[Effects of Hedyotis diffusa Extract on Epidermal Growth Factor Induced Proliferation, Apoptosis, and TNF-α Induced Inflammatory Factors of HaCaT Cells].

Objective To observe the effects of Hedyotis diffusa extract (HDE) on the prolifera- tion, apoptosis, and inflammatory factors of HaCaT cells, and to explore its possible molecular mecha- nisms. Methods HaCaT cells were divided into 3 groups, the vehicle group, the control group, and 3 dose HDE groups. No epidermal growth factor (EGF) or HDE was added in the vehicle group. EGF was added with no HDE treatment in the control group. HDE (25, 50, 100 mg/mL) and EGF were added in the 3 HDE groups to co-culture HaCaT cells. The effects of HDE on EGF-induced proliferation of HaCaT cells were de- tected using CCK-8 assay. The effects of HDE on the growth cycle and apoptosis rate of HaCaT cells were measured using flow cytometry. Moreover, protein expression levels of Ki67, Bcl-xL, clAP1 , procaspase- 3, and cleaved caspase-3 were determined using Western blot. In addition, levels of IL-6, IL-8, and IL-10 in the supernatant were detected using ELISA. The level of phosphoration of NF-κB p65 (p-p65) was meas- ured using Western blot. Results Compared with the vehicle group, the absorbance of HaCaT cells and the expression level of Ki67 increased (P <0. 05, P <0. 01) ; levels of p-p65, IL-6, and IL-8 were elevated (P <0. 05, P <0. 01); IL-10 level was lowered (P <0.01) in the control group. Compared with the control group, the absorbance of HaCaT cells and the expression level of Ki67 decreased (P <0.05, P <0.01) ; levels of p-p65, IL-6, and IL-8 were reduced (P <0. 05, P <0. 01); IL-10 level was elevated (P <0. 05, P < 0.01) in the 3 dose HDE groups. Meanwhile, the apoptosis rate of HaCaT cells increased more in the 3 dose HDE groups than in the control group (P <0. 05, P <0. 01). The percentage of HaCaT cells at G1 phase was 58. 51 %, 73.12%, and 79. 95% in 25, 50, and 100 mg/mL HDE groups, respectively, showing statisti- cal difference when compared with that in the control group (52. 85%; P <0. 05, P <0. 01). The percentage and apoptosis rate of HaCaT cells at G1 phase were elevated more in 50 and 100 mg/mL HDE groups than in 25 mg/mL HDE group (P <0. 01). Besides, the percentage and apoptosis rate of HaCaT cells at G1 phase were elevated more in 100 mg/mL HDE group than in 50 mg/mL HDE group (P <0. 05). Compared with the control group, protein expression levels of Bcl-xL and cIAP1 were reduced in 100 mg/mL HDE group (P < 0. 01). There was no statistical difference in caspase-3 total amount (P >0. 05), but cleaved caspase-3 ex- pression increased with statistical difference (P <0. 01). Conclusion HDE inhibited the proliferation of HaCaT cells possibly by arresting HaCaT cell growth at G1 phase, promoted the apoptosis of HaCaT cells by stressing protein expressions of Bcl-xL and cIAPI , and elevating protein expressions of cleaved caspase-3, and suppressed inflammatory response of HaCaT cells via regulating NF-κB expression.

[Effect of silencing AEG-1 with small interfering RNA on the proliferation and cell cycle of gastric carcinoma SGC-7901 cells].

OBJECTIVE: To explore the effect of down-regulation of astrocyte elevated gene-1 (AEG-1) expression on cell proliferation and cell cycle of gastric carcinoma cells, and its possible molecular mechanism. METHODS: Control siRNA and AEG-1 siRNA were transfected into gastric carcinoma SGC-7901 cells. 48 h after transfection, the cells were divided into 3 groups including untransfected, siRNA control and AEG-1 siRNA transfection groups. Expressions of AEG-1 mRNA and protein in the 3 group cells were detected by real-time quantitative PCR and Western blot. The changes of cell proliferation were examined using CCK-8 kit, and the cell cycle distribution was detected by flow cytometry. Finally, expressions of cell proliferation and cell cycle related proteins were detected by Western blot. RESULTS: Real-time quantitative PCR and Western blot demonstrated that compared with the untransfected and siRNA control groups, expressions of AEG-1 mRNA and protein were significantly down-regulated in the AEG-1 siRNA transfection group (P < 0.05), but there was no significant difference between the untransfected and siRNA control groups (P > 0.05). Furthermore, in vivo experiment confirmed a significant down-regulation of AEG-1 protein in the AEG-1 siRNA transfection group (P < 0.05). In addition, AEG-1 siRNA obviously inhibited the proliferation of SGC-7901 cells at different time points after transfection with AEG-1 siRNA. The percentage of cells in G0/G1 phase in the AEG-1 siRNA transfection group [(61.26 ± 1.25)%] was significantly higher than those in the untransfected group [(46.17 ± 1.91)%] and siRNA control group [(46.46 ± 1.96)%], and there was a significant difference between them (all P < 0.001). Furthermore, the result of Western blotting revealed that down-regulation of AEG-1 expression evoked the down-regulation of cdk2 and cyclin D1 expressions and elevation of p21 expression in vitro and in vivo. CONCLUSIONS: The inhibition of cell proliferation and cell cycle arrest mediated by down-regulation of AEG-1 expression may be closely associated with the changes of expression of cell cycle related proteins including cdk2, cyclin D1 and p21.

