[Clinical practice guideline for postmenopausal osteoporosis with traditional Chinese medicine].

The editorial group of the clinical practice guideline for postmenopausal osteoporosis(PMOP) with traditional Chinese medicine(TCM)(hereinafter referred to as "guideline") is composed of experts specialized in TCM orthopedics, TCM gynecology, clinical epidemiology, etc. The guideline was formulated through registration, collection and selection of clinical issues/outcome indicators, evidence retrieval and screening, preparation of systematic reviews, evaluation of evidence quality, formation of recommendations, drafting, and peer review. The syndromes and treatment of PMOP are elaborated in detail. Specifically, Liuwei Dihuang Pills and Zuogui Pills are recommended for PMOP with Yin deficiency in the liver and kidney, Qing'e Pills for PMOP with kidney deficiency and blood stasis, Yougui Pills and Jingui Shenqi Pills for PMOP with Yang deficiency in the spleen and kidney, and Er'xian Decoction for PMOP with Yin and Yang deficiency in the kidney. In addition, Duhuo Jisheng Decoction can be used to relieve pain. The commonly used Chinese patent medicines include Xianling Gubao Capsules, Qianggu Capsules, Jintiange Capsules, Gushukang Capsules, Hugu Capsules, Jinwu Gutong Capsules, and Guyuling Capsules. Acupuncture and moxibustion are also effective approaches for PMOP. The rehabilitation and daily management were carried out by exercise therapies such as Baduanjin(eight-section brocade), Wuqinxi(five-animal exercises), and Taijiquan(Tai Chi), Chinese medicine diet, health education, and fall prevention. The promotion and application of this guideline will facilitate the implementation of TCM prevention and treatment of PMOP, ensure the quality of life of PMOP patients, provide effective and safe TCM treatment measures for PMOP, and reduce the risk of fracture complications.

[Large- scale prospective clinical study on prophylactic intervention of COVID-19 in community population using Huoxiang Zhengqi Oral Liquid and Jinhao Jiere Granules].

To scientifically evaluate the intervention effect of Chinese medicine preventive administration(combined use of Huo-xiang Zhengqi Oral Liquid and Jinhao Jiere Granules) on community population in the case of coronavirus disease 2019(COVID-19), a large cohort, prospective, randomized, and parallel-controlled clinical study was conducted. Total 22 065 subjects were included and randomly divided into 2 groups. The non-intervention group was given health guidance only, while the traditional Chinese medicine(TCM) intervention group was given two coordinated TCM in addition to health guidance. The medical instructions were as follows. Huoxiang Zhengqi Oral Liquid: oral before meals, 10 mL/time, 2 times/day, a course of 5 days. Jinhao Jiere Granules: dissolve in boiling water and take after meals, 8 g/time, 2 times/day, a course of 5 days, followed up for 14 days, respectively. The study found that with the intake of medication, the incidence rate of TCM intervention group was basically maintained at a low and continuous stable level(0.01%-0.02%), while the non-intervention group showed an overall trend of continuous growth(0.02%-0.18%) from 3 to 14 days. No suspected or confirmed COVID-19 case occurred in either group. There were 2 cases of colds in the TCM intervention group and 26 cases in the non-intervention group. The incidence of colds in the TCM intervention group was significantly lower(P<0.05) than that in the non-intervention group. In the population of 16-60 years old, the incidence rate of non-intervention and intervention groups were 0.01% and 0.25%, respectively. The difference of colds incidence between the two groups was statistically significant(P<0.05). In the population older than 60 years old, they were 0.04% and 0.21%, respectively. The incidence of colds in the non-intervention group was higher than that in the intervention group, but not reaching statistical difference. The protection rate of TCM for the whole population was 91.8%, especially for the population of age 16-60(95.0%). It was suggested that TCM intervention(combined use of Huoxiang Zhengqi Oral Liquid and Jinhao Jiere Granules) could effectively protect community residents against respiratory diseases, such as colds, which was worthy of promotion in the community. In addition, in terms of safety, the incidence of adverse events and adverse reactions in the TCM intervention group was relatively low, which was basically consistent with the drug instructions.

Ca2+ channel subunit α 1D promotes proliferation and migration of endometrial cancer cells mediated by 17ß-estradiol via the G protein-coupled estrogen receptor.

