Tanshinol inhibits trophoblast cell migration and invasion by regulating Gadd45a in preeclampsia.

OBJECTIVE: Tanshinol is an active constituent of Salvia miltiorrhiza that possesses anti-inflammatory, antioxidant, and antibacterial activities. Therefore, this study attempted to detect whether it has a role in the treatment of preeclampsia (PE). METHODS: In this study, we explored the effect of tanshinol on the development of PE at the cellular level. The effect of tanshinol on cell proliferation was measured by colony formation and EdU assays. The migration, invasion, and in vitro angiogenesis of HTR-8/SVneo cells were detected by wound-healing, transwell, and tube formation assays, respectively. In addition, a PE cell model was established by overexpression of Gadd45a, and this cell model was assessed with the optimal concentration of tanshinol. RESULTS: The results show that tanshinol enhanced proliferation, migration, invasion, and tube formation of HTR-8/SVneo cells in vitro. Furthermore, the reduction in proliferation, migration, invasion, and tube formation of cells by Gadd45a overexpression was partially reversed by tanshinol treatment. Tanshinol also inhibited the apoptosis of HTR-8/SVneo cells transfected with Gadd45a. CONCLUSIONS: In summary, tanshinol promoted proliferation, migration, invasion, and tube formation and inhibited the apoptosis of HTR-8/SVneo cells. It may be a novel therapeutic compound to attenuate the development of PE.

Traditional Chinese medicine has maintained the health of people in Asia for thousands of years and is increasingly used worldwide. Tanshinol has been found to be useful in the treatment and prevention of many diseases. Through experiments, we found that tanshinol is a novel therapeutic compound that promotes the proliferation, migration, invasion and tubular formation of HTR-8/SVneo cells. In addition, tanshinol also inhibited the apoptosis rate of preeclampsia cell models. Follow-up experiments will further validate the results of this study.

Babao Dan inhibits lymphangiogenesis of gastric cancer in vitro and in vivo via lncRNA-ANRIL/VEGF-C/VEGFR-3 signaling axis.

Gastric cancer (GC) is one of the most common gastrointestinal malignancies in the world. Growing evidence emphasizes the critical role of long non-coding RNA (lncRNA) in GC tumorigenesis. The aim of the research was to elucidate the effect and mechanism of Babao Dan (BBD) on lymphangiogenesis of GC in vitro and in vivo via lncRNA-ANRIL/VEGF-C/VEGFR-3 signaling axis. The present study investigated BBD significantly decreased the expression of lncRNA-ANRIL and VEGF-C in GC cells (AGS, BGC823, and MGC80-3) by using real-time quantitative polymerasechain reaction (RT-qPCR) and the secretion and expression of VEGF-C by (enzyme linked immunosorbent assay) ELISA and western blot (WB). BBD significantly inhibited the tumor xenograft of GC growth and the expression of lncRNA-ANRIL, VEGF-C, VEGFR-3 and LYVE-1 in vivo. BBD reduced serum VEGF-C level. In vitro, BBD inhibited the tube formation and decreased the cell viability, proliferation and migration of HLECs by using tube formation, MTT, Hoechst and Transwell assays. In addition, WB assay found that BBD decreased the expression levels of VEGF-C, VEGFR-3, matrix metallopeptidase 2 (MMP-2) and matrix metallopeptidase 9 (MMP-9), and RT-qPCR assay found that the mRNA expression levels of lncRNA-ANRIL, VEGF-C, VEGFR-3, MMP-2, MMP-9, CDK4, Cyclin D1, and Bcl-2 were down-regulated, and the expression of p21 and Bax were increased. Taken together, these results demonstrated that BBD inhibited lymphangiogenesis of GC in vitro and in vivo via the lncRNA-ANRIL/VEGF-C/VEGFR-3 signaling axis.

Herbs-Partitioned Moxibustion Combined with Acupuncture Inhibits TGF-ß1-Smad-Snail-Induced Intestinal Epithelial Mesenchymal Transition in Crohn's Disease Model Rats.