Transforming growth factor ß1-509C/T and +869T/C polymorphisms on the risk of upper digestive tract cancer: a meta-analysis based on 10,917 participants.

The aim of this study was to assess the relationships between transforming growth factor ß1-509C/T (rs1800469) and +869T/C (rs1800470) polymorphisms and the risk of upper digestive tract cancer (UDT cancer) by using a meta-analysis. We interrogated the databases of Medline, Embase and Wanfang (Chinese literature database) (latest update; December 15, 2011). Odds ratios (OR) and corresponding 95% confidence intervals (95% CI) were used to assess the strength of the associations. In total, 20 case-control studies were included in this meta-analysis. Overall, both TGF ß1-509C/T and +869T/C polymorphisms were not associated with risk of UDT cancer [-509C/T: OR (95%CI) = 1.10 (0.99-1.22) for TT vs. C carries, P(heterogeneity) = 0.10; +869T/C: OR (95%CI) = 1.04 (0.88-1.23) for CC vs. T carriers, P(heterogeneity) = 0.02]. Subgroup analyses indicated that the -509T allele was associated with increased risk of UDT cancer in population-based studies (OR = 1.16 (1.04-1.31), P(heterogeneity) = 0.31 for TT vs. C carriers) and in small sample-sized studies (OR = 1.45 (1.15-1.84), P(heterogeneity) = 0.56 TT vs. C carriers). All subgroup analyses for the TGF ß1+869T/C polymorphism indicated null association except for hepatocellular carcinoma. Interestingly, both the TGF ß1-509T allele and the +869C allele were associated with decreased risk of hepatocellular cancer based on limited original studies. This meta-analysis indicated that TGF ß1-509C/T rather than +869T/C is a potential risk factor for UDT cancer.

[Effect of down-regulation of histone deacetylase 2 protein expression on cell proliferation and cell cycle in cervical carcinoma].

OBJECTIVE: To study the effect of down-regulation of histone deacetylase 2 (HDAC2) expression on cell proliferation and cell cycle in cervical carcinoma cell lines HeLa. METHODS: HDAC2 siRNA and control siRNA were transfected to HeLa cells. CCK-8 and flow cytometry were used to analyze the changes of cell proliferation and cell cycle, respectively. Western blot was employed to detect the changes of cell proliferation and cell cycle-related proteins. RESULTS: HDAC2 siRNA significantly down-regulated the expression of HDAC2 protein in HeLa cells, resulting in marked inhibition of cell proliferation. In addition, the percentage of cells in G(0)/G(1) phase in HDAC2 siRNA group (63.3% ± 2.0%) was significantly higher than that in untreated group (29.3% ± 1.7%) or control siRNA group (29.4% ± 1.7%), F = 354.181, P = 0.000. Furthermore, Western blot demonstrated that down-regulation of HDAC2 expression decreased the expression of cyclin D1, cyclin E and CDK2 proteins but increased the expression of p21 protein. CONCLUSIONS: Down-regulation of HDAC2 expression mediates proliferation inhibition and cell cycle arrest. It is associated with decrease in cyclin D1, cyclin E and CDK2 protein expression and increase in p21 protein expression.

[Effects of KIAA0101 expression on proliferation and invasion of gastric carcinoma MKN-45 cells].