Calcium and calcium channels are closely related to the estrogen-induced nongenomic effect of endometrial carcinoma, but the specific role of calcium channels is unknown. This study aimed to explore the expression and the biologic effect of the L-type calcium channel in endometrial carcinoma cells and to clarify the molecular mechanism of the relationship between L-type calcium channels and estrogen. The immunohistochemical results showed that Ca(2+) channel subunit α 1D (Cav1.3) expression was high in atypical hyperplasia (1.90 ± 0.35) and endometrial carcinoma tissues (2.05 ± 0.82) but weak (0.80 ± 0.15) in benign endometrial tissues (P < 0.05). Treatment with 17ß-estradiol rapidly increased Cav1.3 expression in a dose- and time-dependent manner, and 100 nM cell-impermeable ß-estradiol-6-(O-carboxymethyl)oxime:bovine serum albumin also promoted Cav1.3 expression. Transfection with small interfering RNA against G protein-coupled estrogen receptor (GPER) suppressed estrogen-induced up-regulation of Cav1.3 compared with control cells and markedly reduced the estrogen-induced phosphorylation of ERK1/2 and CREB. Knocking down the Cav1.3 significantly suppressed estrogen-stimulated Ca(2+) influx, cell proliferation, and migration in endometrial cancer cells. Taken together, Cav1.3 was overexpressed in atypical hyperplasia and endometrial carcinoma, and the estrogen-induced phosphorylation of downstream molecular ERK1/2 and CREB is the result of activation of the GPER pathway. L-type channel Cav1.3 is required for estrogen-stimulated Ca(2+) influx and contributes broadly to the development of endometrial cancer. The Cav1.3 channel may be a new target for endometrial carcinoma treatment.

Epidemiological and etiological investigations of hand, foot, and mouth disease in Jiashan, northeastern Zhejiang Province, China, during 2016 to 2022.

Background: Hand, foot, and mouth disease (HFMD) is a common infectious disease in children. Enterovirus A71 (EV71) and coxsackievirus A16 (CA16) have been identified as the predominant pathogens for several decades. In recent years, coxsackievirus A6 (CA6) and coxsackievirus A10 (CA10) have played increasingly important roles in a series of HFMD outbreaks. We performed a retrospective analysis of the epidemiology of HFMD and the spectrum of different viral serotypes, to elucidate the genetic and phylogenetic characteristics of the main serotypes in the Jiashan area during 2016 to 2022. Methods: Descriptive epidemiological methods were used to analyze the time and population distribution of HFMD in Jiashan during 2016 to 2022 based on surveillance data. Molecular diagnostic methods were performed to identify the viral serotypes and etiological characteristics of HFMD. Phylogenetic analyses was based on VP1 region of CA16 and CA6. Results: The average annual incidence rate of HFMD fluctuated from 2016 to 2022. Children aged 1-5 years accounted for 81.65% of cases and boys were more frequently affected than girls. Except when HFMD was affected by the COVID-19 epidemic in 2020 and 2022, epidemics usually peak in June to July, followed by a small secondary peak from October to December and a decline in February. Urban areas had a high average incidence and rural areas had the lowest. Among 560 sample collected in Jiashan, 472 (84.29%) were positive for enterovirus. The most frequently identified serotypes were CA6 (296, 52.86%), CA16 (102, 18.21%), EV71 (16, 2.86%), CA10 (14, 2.50%) and other enteroviruses (44, 7.86%). There were 71 and 142 VP1 sequences from CA16 and CA6, respectively. Substitution of N218D, A220L and V251I was detected in CA16 and may have been related to viral infectivity. Phylogenetic analysis showed that CA16 could be assigned to two genogroups, B1a and B1b, while all the CA6 sequences belonged to the D3a genogroup. Conclusion: CA6 and CA16 were the two major serotypes of enteroviruses circulating in the Jiashan area during 2016 to 2022. Continuous and comprehensive surveillance for HFMD is needed to better understand and evaluate the prevalence and evolution of the associated pathogens.

[Impact of calcium channel antagonists for estrogen action on the endometrial carcinoma HEC-1A cells].

