Zishen Qingre Lishi Huayu recipe promotes proliferation and inhibits apoptosis of GCs of PCOS via KLF4-C/EBPß pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Zishen Qingre Lishi Huayu recipe (ZQLHR) is a herbal recipe created on the basis on the theory of traditional Chinese medicine and clinical practice, and is mainly used in the treatment of polycystic ovary syndrome (PCOS). However, the underlying mechanism for this fact has not been clearly elucidated. AIM OF THE STUDY: To verify whether ZQLHR regulates granulosa cells (GCs) proliferation and apoptosis through the Krüppel-like factor 4 (KLF4) - CCATT enhancer-binding proteinß (C/EBPß) pathway, and to provide in vitro molecular mechanism supporting for the effects of ZQLHR to enhance follicular development and treat patients with PCOS. MATERIALS AND METHODS: Based on previous experiments, we performed the following experiments. Firstly, we treated KGN cells (a steroidogenic human granulosa-like tumor cell line) for 48 h using different concentrations of ZQLHR in order to observe apoptosis in each group. Secondly, the mRNA and protein expression levels of KLF4 and C/EBPß in KGN cells after administrated with ZQLHR were examined by quantitative real-time PCR(q-PCR) and Western blot assay. Thirdly, after knocking down KLF4 and C/EBPß using siRNAs, the relationship between KLF4 and C/EBPß in KGN cells was detected. Further, cell counting kit-8 assay, colony formation assay and flow cytometry were used to verify whether ZQLHR promotes proliferation and facilitates apoptosis in KGN cells through the KLF4-C/EBPß pathway. Finally, q-PCR and Western blot were used to test whether ZQLHR mediated proliferation and apoptosis-related factors such as B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (BAX), proliferating cell nuclear antigen (PCNA) and cleaved caspase-3 to affect the proliferation and apoptosis of KGN cells through the KLF4-C/EBPß pathway. CONCLUSIONS: ZQLHR, containing 0.2% by volume, administered to KGN cells resulted in the lowest rate of apoptosis. The expression levels of KLF4 and C/EBPß were increased in KGN cells following ZQLHR treatment. Additionally, ZQLHR promoted proliferation and inhibited apoptosis of KGN cells by modulating proliferation and apoptosis-related factors via the KLF4-C/EBPß pathway. Furthermore, we confirmed that KLF4 and C/EBPß regulate each other in KGN cells. These findings indicate that ZQLHR enhances the proliferation of GCs and suppresses their apoptosis, which constitutes a therapeutic mechanism for treating patients with PCOS.

Transcriptomic analysis reveals that the anti-PCOS effects of Zishen Qingre Lishi Huayu recipe may involve pathways and genes related to autophagy, steroidogenesis, oxidative stress, and inflammation in granulosa cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Zishen Qingre Lishi Huayu recipe (ZQLHR) is a Chinese medicine compound composed of nine herbs for the treatment of polycystic ovary syndrome (PCOS). It is used to nourish kidneys, clear heat, reduce dampness and dissipation blood stasis by promoting diuresis and blood circulation, dredging the meridians and harmonizing menstruation in the treatment of PCOS. Several clinical studies have shown that ZQLHR is effective in the treatment of PCOS, but the underlying mechanism remains unclear. AIM OF THE STUDY: In this study, we researched on the effects and mechanism of action of ZQLHR during treatment of human granulosa cells (hGCs) obtained from PCOS patients in order to provide a scientific basis for the clinical application of ZQLHR in the treatment of PCOS, emphasize the importance of some genes that have been reported to play a role in the pathogenesis or therapeutic mechanisms of PCOS from the perspective of disease treatment, and identify some new genes and signaling pathways that may play an important role in the treatment of PCOS. MATERIALS AND METHODS: KGN cells (a granulosa cell-like tumor cell line) were subjected to a cell counting kit-8 assay to explore the appropriate intervention concentration and duration of ZQLHR. Treated with or without ZQLHR (ZQLHR and control groups), the hGCs obtained from PCOS patients were sequenced using RNA sequencing, and the genes thus detected were further analyzed through Kyoto encyclopedia of genes and genomes enrichment analysis, gene set enrichment analysis, and individuation gene analysis. These genes were also compared with PCOS-related genes in other databases. To further verify the authenticity of the differentially expressed genes between the two groups, the expression of eight randomly selected vital genes and three proteins of interest was verified through real time quantitative polymerase chain reaction and Western blot experiment respectively. RESULTS: The best intervention concentration and duration for ZQLHR to promote the proliferation of KGN cells were 0.2% and 48 h respectively in this experiment. Multiple signaling pathways and 55 focus differentially expressed genes, both related to autophagy, steroidogenesis, oxidative stress-related longevity, inflammation, and complications of PCOS, may play an important role in the therapeutic mechanism of action of ZQLHR. The expression of eight genes is consistent with the result of RNA sequencing, and the expression of three proteins of interest is the same as expected. CONCLUSIONS: The promotion of hGCs proliferation upon treatment with ZQLHR may be a manifestation of ZQLHR in the treatment of PCOS patients. The positive effects of ZQLHR against PCOS may involve pathways and genes related to autophagy, steroidogenesis, oxidative stress-related longevity, and inflammation in hGCs. Some components of ZQLHR applied for the treatment of PCOS may also be effective for the treatment of some complications of PCOS.