OBJECTIVE: To investigate the expression of KIAA0101 protein in gastric carcinoma cells, and to explore the effects of its down-regulation on the cell proliferation, cell cycle and invasion. METHODS: Western blot was used to detect KIAA0101 protein expression in three gastric carcinoma cell lines including MKN-28, SGC-7901 and MKN-45. KIAA0101 siRNA and control siRNA were utilized to transfect MKN-45 cells, respectively. CCK-8 was used to analyze the changes of cell proliferation, and flow cytometry to examine the changes of cell cycle distribution. Finally, Boyden chamber was used to detect the ability of cell invasion. RESULTS: Relative level of KIAA0101 protein in MKN-45 cells was significantly higher than those in MKN-28 and SGC-7901 cells, and there was significant difference among the three cell lines (P < 0.05). The result of CCK-8 study demonstrated that, compared with untreated group and control siRNA group, the proliferation of MKN-45 cells in KIAA0101 siRNA group was significantly inhibited (P < 0.05). Additionally, the result of cell cycle analysis revealed that the percentage of cell number in G(0)/G(1) phase in KIAA0101 siRNA group [(61.47 ± 0.89)%] was significantly higher than those in untreated group [(47.43 ± 0.85)%] and control siRNA group [(48.43 ± 0.73)%; F = 271.653, P = 0.000]. Further, Boyden chamber assay showed that the cell numbers migrated to Matrigel in KIAA0101 siRNA group (61.51 ± 4.76) were significantly lower than those in untreated group (138.74 ± 10.16) and control siRNA group (132.93 ± 11.25; F = 65.949, P = 0.000). CONCLUSIONS: Down-regulation of KIAA0101 expression leads to an inhibition of cell proliferation, cell cycle and cell invasion. It may provide a novel target for the treatment of patients with gastric carcinoma.

[Clinical observation on treatment of premature ovarian failure patients of shen deficiency gan stagnation syndrome by combination of bushui roumu recipe and medroxyprogesterone acetate tablet HUA].

OBJECTIVE: To study the therapeutic efficacy of bushui roumu recipe (BRR) combined medroxyprogesterone acetate tablet (MAT) in treating premature ovarian failure (POF). METHODS: Totally 90 POF patients of Shen deficiency Gan stagnation syndrome were assigned to 3 groups by random number table, 30 in each group. Patients in the treatment group were treated with BRR and MAT, those in the Chinese medicine group were treated with BRR, and those in the Western medicine group were treated with artificial period method. All patients were treated for 3 months. The menstrual improvement was observed before and after treatment. The therapeutic efficacy was assessed using modified Kupperman scoring standard. The serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) were observed before and after treatment. RESULTS: (1) In aspect of the menstrual improvement: In the treatment group 20 patients had menstrual onset during the treatment course. Ten had normal menstruation after discontinued medication. Of them one got pregnancy one month after treatment. In the Chinese medicine group 6 patients had menstrual onset during the treatment course. Two had normal menstruation after discontinued medication. In the Western medicine group 26 patients had menstrual onset during the treatment course. Twelve had normal menstruation after discontinued medication. Better effects on the menstrual improvement were obtained in the treatment group than in the Chinese medicine group (P < 0.01), but with no statistical difference when compared with the Western medicine group (P > 0.05). (2) There was statistical difference in modified Kupperman scores of the 3 groups between before and after treatment (P < 0.01). The improvement of total modified Kupperman score was better in the treatment group than in the other two groups (P < 0.01). The improvement of palpitation was better in the treatment group than in the other two groups (P < 0.05). The improvement of tidal fever and sweat was better in the treatment group and the Chinese medicine group than in the Western medicine group (P < 0.05). (3) After treatment all patients' serum E2 was higher than before treatment, serum levels of FSH and LH were lower than before treatment. Compared pre- and post-treatment, there was statistical difference (P < 0.01). The serum E2 level in the 3 groups was higher after treatment than before treatment with statistical difference (P < 0.01). The levels of FSH and LH were lower in the 3 groups after treatment than before treatment with statistical difference (P < 0.01). The improvement of E2 was better in the treatment group than in the Chinese medicine group (P < 0.05). The improvement of FSH and LH was better in the treatment group than in the Western medicine group (P < 0.05). CONCLUSION: Combination of BRR and MAT could improve the clinical symptoms, menstruation, and serum reproductive hormones in POF patients of Shen deficiency Gan stagnation syndrome.

[Effect of blockade of NF-kappaB signaling pathway on cell apoptosis in cutaneous squamous cell carcinoma].

