale value. V. Statistical methods SPSS12.0 statistical software was used to analyze the results. Measurement data were expressed as mean±standard deviation (±s), and the variance chi-square test was used first, followed by q-test and rank-sum test to compare the differences between groups, and x2 test for the counting data. Results I. General conditions After statistical analysis, the differences between the four groups in terms of age, fertility, days of menopause and size of gestational sacs were not statistically significant and were comparable. Ultrastructural observation of chorionic villi and metaphase cells (Figure 1) Group D. The surface of chorionic trophoblasts and metaphase cells were rich in microvilli, the basement membranes of the trophoblasts were normal, the nuclei of trophoblasts and metaphase cells were normal in morphology, the chromatin in the nucleus was uniformly distributed, and the organelles were normal, and there were a large number of secretory particles with high electron density in granulosa cells, and the inter-cellular connection was tight. Group C. The surface microvilli of trophoblasts and metaphase cells were detached, the basement membrane of trophoblasts was thickened, the nuclei of trophoblasts and metaphase cells were irregular, the nuclear membrane was swollen, and apoptotic morphological changes, such as clumping of euchromatin and heterochromatin, and nuclear consolidation, were observed. There was a marked decrease in the number of endocrine granules in granulosa cells, and widening of intercellular junctions. Group B. The ultrastructural changes of syncytiotrophoblasts, cytotrophoblasts, metaphase cells and granulosa cells were similar to those of group C. Group B showed apoptotic morphological changes. Group A showed apoptotic morphological changes, which were more serious than those in Groups C and B. Typical apoptotic vesicles were found in syncytiotrophoblasts, cytotrophoblasts, metaphase cells and granulosa cells, which contained organelles with a basically normal structure. Changes of AR, [Ca2+] i and PKC in group A, AR and [Ca2+] i of chorionic trophoblasts and metaphase cells were increased, and the gray value of PKC of chorionic and metaphase cells was increased, and the differences were statistically significant compared with those of other three groups (P<0.05), which showed that the expression of PKC protein in group A was lower than that in the other groups. Table 1 Changes of AR, [Ca2+]i and PKC in chromaffin trophoblast cells(±s) Group Number of cases AR(%) [Ca2+]i PKC gray value Group A 30 3.02±0.20*▲▲△ 125.37±6.40*▲△△ 135.71±3.82*▲△ Group B 30 2.89±0.19* 120.33±7.58* 133.73± 3.81* Group C 30 2 2.89±0.19* 120.33±7.58* 133.73± 3.81* Group B 30 2.89±0.19* 120.33±7.58* 133.73± 3.81 3.81* Group C 30 2.87±0.21* 119.91±6.80* 133.17±3.70* Group D 30 2.17±0.23 109.03±7.84 127.51±3.73 *P<0.01 comparing with Group D ▲P<0.05 comparing with Group C ΔΔΔ comparing with Group B ΔΔΔ comparing with Group C ΔΔΔ comparing with Group B ΔΔΔ comparing with Group B ΔΔ comparing with Group C ΔΔ comparing with Group C ΔΔΔ comparing with Group B P<0.01 compared with group B. Table 2 Changes in AR, [Ca2+] i and PKC of decidua tissue cells ( ±s) Groups Number of cases AR (%) [Ca2+] i PKC Gray scale value Group A 20 3.13±0.18*▲△ 130.65±7.46*▲△ 142.80±4.93*▲△ Group B 20 3.02±0.14* 125.14±8.04 * 139.17±4.94* Group C 20 2.99±0.19* 124.42±7.96* 138.25±5.18* Group D 30 2.83±0.15 110.31±7.53 131.45±4.03 *P<0.01 vs. Group D ▲P<0.05 vs. Group C △P<0.05 vs. Group B DISCUSSION Recent studies have concluded that Abnormal uterine bleeding after drug abortion is related to the lack of apoptosis of chorionic villus and meconium cells, and the cells of chorionic villus and meconium tissues after the action of mifepristone showed apoptotic tendency, and the results of the present study are consistent with the report. It has been proved that traditional Chinese medicines can regulate apoptosis, activating blood circulation and removing blood stasis can induce apoptosis and regulate the balance between cell proliferation and apoptosis, and heat-clearing medicines and tonic medicines can induce apoptosis in tumor cells, but there is no research report on traditional Chinese medicines inducing apoptosis in chorionic villi and ecchymotic tissue cells of early pregnancy for prevention of hemorrhage after medication abortion. In this study, we observed the ultrastructural changes of human early pregnancy chorionic villus and meconium cells after abortion in 4 groups of women, and found that the apoptosis degree of chorionic villus and meconium cells was higher than that of the other 3 groups, and the chorionic villus and meconium AR in this group was significantly higher than that of the other groups, suggesting that Gongqing granules had the effect of inducing apoptosis of human