Kidney Mesenchymal stem cells alleviate cisplatin-induced kidney injury and apoptosis in rats.

OBJECTIVE: This experiment was designed to demonstrate Mesenchymal stem cells (MSCs) derived from kidney can alleviate cisplatin-induced kidney injury and renal cell apoptosis through paracrine pathway. METHODS: Firstly, MSCs were isolated from kidney of young rats, and their surface-specific markers were identified by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and immunofluorescence staining. Self-renewal ability of Kidney Mesenchymal Stem Cells (KMSCs) was observed by cell counting and 5-Bromo-2'-deoxyuridine (BrdU) fluorescence staining. KMSCs at logarithmic growth stage were traced and injected into rat through tail vein. RESULTS: The results showed that KMSCs homed in the kidney tissues, decreased the secretion of inflammatory factors (CRP, TNFα, IL-1ß, IL-6), and alleviated renal function. Hematoxylin and Eosin (H＆E), Masson and Periodic Acid-silver Methenamine (PASM) staining showed that KMSCs could alleviate pathological damage in rats. Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling (TUNEL) assay showed that KMSCs could reduce the apoptosis of rat kidney cells induced by cisplatin. Finally, Immunohistochemistry (IHC) results showed that cisplatin could induce higher expression of the pro-apoptotic protein Bax and lower expression of anti-apoptotic Bcl-2 in kidney tissues. However, KMSCs could reverse the pro-apoptotic effect of cisplatin on kidney cells and improve the survival rate of rats. CONCLUSIONS: In conclusion, KMSCs were successfully isolated from kidney tissues, and KMSCs have therapeutic effects on rat kidney injury induced by cisplatin.

Effects of Acupuncture Combined with Moxibustion on Reproductive and Metabolic Outcomes in Patients with Polycystic Ovary Syndrome: A Systematic Review and Meta-Analysis.