Crohn's disease may cause excessive damage and repair in the intestinal epithelium due to its chronic relapsing intestinal inflammation. These factors may initiate the TGF-ß 1-Smad pathway to activate the transcription factor of Snail, and the Snail-mediated pathway promotes the transformation of intestinal epithelial cells to mesenchymal cells, leading to intestinal fibrosis. Acupuncture and moxibustion have been demonstrated to prevent intestinal fibrosis in Crohn's disease. However, it is not clear whether acupuncture and moxibustion can inhibit intestinal epithelial mesenchymal transformation in Crohn's disease by affecting the TGF-ß 1-Smad-Snail pathway. This study indicated that abnormal increased expressions of TGFß1, TßR2, Smad3, and Snail were significantly downregulated by herbs-partitioned moxibustion at Tianshu (ST25) and Qihai (RN6) and acupuncture at Zusanli (ST36) and Shangjuxu (ST37). In addition, protein and mRNA levels of E-cadherin, the epithelial cell marker, were significantly increased. Protein and mRNA levels of fibronectin, the mesenchymal cell marker, were decreased in the intestinal tissue. Moreover, the number of mesenchymal cells in the intestinal mucosa can be reversely transformed to intestinal epithelial cells. Therefore, herbs-partitioned moxibustion combined with acupuncture can prevent intestinal epithelial mesenchymal transition by inhibiting abnormal expression of TGFß1, TßR2, Smad3, and Snail in the TGF-ß1-Smad-Snail pathway in Crohn's disease.

Qingjie Fuzheng granules inhibit colorectal cancer cell growth by the PI3K/AKT and ERK pathways.

BACKGROUND: Qingjie Fuzheng granules (QFGs) are part of a traditional Chinese medicine formula, which has been widely used and found to be clinically effective with few side effects in various cancer treatments, including colorectal cancer (CRC). However, the precise mechanisms and molecular signaling pathways involved in the activity of QFGs' anticancer effect have not been reported in the literature. In this study, we hypothesized that QFGs can inhibit the growth of colorectal cancer cells, and that its mechanism is closely related to one or more intracellular signal transduction pathways. AIM: To better evaluate the mechanism underlying the anti-cancer effect of QFGs on the CRC cell lines HCT-116 and HCT-8. METHOD: First, we measured cell viability and cytotoxicity by performing MTT and lactate dehydrogenase (LDH) assays. We evaluated the role of QFGs in cell proliferation and apoptosis by assessing colony formation and analyzing Hoechst 33258 staining. Second, cell cycle and apoptosis rates were measured by fluorescence activated cell sorting, and the expression levels of survivin, cyclin D1, CDK4, p21, Bax, Bcl-2, Fas, FasL, and cleaved-caspase-3/-8/-9 were measured by performing western blots and caspase activity assays. Furthermore, inhibitors of caspase-3/-8/-9 were used to elucidate the specific apoptosis pathway induced by QFGs in cancer cells. Finally, activation of the PI3K/AKT and ERK signaling pathways was examined using the western blot assay to investigate the possible mechanism. RESULTS: MTT and LDH assays revealed that after 0.5-2.0 mg/mL of QFGs treatment, cell viability was reduced by (6.90% ± 1.03%)-(59.70% ± 1.51%) (HCT-116; P < 0.05) and (5.56% ± 4.52%)-(49.44% ± 2.47%) (HCT-8; P < 0.05), and cytotoxicity was increased from 0.52 ± 0.023 to 0.77 ± 0.002 (HCT-116; P < 0.01) and from 0.56 ± 0.054 to 0.81 ± 0.044 (HCT-8; P < 0.01) compared with the non-QFGs treatment groups. Additionally, colony formation and Hoechst 33258 staining assays showed that QFGs inhibited proliferation and induced apoptosis in CRC cells. QFGs also increased the expression levels of Bax, Fas and FasL, decreased the level of Bcl-2, and stimulated the activation of caspase-3/-8/-9, which were revealed by western blot and caspase activity assays. In contrast, when adding the three caspase inhibitors, the suppression effect of QFGs on cell viability and apoptosis were markedly inhibited. Moreover, QFGs suppressed the phosphorylation levels of PI3K, AKT and ERK. CONCLUSION: These results demonstrated that QFGs can inhibit CRC cell proliferation and induce apoptosis by suppressing the PI3K/AKT and ERK signaling pathways.

Comparison of the analgesic effects between electro-acupuncture and moxibustion with visceral hypersensitivity rats in irritable bowel syndrome.