early pregnancy chorionic villus and meconium cells in medication abortion. In order to further explore this mechanism of action of Gongqing granules, we have studied the second messenger Ca2+ and its important target molecular protein PKC, which are the key to cellular life activities. Ca2+ is the hub of various cell signaling pathways, and there is evidence that elevated [Ca2+]i can cause apoptosis. In recent years, the study of apoptosis in drug-induced abortions has been gradually deepened, but the study of Ca2+ overload, which is an important factor involved in apoptosis signaling, has not been reported. We included [Ca2+]i for the first time in the study of the effect of abortifacient drugs on apoptosis, and found that the [Ca2+]i of chorionic villi and meconium was elevated by Gongqing granules, suggesting that Gongqing granules may induce apoptosis in human early pregnancy chorionic villi and meconium by affecting Ca2+-mediated signal transduction. In the signaling pathway, PKC is an important target protein for intracellular Ca2+ action, and it plays an important role in cell growth, differentiation and apoptosis. PKC activation mediates a variety of biological effects by phosphorylating serine/threonine residues of target protein molecules, which can down-regulate G protein activation due to phosphorylation, causing an increase in cyclic adenosine monophosphate (cAMP) levels and inducing apoptosis. It has also been reported that PKC activation causes an increase in intracellular cAMP level, which promotes transcription leading to cell proliferation and differentiation. It was found that mifepristone inhibited the PKC signaling pathway and raf-1 waterfall kinase chain and division-activating protein in early pregnancy trophoblast cells, inhibited proliferation and division, and induced apoptosis. In this study, we found that the expression of PKC protein in chorionic trophoblast cells and metaphase tissue cells was reduced after drug abortion, suggesting that Gongqing pellets may inhibit cell differentiation and value-added and induce apoptosis by reducing the expression of PKC protein. Chinese medicine believes that medication abortion is artificial termination of pregnancy, medication abortion yin and blood suddenly weak, gas does not take blood; or abortion is incomplete, the residual blood stagnation in the uterus, blood stasis does not go, the new blood is difficult to be safe; or the external evils attacked by the brewing of heat, heat injuries to the blood; or the three are intertwined with each other, coexisting in the real and the real, resulting in the unclean discharge. "Blood stasis, deficiency and heat are the main mechanisms of this disease, and the key to treating this disease is to activate blood circulation and eliminate blood stasis, while blood circulation and nourishment of blood and qi are the basis for treating this disease. Materia Medica, the principal medicine of Gongqing Granules, activates blood circulation and removes blood stasis, clears heat and stops bleeding; the subject medicines, Amaranthus, Radix et Rhizoma Gastrodiae, Radix Achyranthis Bidentatae, Radix Bupleurum officinale help the principal medicine to activate blood circulation and remove blood stasis, cools blood circulation and stops bleeding; the adjuvant medicines Angelica Sinensis and Codonopsis Pilosulae nourish blood and benefit the vital energy to activate blood circulation and remove blood stasis; and the medicine, Glycyrrhiza Glutinosa, replenishes the middle Jiaozhong and benefit the vital energy to regulate the medicines; experiments have confirmed that this medicine can synergize with mifepristone in anti-early pregnancy to increase the rate of complete miscarriage, shorten the time of vaginal bleeding, reduce the quantity of vaginal bleeding, and do not influence the function of ovulation of the ovary or the restoration of menses. Pharmacodynamic experiments showed that Gongqing Granules could significantly stimulate the uterine smooth muscle, shorten the coagulation time, increase the content of estrogen, have antibacterial and analgesic effects, and found that the drug has a tendency to induce apoptosis of chorionic cells. The present study found that this formula can induce apoptosis of human early pregnancy chorionic villus and ecdysis tissue cells, promote the complete discharge of chorionic villus and ecdysis tissue, thus preventing and controlling the bleeding after medical abortion. The regulation of Ca2+ and PKC-mediated cell signaling may be one of the mechanisms of action. However, the source of intracellular Ca2+ and its exact mechanism in inducing apoptosis of chorionic villi and metaphase tissue has not yet been clarified, and in-depth research in this direction will further reveal the pathogenesis of this disease and the mechanism of drug prevention and treatment.