Objectives: In this systematic review, the effects of acupuncture combined with moxibustion on reproductive and metabolic outcomes in patients with polycystic ovary syndrome (PCOS) were evaluated. Methods: Randomized controlled trials (RCTs) assessing acupuncture combined with moxibustion + basic treatment (experimental group) versus basic treatment alone (control group) for treating PCOS were identified from English and Chinese databases up to November 3, 2021. Outcomes related to pregnancy, ovulation, miscarriage, sex hormones, and metabolic disorders were of interest. In the meta-analysis, risk ratios (RRs) and mean differences (MDs) and their 95% confidence intervals (CIs) were used as effect measures. Results: Twenty-five RCTs (n = 1991) were included. The pooled results showed that the experimental group had significant increases in the pregnancy rate (RR 1.81, 95% CI 1.58 to 2.08) and ovulation rate (RR 1.31, 95% CI 1.22 to 1.40) and decreases in the miscarriage rate (RR 0.45, 95% CI 0.28 to 0.73), and ovarian volume (MD -0.75 cm3, 95% CI -1.30 to -0.20). In the experimental group, improvements in the luteinizing hormone (LH) level, the LH-to-follicle-stimulating hormone (FSH) ratio, total testosterone level, fasting insulin level, and body mass index, but not in FSH, oestradiol, or dehydroepiandrosterone sulfate levels, were significantly greater. All reported adverse events were mild. Based on the limitations of risk of bias, inconsistency, imprecision, and/or publication bias, the level of evidence was judged to be moderate for the pregnancy rate, ovulation rate, miscarriage rate, LH level, and LH/FSH ratio and very low for the other outcomes. Conclusion: Among patients with PCOS, using acupuncture combined with moxibustion as a complementary therapy to basic treatments can improve pregnancy, ovulation, and miscarriage rates, the levels some sex hormones, and metabolic indicators, with good safety. Additionally, this combination therapy may have no effect on the FSH, oestradiol, or dehydroepiandrosterone sulfate level.

Kuntai capsules improve glucolipid metabolism in patients with polycystic ovary syndrome: A randomized, double-blind, placebo-controlled trial.

BACKGROUND: The aim of this study was to observe the effect and safety of Heyan Kuntai Capsule (HYKT) on glucose and lipid metabolism in patients with polycystic ovary syndrome (PCOS). METHODS: Hundred patients with PCOS were randomly divided into HYKT group (n = 50) and placebo groups (n = 50) in which the individuals were treated with HYKT and its placebo continuously for 6 months. Meanwhile, all participants received health education (such as exercise and diet). The primary outcomes were serum sex hormone levels, a series of blood lipid, fasting and postprandial 2 hours blood glucose. Body mass index (BMI), waist-hip ratio (WHR), insulin, homeostatic model assessment of insulin resistance (HOMA-IR), and insulin-sensitive index (ISI) were also observed. In addition, adverse events were recorded to evaluate the drug safety. RESULTS: After treatment, the BMI and WHR of all the patients were decreased. The fasting and postprandial 2 hours blood glucose levels were significantly declined when treated with HYKT, which were not observed in the placebo group. Similarly, serum sex hormones including luteinizing hormone (LH), LH/follicle-stimulating hormone (FSH), and testosterone were lowered after treated with HYKT instead of the placebo. Besides, blood lipids outcomes such as total cholesterol, triglyceride, and low-density lipoprotein cholesterol, as well as insulin and HOMA-IR were decreased with significance in HYKT group when compared with those in the placebo group, whereas high-density lipoprotein cholesterol and ISI increased obviously. CONCLUSION: HYKT showed the effect on ameliorating the glucose and lipid metabolism disorder and improving insulin resistance and increase insulin sensitivity of PCOS patients, which is similar to insulin sensitizing agent.

Efficacy of acupuncture on pelvic pain in patients with endometriosis: study protocol for a randomized, single-blind, multi-center, placebo-controlled trial.

BACKGROUND: Endometriosis is a chronic gynecological disease that is characterized by the presence of endometrial tissue outside the uterine cavity. The main symptoms include dysmenorrhea, dyspareunia, chronic pelvic pain, and infertility. These symptoms impair the lives of most of the women suffering from the disease. Surgical resection of endometriotic lesions is an effective means of treating dysmenorrhea, but the risk of recurrence is high. Western medicine has limited use for treating it due to side effects and ineffectiveness. The purpose of this study is to verify the effectiveness and safety of acupuncture. METHODS/DESIGN: This trial will be carried out in four parts. A total of 106 eligible patients with pelvic pain related to endometriosis will be randomly assigned into two groups, in a 1:1 ratio, as the treatment group or the control group. The participants assigned to the treatment group will be treated with acupuncture treatment at Guanyuan (CV4), Sanyinjiao (SP6), Taichong (LR3), Zhaohai (KI6) and Qichong (ST30) while the control group will receive acupuncture at non-acupoints. The trial will include three menstrual cycles of treatment and three menstrual cycles of follow-up. The primary outcome is pelvic pain that will be assessed by means of a 10-cm visual analog scale (VAS). At each stage, we will evaluate the safety of the acupuncture treatment. DISCUSSION: The study will compare the effectiveness and safety of acupuncture with comfort needles on pelvic pain related to endometriosis in the hope of providing significant evidence for using acupuncture on pelvic pain related to endometriosis. TRIAL REGISTRATION: ClinicalTrials.gov, ID: NCT03125304 . Registered on 30 April 2017.