AIM: To observe whether there are differences in the effects of electro-acupuncture (EA) and moxibustion (Mox) in rats with visceral hypersensitivity. METHODS: EA at 1 mA and 3 mA and Mox at 43 °C and 46 °C were applied to the Shangjuxu (ST37, bilateral) acupoints in model rats with visceral hypersensitivity. Responses of wide dynamic range neurons in dorsal horns of the spinal cord were observed through the extracellular recordings. Mast cells (MC) activity in the colons of rats were assessed, and 5-hydroxytryptamine (5-HT), 5-hydroxytryptamine 3 receptor (5-HT3R) and 5-HT4R expressions in the colons were measured. RESULTS: Compared with normal control group, responses of wide dynamic range neurons in the dorsal horn of the spinal cord were increased in the EA at 1 mA and 3 mA groups (1 mA: 0.84 ± 0.74 vs 2.73 ± 0.65, P < 0.001; 3 mA: 1.91 ± 1.48 vs 6.44 ± 1.26, P < 0.001) and Mox at 43 °C and 46 °C groups (43 °C: 1.76 ± 0.81 vs 4.14 ± 1.83, P = 0.001; 46 °C: 5.19 ± 2.03 vs 7.91 ± 2.27, P = 0.01). MC degranulation rates and the expression of 5-HT, 5-HT3R and 5-HT4R in the colon of Mox 46 °C group were decreased compared with model group (MC degranulation rates: 0.47 ± 0.56 vs 0.28 ± 0.78, P < 0.001; 5-HT: 1.42 ± 0.65 vs 7.38 ± 1.12, P < 0.001; 5-HT3R: 6.62 ± 0.77 vs 2.86 ± 0.88, P < 0.001; 5-HT4R: 4.62 ± 0.65 vs 2.22 ± 0.97, P < 0.001). CONCLUSION: The analgesic effects of Mox at 46 °C are greater than those of Mox at 43 °C, EA 1 mA and EA 3 mA.

Comparison of Electroacupuncture and Moxibustion for Relieving Visceral Hypersensitivity in Rats with Constipation-Predominant Irritable Bowel Syndrome.

Aim. To compare whether there is different effect between electroacupuncture (EA) and moxibustion (Mox) on visceral hypersensitivity (their analgesic effects) in constipation-predominant irritable bowel syndrome (C-IBS). Methods. EA at 1 mA and 3 mA and Mox at 43°C and 46°C were applied to the Shangjuxu (ST37, bilateral) acupoint in rats with C-IBS and normal rats. An abdominal withdrawal reflex (AWR) score was used to assess visceral hypersensitivity. Toluidine blue staining was used to assess mast cell (MC) activity in colon of rats. Immunochemistry was used to measure 5-HT and 5-HT4 receptor expression in the colon. Results. AWR scores in all EA (1 mA and 3 mA) and Mox (43°C and 46°C) treatment groups after colorectal distention (CRD) stimulation pressure of 20, 40, 60, and 80 mmHg were significantly lower than those of the model (MC) group (P all < 0.01). The MC counts and degranulation rates in the colon of all EA and Mox treatment groups and the MC group were significantly higher than those of the NC group (P all < 0.01). MC degranulation rates in the colon of all EA and Mox treatment groups were lower than those of the MC group (P all < 0.05). 5-HT expression in colon of all EA and Mox treatment groups was significantly lower than that of the MC group (P all < 0.01), and 5-HT4R expression in colon of both EA groups was significantly higher than that of the MC group (P both < 0.01). Conclusion. EA and Mox treatments may both ameliorate visceral hypersensitivity at different degree in rats with C-IBS, and EA treatment was better than Mox.

[Analysis on intestinal disorders in Jiujing Tu (Illustration of Moxiustion) found from Dunhuang].

Jiujing Tu (Illustration of Moxibustion), excavated from Mo Kao Grotto at Dunhuang, is one of the earliest existing monographs on moxibustion. The medical masters from different schools have focused on this book because it is different from the existing ancient medical works and have not been collected in the medical works of different dynasties. In this study, the literature of Jiujing Tu on five acupoints (Dachangshu, Pangguangshu, Daxiaochangshu, Nieshu and Cigong) relevant with intestinal disorders is collected. It is intended to discuss and analyze the acupoint location, main intestinal disorder, moxibustion characters, recognition on the literature of different dynasties and modern clinical applications. It is believed that the thought of strong moxibustion in the treatment of intestinal disorders advocated in Jiujing Tu has profound impact on the medical development in later generations. It deserves us to have a further digging, collection and promotion of this thought in the modern time.

Herb-partitioned moxibustion upregulated the expression of colonic epithelial tight junction-related proteins in Crohn's disease model rats.