OBJECTIVE: To study the effects of nifedipine and mibefradil on the cell proliferation, apoptosis, migration on the HEC-1A in vitro and also study the mRNA and protein expression levels of the calcium channel alpha1D (Cav1.3) and calcium channel alpha1G (Cav3.1) to discuss the effects of the calcium antagonists on the mechanisms of the endometrial carcinoma. METHODS: (1) Add 10 µmol/L nifedipine and mibefradil at 15 minutes before adding 10 µmol/L 17ß-estradiol (17ß-E(2)) and 100 µmol/L b-estradiol-6-(O-carboxymethyl)oxime (E(2)-BSA) to the HEC-1A in different time including 0, 5, 15, 30, 60, 120 minutes. Then the changes of mRNA and protein were detected by reverse transcripiton (RT)-PCR and western-blot. (2) Add 1.25, 2.5, 5, 10, 20, 40, 80, 100 µmol/L nifedipine and mibefradil to the HEC-1A at 24, 48, 96 hours to detect the cell proliferation by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) method. (3) Add 10 µmol/L nifedipine and mibefradil to the HEC-1A, then detect the apoptosis at 0 minute, 30 minutes, 1 hour, 6 hours, 24 hours and migration in vitro at 36 hours with transwell methods. RESULTS: (1) After the pretreated effect of the nifedipine before 17ß-E(2), the mRNA express of Cav1.3 genes was lowest at 15 minutes, and returned to the control level after 30 minutes. The protein level didn't change very much in 30 minutes, but rose after 60 minutes. The Cav3.1 genes mRNA express was lowest at 5 minutes, rose at 30 minutes and returned to the 0 minute level gradually. (2) After the pretreated effect of the nifedipine before E(2)-BSA, the Cav1.3 genes mRNA was lowest at 5 minutes and returned at 15 minutes. The protein level rose gradually in 15 minutes but reduced after 15 minutes. The Cav3.1 mRNA and protein level were reduced at every time point. (3) After the pretreated effect of the mibefradil before 17ß-E(2), there was no change of mRNA expression of Cav1.3 genes. The protein level rose at 15 and 60 minutes, there was no change in any other time. The Cav3.1 genes mRNA were gradually reduced and the protein level rose at 15 minutes, and there was no change in any other times. (4) After the pretreated effect of the mibefradil before E(2)-BSA, the mRNA and protein of Cav1.3 levels were reduced after 15 minutes. There was no mRNA expression of Cav3.1, while the protein level was lowest at 15 minutes. (5) Nifedipine and mibefradil affected HEC-1A proliferation depended on the different concentration and interval time points. There was significant difference than those in control group (P < 0.05). (6) There were statistical differences in apoptosis rate after adding nifedipine (P < 0.05), while rose at mibefradil treated the same time (24 hours: 8.41 ± 0.07, 0 minute: 3.74 ± 0.18; P < 0.05). (7) The numbers of stained cells after both nifedipine and mibefradil treated reduced more than control group. CONCLUSIONS: (1) Nifedipine and mibefradil could inhibit both the effect of the estrogen on the L-type and T-type calcium channel in short time, meanwhile the mibefradil effects last long time. (2) The inhibited effect of the mibefradil on the proliferation, apoptosis and migration of HEC-1A cells in vitro is more significant than that by nifedidipine.

Dedifferentiation derived cells exhibit phenotypic and functional characteristics of epidermal stem cells.

Differentiated epidermal cells can dedifferentiate into stem cells or stem cell-like cells in vivo. In this study, we report the isolation and characterization of dedifferentiation-derived cells. Epidermal sheets eliminated of basal stem cells were transplanted onto the skin wounds in 47 nude athymic (BALB/c-nu/nu) mice. After 5 days, cells negative for CK10 but positive for CK19 and beta1-integrin emerged at the wound-neighbouring side of the epidermal sheets. Furthermore, the percentages of CK19 and beta1-integrin+ cells detected by flow cytometric analysis were increased after grafting (P < 0.01) and CK10+ cells in grafted sheets decreased (P < 0.01). Then we isolated these cells on the basis of rapid adhesion to type IV collagen and found that there were 4.56% adhering cells (dedifferentiation-derived cells) in the grafting group within 10 min. The in vitro phenotypic assays showed that the expressions of CK19, beta1-integrin, Oct4 and Nanog in dedifferentiation-derived cells were remarkably higher than those in the control group (differentiated epidermal cells) (P < 0.01). In addition, the results of the functional investigation of dedifferentiation-derived cells demonstrated: (1) the numbers of colonies consisting of 5-10 cells and greater than 10 cells were increased 5.9-fold and 6.7-fold, respectively, as compared with that in the control (P < 0.01); (2) more cells were in S phase and G2/M phase of the cell cycle (proliferation index values were 21.02% in control group, 45.08% in group of dedifferentiation); (3) the total days of culture (28 days versus 130 days), the passage number of cells (3 passages versus 20 passages) and assumptive total cell output (1 x 10(5) cells versus 1 x 10(12) cells) were all significantly increased and (4) dedifferentiation-derived cells, as well as epidermal stem cells, were capable of regenerating a skin equivalent, but differentiated epidermal cells could not. These results suggested that the characteristics of dedifferentiation-derived cells cultured in vitro were similar to epidermal stem cells. This study may also offer a new approach to yield epidermal stem cells for wound repair and regeneration.