Effect of Acupuncture and Clomiphene in Chinese Women With Polycystic Ovary Syndrome: A Randomized Clinical Trial.

Importance: Acupuncture is used to induce ovulation in some women with polycystic ovary syndrome, without supporting clinical evidence. Objective: To assess whether active acupuncture, either alone or combined with clomiphene, increases the likelihood of live births among women with polycystic ovary syndrome. Design, Setting, and Participants: A double-blind (clomiphene vs placebo), single-blind (active vs control acupuncture) factorial trial was conducted at 21 sites (27 hospitals) in mainland China between July 6, 2012, and November 18, 2014, with 10 months of pregnancy follow-up until October 7, 2015. Chinese women with polycystic ovary syndrome were randomized in a 1:1:1:1 ratio to 4 groups. Interventions: Active or control acupuncture administered twice a week for 30 minutes per treatment and clomiphene or placebo administered for 5 days per cycle, for up to 4 cycles. The active acupuncture group received deep needle insertion with combined manual and low-frequency electrical stimulation; the control acupuncture group received superficial needle insertion, no manual stimulation, and mock electricity. Main Outcomes and Measures: The primary outcome was live birth. Secondary outcomes included adverse events. Results: Among the 1000 randomized women (mean [SD] age, 27.9 [3.3] years; mean [SD] body mass index, 24.2 [4.3]), 250 were randomized to each group; a total of 926 women (92.6%) completed the trial. Live births occurred in 69 of 235 women (29.4%) in the active acupuncture plus clomiphene group, 66 of 236 (28.0%) in the control acupuncture plus clomiphene group, 31 of 223 (13.9%) in the active acupuncture plus placebo group, and 39 of 232 (16.8%) in the control acupuncture plus placebo group. There was no significant interaction between active acupuncture and clomiphene (P = .39), so main effects were evaluated. The live birth rate was significantly higher in the women treated with clomiphene than with placebo (135 of 471 [28.7%] vs 70 of 455 [15.4%], respectively; difference, 13.3%; 95% CI, 8.0% to 18.5%) and not significantly different between women treated with active vs control acupuncture (100 of 458 [21.8%] vs 105 of 468 [22.4%], respectively; difference, -0.6%; 95% CI, -5.9% to 4.7%). Diarrhea and bruising were more common in patients receiving active acupuncture than control acupuncture (diarrhea: 25 of 500 [5.0%] vs 8 of 500 [1.6%], respectively; difference, 3.4%; 95% CI, 1.2% to 5.6%; bruising: 37 of 500 [7.4%] vs 9 of 500 [1.8%], respectively; difference, 5.6%; 95% CI, 3.0% to 8.2%). Conclusions and Relevance: Among Chinese women with polycystic ovary syndrome, the use of acupuncture with or without clomiphene, compared with control acupuncture and placebo, did not increase live births. This finding does not support acupuncture as an infertility treatment in such women. Trial Registration: clinicaltrials.gov Identifier: NCT01573858.

Ping-Chong-Jiang-Ni Formula Induces Apoptosis and Inhibits Proliferation of Human Ectopic Endometrial Stromal Cells in Endometriosis via the Activation of JNK Signaling Pathway.