BACKGROUND: Herb-partitioned moxibustion (HPM) at Tianshu (ST25) and Qihai (RN6) has been used to treat Crohn's disease (CD). Injury to intestinal epithelial tight junctions (TJs) is the leading cause of CD onset with under expression of TJ-related proteins such as occludin, claudin-1, and zonula occludens protein-1 (ZO-1). This study aimed to investigate whether HPM can change the permeability of the intestinal epithelial barrier by affecting the expression of colonic epithelial TJ-related proteins in vitro. METHODS: Forty-eight male Sprague-Dawley rats were randomly divided into four groups of twelve rats: normal control (NC) group; model control (MC) group; herb-partitioned moxibustion (HPM) group; and mesalazine control (MESA) group. The rats in the latter three groups were given trinitrobenzene sulfonic acid (TNBS) enemas to establish CD models. The HPM group was treated with HPM at Tianshu (ST25) and Qihai (RN6) once daily for 14 consecutive days, while the MESA group was given mesalazine solution (at the proportion of 0.018:1) by lavage twice daily for the same period. After the treatment period, the colon tissues from all groups were partly processed for macroscopic damage assessment and histological observation, and partly purified and cultured in vitro to examine the permeability of the intestinal epithelial cell barrier by trans-epithelial electrical resistance (TEER). Western blot and fluorescence quantitative polymerase chain reaction (FQ-PCR) analyses were performed to observe the expression of occludin, claudin-1, and ZO-1 proteins and mRNAs, respectively. RESULTS: In the HPM and MESA groups, the typical CD macroscopic damage, i.e., inflammatory cell infiltration in colonic mucosa and submucosa, submucosal lymphoid follicular hyperplasia, hyperemia and edema, and morphological changes were improved to different degrees in the colonic tissues (HPM, MESA vs. MC for macroscopic score of colonic damage: all P < 0.001). The decreasing tendencies were minor for colonic TEER values (HPM, MESA vs. MC: all P < 0.001), and expression of intestinal epithelial TJ-related proteins (HPM, MESA vs. MC: all P < 0.05) and mRNAs (HPM, MESA vs. MC: all P < 0.05), especially in the HPM group (HPM vs. MESA for TEER values: P < 0.001). CONCLUSIONS: HPM at Tianshu (ST25) and Qihai (RN6) upregulated the expression of occludin, claudin-1, and ZO-1 in TNBS-induced CD model rats.

[Effect of Electroacupuncture and Moxibustion on Visceral Pain and Expression of VR 1 and HSP 70 of "Tianshu" (ST 25) Region in Colorectal Distension-induced Visceral Hypersensitivity Rats].

OBJECTIVE: To compare the effects of electroacupuncture (EA) and moxibustion (Moxi) on visceral pain and expression of vanilloid receptor subtype 1 (VR 1) and heat shock protein (HSP)70 in "Tianshu" (ST 25) region in colorectal distension (CRD)-induced visceral hypersensitivity (VHS) rats. METHODS: Fifty male SD rats were randomly divided into normal control, VHS model, 43℃-moxi, 46℃-moxi, 1 mA-EA and 3 mA-EA groups (n=10 in each group). The VSH model was established by CRD once daily for 14 days. EA or Moxi stimulation was applied to bilateral "Tianshu" (ST 25) for 10 min, once daily for consecutive 10 days. The abdominal withdrawal reflex (AWR) scores (0-4 points) were rated according to Al-Chaer's and coworkers' standards (2000) and the expression levels of VR 1 and HSP 70 in bilateral ST 25 area tissues detected by immunohistochemistry. RESULTS: The AWR scores for 20, 40, 60 and 80 mmHg CRD pressures were significantly increased compared to the normal control group (P<0.01) and notably decreased after 43℃- and 46℃-moxi, and 1 mA- and 3 mA-EA stimulation of bila-teral ST 25 in comparison with the model group (P<0.05, P<0.01), and the effect of 46℃-moxi was apparently superior to those of 1 mA-EA at 40 and 80 mmHg, and 3 mA-EA at 40 mmHg (P<0.05). After modeling, the expression of both VR 1 and HSP 70 (percentages of area of positive-cells) in ST 25 region had no significant changes (P>0.05). Compared to the model group, the expression levels of VR 1 in the 43℃-moxi and 46℃-moxi groups, and HSP 70 in the 43℃-moxi and 46℃-moxi, 1 mA-EA and 3 mA-EA groups were significantly up-regulated (P<0.01), but without obvious changes in the expression of VR 1 in the 1 mA-EA and 3 mA-EA groups (P>0.05). The effects of 46℃-moxi were considerably better than those of 43℃-moxi, 1 mA-EA and 3 mA-EA in up-regulating VR 1 and HSP 70 expression (P<0.05, P<0.01). No significant differences were found among the 43℃-moxi, 1 mA-EA and 3 mA-EA groups in the expression of VR 1 and HSP 70 (P>0.05). CONCLUSIONS: Moxibustion at 43℃ and 46℃ and EA at 1 mA and 3 mA, especially the 46℃-moxi, can relieve visceral pain in visceral hypersensitivity rats, which may be related to their effects in up-regulating expression of VR 1 and HSP 70 in "Tianshu" (ST 25) area.