Flavonoids from Scutellaria barbata inhibit activation of tumor-associated macrophages by blocking the Toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-κB signaling pathway.

OBJECTIVE: To determine the efficacy of Scutellaria barbata flavonoids and polysaccharides on Ishikawa endometrial carcinoma cells co-cultured with U937 macrophages. METHODS: The presence of CD163 and CD206 was determined by flow cytometry. Thiazolyl Blue Tetrazolium Bromide assays were used to assess the proliferation effect of tumor-associated macrophages (TAMs) on Ishikawa cells. The secretion of interleukin (IL)-10 in the co-culture conditioned media was examined using an enzyme-linked immunosorbent assay. The protein expression levels of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and nuclear factor (NF)-κB p65 were detected by Western blot. The mRNA expression levels of TLR4 and MyD88 were analyzed by real-time polymerase chain reaction (PCR). The expression levels of IL-12, IL-1ß and tumor necrosis factor-α (TNF-α) were evaluated with real-time PCR. RESULTS: Compared with the U937 control group, the expression levels of CD163 and CD206 in the TAM group were higher (P < 0.05). TAMs co-cultured with Ishikawa cells for 24 or 48 h showed higher proliferation rates (P < 0.05). The expression levels of IL-12 decreased than compared with those in the U937 untreated group (P < 0.05) and those of the Scutellaria barbata flavonoids group (P < 0.05). The expression levels of CD206, CD163, IL-10, IL-1ß and TNF-α, NF-κB p65 and TLR4/MyD88 in the TAMs control group were greater than those in the U937 untreated group (P < 0.05) and those of the Scutellaria barbata flavonoids group (P < 0.05). CONCLUSION: Scutellaria barbata flavonoids may inhibit TAM activation by blocking the TLR4/MyD88/NF-κB signaling pathway.

Effects of acupuncture on rates of ovulation and pregnancy in women with unruptured follicular luteinization syndrome: A protocol for systematic review and meta-analysis.

BACKGROUND: As a fabulous part of Oriental Medcine, acupuncture and moxibustion possesses the advantage of high safety, convenience and less adverse effects. Unruptured follicular luteinization syndrome is a common cause of infertility in women of reproductive age, which seriously affects the physical and mental health of patients. Certain studies have reported that acupuncture can improve the rate of pregnancy in women with unruptured follicular luteinization syndrome. In this protocol, the effects of acupuncture on rates of ovulation and pregnancy among women with unruptured follicular luteinization syndrome will be further explored. METHODS: Electronic bibliographic databases such as: MEDLINE, EMBASE, PsycINFO, Global Health, The Cochrane Library (Cochrane Database of Systematic Reviews, Cochrane Central Register of Controlled Trials (CENTRAL), Cochrane Methodology Register), Health Technology Assessment Database, and Web of Science (Science and Social Science Citation Index), PubMed, Chinese Biomedical Databaseare, Chinese VIP Information, Chinese National Knowledge Infrastructure (CNKI), all helpful to identify relevant randomized controlled trials (RCTs) of effects of acupuncture on rates of ovulation and pregnancy among women with unruptured follicular luteinization syndrome. The pooled odds ratio of achieving a clinical pregnancy, ongoing pregnancy, or live birth were used as the main outcome and the secondary outcome includes the changes of ovarian artery dynamics before and after treatment, so as to the adverse reactions of treatment. We will use RevMan 5.3 software to help us to analyze all data and use the Cochrane evaluation manual 5.1.0 to help us to assess the methodological quality for incorporated RCTs. RESULT: This systematic review will provide evidence for assessing the effects of acupuncture on rates of ovulation and pregnancy in women with unruptured follicular luteinization syndrome. CONCLUSION: The results of this study will be a useful reference for clinical treatment with acupuncture to improve rates of ovulation and pregnancy among women with unruptured follicular luteinization syndrome.

Nifedipine induced autophagy through Beclin1 and mTOR pathway in endometrial carcinoma cells.