Endometriosis is a common gynecological condition in childbearing age women and its therapy in modern medicine achieves usually temporary cure. Ping-Chong-Jiang-Ni formula (PCJNF), a Chinese herbal medicine (CHM), was shown to be clinically effective on endometriosis. Meanwhile, c-Jun N-terminal kinase (JNK) signaling pathway was involved in the therapeutic process of CHM on endometriosis. Here, we explored the effect of PCJNF on the ectopic endometrial stromal cells (EESCs) from endometriosis and test whether JNK signaling was involved. After being treated with PCJNF-containing serum obtained from Sprague Dawley rat, cell proliferation, migration, invasion, and apoptosis were evaluated in EESCs, and the total and phosphorylated JNK, ERK, and p38 proteins were detected. Our results showed that PCJNF could suppress cell proliferation, migration, and invasion and induce apoptosis in EESCs. The suppressed proliferation and increased apoptosis were dependent on JNK activation. Additionally, PCJNF caused cell cycle arrest at G2/M phase and this effect was mediated by JNK signaling, while the decreased cell migration and invasion treated by PCJNF were independent of JNK signaling. In summary, our results provided the first evidence that PCJNF could suppress cell proliferation, migration, and invasion, while increasing apoptosis in EESCs, and the suppressed proliferation and enhanced apoptosis were mediated by JNK signaling.

Randomized controlled trial of letrozole, berberine, or a combination for infertility in the polycystic ovary syndrome.

OBJECTIVE: To study whether a combination of berberine and letrozole results in higher live births than letrozole alone in infertile women with polycystic ovary syndrome (PCOS). DESIGN: A multicenter randomized double-blinded placebo-controlled trial. SETTING: Reproductive and developmental network sites. PATIENT(S): Eligible women had PCOS as defined by the Rotterdam criteria. We enrolled 644 participants randomized 1:1:1 among letrozole, berberine, and combination groups. INTERVENTIONS(S): Berberine or berberine placebo were administrated orally at a daily dose of 1.5 g for up to 6 months. Patients received an initial dose of 2.5 mg letrozole or placebo on days 3-7 of the first three treatment cycles. This dose was increased to 5 mg on the last three cycles if not pregnant. MAIN OUTCOMES MEASURE(S): Cumulative live births. RESULTS: The cumulative live births were similar between the letrozole and combination groups after treatment (36% and 34%), and were superior to those in the berberine group (22%). Likely, conception, pregnancy, and ovulation rates were similar between the letrozole and combination groups, and these were significantly higher than in the berberine group. There was one twin birth in the letrozole group, three twin births in the combination group, and none in the berberine group. CONCLUSION(S): Berberine did not add fecundity in PCOS when used in combination with the new ovulation agent letrozole. CLINICAL TRIAL REGISTRATION NUMBER: ChiCTR-TRC-09000376 (http://apps.who.int/trialsearch/).

[Treatment of refractory polycystic ovary syndrome by bushen huoxue method combined with ultrasound-guided follicle aspiration].

OBJECTIVE: To observe the clinical effect of traditional Chinese medicine Bushen Houxue (BSHX) method combined with ultrasound-guided follicle aspiration (MFA) in treating refractory polycystic ovary syndrome (PCOS). METHODS: Forty-four patients with PCOS were randomly assigned to two groups by randomizing digital table, 20 in the observation group and 24 in the control group. MFA was performed on both groups, and the decoction of BSHX, which consisted of dodder seed 20 g, prepared rehmannia root 10 g, mulberry mistletoe 20 g, epimedium 15 g, psoralea fruit 10 g, solomonseal rhizome 10 g, honeylocust thorn 15 g, peach kernel 10 g, pleione bulbocodioides 10 g, red sage root 20 g, and licorice root 6 g, was given to the observation group one dose every day for 14 days every menstrual cycle. Changes of follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) were determined before and after MFA. The impacts on quantity of HMG used, number of sinus follicle, mature follicle, incidence of ovarian hyperstimulating syndrome (OHSS), luteinized unrupture follicular syndrome (LUFS) and pregnance rate were also observed. RESULTS: MFA had been performed for 42 cycles in the observation group and 56 cycles in the control group. Levels of T, LH and LH/FSH ratio markedly reduced after aspiration, showing significant difference as compared with those of before treatment in both groups (P < 0.01), and difference of LH/FSH between groups was of statistical significance (P <0.01). In the observation group, 18 patients (90.0%) had their sinus follicle decreased to < 10 after MFA, while in the control group, it reached to <10 in 22 patients (91.70%), all were different to those before treatment (P <0.01). In the observation group, the quantity of HMG used for promoting ovulation was (585.0 +/- 195.0) IU, number of mature follicle at the day of HCG injection was 1.1 +/- 0.3, while in the control group, the corresponding data were (1470.0 +/- 532.5) IU and 3.1 +/- 1.4, all with significant difference between groups (P <0.01). None of OHSS and 1 case of LUFS occurred in the former group, while 1 mild OHSS and 2 LUFS in the latter. After ovulation promoting therapy and in the 3-month secutive follow-up period, pregnancy was found in 8 out of the 18 patients in the observation group (one twins and 7 single), with the pregnancy rate of 44.4%; while in the control group, 7 in 22 (2 twins and 5 single) was found, the pregnancy rate being 31.8%. CONCLUSION: BSHX method combined with MFA is a safe and effective treatment for refractory PCOS, with few trauma. The combined usage of Chinese herbal medicine could significantly reduce dosage of HMG used for promoting follicle and the production of multiple mature follicles, thus to avoid the risk of OHSS.