[Professor SHI Yin's experience of staging, classification and sorting method for polycystic ovary syndrome].

The clinical experience of professor SHI Yin for polycystic ovary syndrome (PCOS) was summarized. According to the main pathogenesis of PCOS, the tonifying kidney should be taken as essence with synchronous treatment on liver, spleen and heart, presenting staging, classification and sorting method for PCOS. In the staging method, the regulation on follicle development should be taken as treatment core to comply with the rules of yin and yang. A four-stage method was proposed, where "regulating method" was suitable in menstrual period, "tonifying method" in follicular phase;"dredging method" in ovulatory period and "adjustment and tonifying " in luteal phase. In the classification and sorting method, attention was paid on individualized treatment, and treatment was based on fat type, thin type and non-fat type as well as childbearing. Besides, psychological counseling and life adjustment for patient was essential, and the unity of body and mind could enhance curative effect.

Moxibustion combined with acupuncture increases tight junction protein expression in Crohn's disease patients.

AIM: To investigate the effect of herb-partitioned moxibustion combined with acupuncture on the expression of intestinal epithelial tight junction (TJ) proteins. METHODS: Sixty patients diagnosed with mild to moderate Crohn's disease (CD) were allocated into the herb-partitioned moxibustion combined with acupuncture (HMA) group (n = 30) or the mesalazine (MESA) group (n = 30) using a parallel control method. There were 2 sets of acupoints used alternately for HMA treatment. The following points were included in Set A: ST25 (Tianshu), RN6 (Qihai), and RN9 (Shuifen) for herb-partitioned moxibustion and ST36 (Zusanli), ST37 (Shangjuxu), LI11 (Quchi), and LI4 (Hegu) for acupuncture. The points for Set B included BL23 (Shenshu) and BL25 (Dachangshu) for herb-partitioned moxibustion and EX-B2 of T6-T1 (Jiajixue) for acupuncture. The patients received the same treatment 6 times a week for 12 consecutive weeks. The MESA group received 1 g of mesalazine enteric coated tablets 4 times daily for 12 consecutive weeks. Intestinal tissues were stained and examined to compare the morphological and ultrastructural changes before and after the treatment session. Immunohistochemistry and in situ hybridization assays were used to detect the expression of intestinal epithelial TJ proteins zonula occludens-1 (ZO-1), occludin, and claudin-1. The mRNA levels were also evaluated. RESULTS: After the treatment, both herb-partitioned moxibustion combined with acupuncture and mesalazine improved intestinal morphology and ultrastructure of CD patients; the patients treated with HMA showed better improvement. HMA significantly increased the expression of ZO-1 (P = 0.000), occludin (P = 0.021), and claudin-1 (P = 0.016). MESA significantly increased the expression of ZO-1 (P = 0.016) and occludin (P = 0.026). However, there was no significant increase in the expression of claudin-1 (P = 0.935). There was no statistically significant difference between the two groups for the expression of occludin and claudin-1 (P > 0.05). The HMA group showed a significant improvement in ZO-1 expression compared to the MESA group (2333.34 ± 352.51 vs 2160.38 ± 307.08, P = 0.047). HMA significantly increased the expression of ZO-1 mRNA (P = 0.000), occludin mRNA (P = 0.017), and claudin-1 mRNA (P = 0.017). MESA significantly increased the expression of ZO-1 mRNA (P = 0.000), occludin mRNA (P = 0.042), and claudin-1 mRNA (P = 0.041). There was no statistically significant difference between the two groups in the expression of occludin and claudin-1 mRNA (P > 0.05). However, the HMA group showed a significant improvement in ZO-1 mRNA expression compared with the MESA group (2378.17 ± 308.77 vs 2200.56 ± 281.88, P = 0.023). CONCLUSION: HMA can repair intestinal epithelial barrier lesions and relieve inflammation by upregulating the expression of TJ proteins and their mRNAs.