BACKGROUND: Endometrial carcinoma is one of the most common female tract genital malignant tumors. Nifedipine, an L-type calcium channel antagonist can inhibit cell proliferation of carcinomas. Recent studies indicated that a rise in the free cytosolic calcium ([Ca(2+)](c)) was a potent inducer of autophagy. Here, we investigated the relationship between nifedipine and autophagy in Hec-1A cells. METHODS: Cells were cultured with nifedipine (10 µmol/L) and harvested at different times for counting cell number. MTT assay was applied to evaluate the cell viability and transwell assay to reveal cell migration. Apoptotic cells were detected with annexin V/PI assay. Then cells were treated with 3-methyladenine (3-MA) (2.5 mmol/L) for 0, 5, 15, 30, 60, and 120 minutes and the expression of the L-type calcium channel alpha1D (Cav1.3) protein was detected. At last, cells were cultured and assigned to four groups with different treatment: untreated (control group), 10 µmol/L nifedipine (N group), 2.5 mmol/L 3-MA (3-MA group), and 10 µmol/L nifedipine plus 2.5 mmol/L 3-MA (N+3MA group). Autophagy was detected with GFP-LC3 modulation by fluorescent microscopy, and expression of the autophagy-associated proteins (LC3, Beclin1 and P70s6K) by Western blotting and monodansylcadaverine (MDC) labeled visualization. RESULTS: Proliferation of Hec-1A cells was obviously suppressed by nifedipine compared with that of the untreated cells for 24, 48, and 96 hours (P = 0.000 for each day). The suppression of migration ability of the nifedipine-treated cells (94.0 ± 8.2) was significantly different from that of the untreated cells (160.00 ± 9.50, P = 0.021). The level of early period cell apoptosis induced by nifedipine was (2.21 ± 0.19)%, which was (2.90 ± 0.13)% in control group (P = 0.052), whereas the late period apoptosis level reached (10.38 ± 0.96)% and (4.40 ± 0.60)% (P = 0.020), respectively. The 3-MA group induced a slight increase in the Cav1.3 levels within 15 minutes, but significantly attenuated the Cav1.3 levels after 30 minutes. There were more autophagic vacuoles labeled by MDC in the N group (20.63 ± 3.36) than the control group (6.29 ± 0.16, P = 0.015). GFP-LC3 localization revealed that the LC3 levels of cells in 3-MA group, N+3MA group, 3-MA group were 2.80 ± 0.29, 2.30 ± 0.17, and 1.80 ± 0.21, respectively. Cells in the N group showed significant augmentation of autophagy (P < 0.05). Western blotting analysis confirmed the down-regulation of LC3 levels in 3-MA group (0.85 ± 0.21) and N+3MA group (1.21 ± 0.12) compared with nifedipine treatment (2.64 ± 0.15, P < 0.05). The annexin-V-FITC/PI assay showed that the level of early period cell apoptosis induced in the N+3-MA group ((11.22 ± 0.91)%) differed significantly from that of the control group ((2.51 ± 0.70)%) and N group ((3.47 ± 0.39)%). Similarly, the late period level of the N+3-MA group ((55.19 ± 2.51)%) differed significantly from that of the control group ((15.81 ± 1.36)%) and the N group ((22.09 ± 2.48)%, P < 0.05). The down-regulated expression of P70s6k and up-regulated expression of the Beclin1 revealed significant differences between the N+3-MA group and control group (P = 0.025; Beclin1: P = 0.015). CONCLUSIONS: Proliferation and migration in vitro of endometrial carcinoma Hec-1A cells are significantly suppressed by nifedipine. The nifedipine leads autophagy to oppose Hec-1A cells apoptosis. Autophagy inhibition by 3-MA leads down-regulation of Cav1.3 and enhances nifedipine-induced cell death. The nifedipine-induced autophagy is linked to Beclin1 and mTOR pathways.

[A rapid multi-residual analysis for organophosphorus pesticides in the products of animal origin using gas chromatography coupled with accelerated solvent extraction and gel permeation chromatographic purification].

A rapid method has been developed to determine the multi-residues of 36 organophosphorus pesticides in the products of animal origin using capillary gas chromatography with flame photometric detector (GC-FPD (P)). The organophosphorus pesticides were extracted with acetonitrile by accelerated solvent extraction, and cleaned up by auto gel permeation chromatography and primary secondary amine (PSA) packing material. The collected solution was analyzed by the GC-FPD (P) and quantified by internal standard method. The 36 organophosphorus pesticides were separated efficiently from impurity in high sensitivity and reproducibility by GC-FPD (P). The limits of detection (LODs) ranged from 0.0012 mg/kg (phorate) to 0.014 mg/kg (pyraclofos), and the limits of quantitation (LOQs) ranged from 0.004 mg/kg (phorate) to 0.047 mg/kg (pyraclofos). The recoveries ranged from 58.2% to 106.3% in blank samples spiked with 0.05, 0.1 and 0.2 mg/kg of 36 organophosphorus pesticides. The LODs, LOQs and the recoveries of the method all satisfy the requirement of pesticide residue analysis.