[Effects of combined therapy of salpingostomy and Bushen Huoxue drugs on fallopian tube obstructive infertility].

OBJECTIVE: To compare the effect of treatment of fallopian tube obstructive infertility by salpingostomy alone and in combination with TCM drugs for Bushen Huoxue (invigorating Shen and promoting blood circulation). METHODS: To all the patients, salpingostomy was performed 3 to 7 days after menstruation and hydrotubation with Xiangdan Injection (XI) was applied once in the next menstrual cycle. Bushen Huoxue Decoction (BHD) was administrated additionally to patients in the treated group, 1 dose every day starting from the 5th day of menstrual cycle for 14 days. Three months' treatment was taken as one therapeutic course and the observation lasted for 4 courses. RESULTS: The condition of follicular development, thickness of endometrium and level of serum estradiol in the preovulatory phase after treatment were all significantly increased in the two groups (P < 0.05 and P < 0.01); but the improvement in the treated group was significantly superior to that in the control group, showing significant difference. Moreover, the pregnancy rate was also higher in the former than in the latter (P < 0.05). CONCLUSION: The combined therapy of salpingostomy and TCM drugs for invigorating Shen and promoting blood circulation is an effective therapy for fallopian tube obstructive infertility, it has the effect of enhancing follicular development and increasing thickness of endometrium, and could elevate the pregnancy rate in patients.

[Effect of dandi tablet on blood lipids and sex hormones in women of postmenopausal stage].

OBJECTIVE: To explore the effect of Dandi Tablet (DDT), a TCM preparation for tonifying Shen and activating blood circulation, on blood lipids and sex hormones in women of postmenopausal stage. METHODS: Seventy-three women were randomly divided into two groups. The 46 subjects in the DDT group were treated with DDT (consisted of prepared Rehmannia Root 24 g, Dogwood Fruit 9 g, Poria 9 g, Water-plantain 9 g, Tuber Peony bark 9 g, Chinese Yam 12 g and Red Sage 15 g, etc.) twice a day, 4 tablets each time. The 27 subjects in the control group were treated with nylestriol (each tablet contains 2.5 mg of 17 alpha-ethynylestriol-3-cyclopentyl ether) 5 mg every month, for 3 successive months. Levels of blood lipids, including total cholesterol (TC), triglyceride (TG), high and low density lipoprotein cholesterol (HDL-C, LDL-C), apoprotein A and B (apoA, apoB) and sex hormones, including follicular stimulating hormone (FSH) and estradiol (E2) were determined before and after treatment. RESULTS: Compared with pre-treatment, after treatment, levels of TC/HDL-C and FSH significantly lowered, HDL-C and E2 increased in both groups (P < 0.05 or P < 0.01), levels of TG lowered and apoA increased significantly in the DDT group (P < 0.05). CONCLUSION: DDT has the effect of elevating levels of estrogen and improving blood lipid metabolism, therefore shows a coronary heart disease preventive effect in women of postmenopausal stage.