OBJECTIVE: To evaluate the siRNA-mediated inhibitory effect of nuclear factor-kappaB (NF-kappaB) p65 on expression of p65, and explore the effect of blockade of NF-kappaB signal pathway on cell apoptosis in cutaneous squamous cell carcinoma (cutaneous SCC). METHODS: Cutaneous SCC cell line SCL-1 cells were transfected with 50 nmol/L p65 siRNA. The expression level of p65 mRNA was measured using RT-PCR method at 0, 24, 48 and 72 h . Expressions of p65, bcl-2 and bax proteins were determined using Western blotting. Activities of caspase-3/9 was detected by Caspase-Glo®-3/7, 8 and 9 kit. Finally, cell apoptosis was detected using flow cytometry. RESULTS: The expression level of p65 mRNA in Cutaneous SCC SCL-1 cells was obviously down-regulated 48 h after transfection with p65 siRNA, and a significant difference was detected, as compared with 0 h after (0.23 ± 0.10 vs. 0.66 ± 0.05, P<0.05). The protein levels of p65 and bcl-2 decreased, and the bax protein level and activities of caspase-3/9 increased after transfection with p65 siRNA at h 48 . Further, the results of flow cytometry demonstrated that p65 siRNA could induce apoptosis of SCL-1 cells, and cell apoptosis ratio (20.28% ± 1.87%) in p65 siRNA group was significantly higher than that in the untreated group and control siRNA group (9.13% ± 1.51% and 9.37% ± 1.38%, respectively, F=47.532, P<0.01). CONCLUSION: p65 siRNA can block NF-kappaB signal pathway, down-regulate expression of bcl-2, elevate the bax level and increase the activities of caspase-3/9, suggesting that NF-kappaB signal pathway may be a key molecular target for therapy of cutaneous SCC.

Usage of a Reward System for Dealing with Pediatric Dental Fear.

BACKGROUND: Pediatric dental fear, if left unchecked, can persist for a lifetime and adversely impact the physical and psychological health of a patient. In this study, a feasible nonmedical method for relieving pediatric dental fear was investigated. METHODS: A randomized, single-blind, controlled trial model was applied. The juvenile patients experiencing dental fear, whose parents or guardian had signed an informed consent form, were randomly divided into two groups. Group A (n = 50) was the control group, while Group B (n = 50) was the reward group. Participants in Group A accepted routine treatment. Participants in Group B were told that they would obtain a gift as a rewarda for their good behavior if they were compliant during their dental treatments. The Chinese version of the Children's Fear Survey Schedule-Dental Subscale (CFSS-DS) was used to evaluate the level of dental fear of each patient both before and after each treatment. A contrast analysis and a correlation analysis of the results were used to assess the efficacy of the reward mechanism. RESULTS: All participants in Group B, were obedient during the dental treatment, and they also successfully chose the present they wanted at the end of their dental treatment. Children at different ages showed different reward preferences. Significant difference in the fear scores of the participants in Group B before the treatment and after receiving the reward was found (independent samples t-test, t = 14.72, P < 0.001). In Group A, 86% children's fear score did not undergo a noticeable change. CONCLUSIONS: A reward system is proved feasible to relieve pediatric dental fear, and the form of reward should meet the demand of patients.

[Efficacy of probiotics on ulcerative colitis and its mechanism].

OBJECTIVE: To evaluate the efficacy of probiotics on the treatment outcomes of ulcerative colitis, and explore its possible mechanism. METHODS: According to the table of random number, 60 ulcerative colitis patients in our hospital were enrolled prospectively and divided into 3 groups: Golden Bifid group, Changmei group, combination group (Golden+ Changmei). Patients in Golden Bifid group received Golden Bifid 2.0 g, bid, those in Changmei group received Changmei 1.0 g, tid, and those in combination group received the above two drugs for 24 months. The clinical symptom score, colon mucosa inflammation score and endoscopic grade score were calculated and compared. IL-10 in mucosa and serum was determined by immunohistochemistry and double-antibody sandwich ELISA. RESULTS: In combined group after 24 months of treatment, clinical symptom score (12.5±2.1 vs. 2.3±0.8, P=0.016), endoscopic classification score (3.02±0.17 vs. 0.25±0.13, P=0.032), inflammatory reaction score (2.63±0.19 vs. 0.77±0.16, P=0.028) were significantly higher than those before treatment. While the scores differences of before and after treatment in Gold Bifid group and Changmei group were not statistically significant (P>0.05). IL-10 in serum [(17.4±2.2) ng/L vs. (12.8±2.2) ng/L, P=0.015] and colon mucosa [85% (17/20) vs. 55% (11/20), P=0.026] in combination group were significantly higher than those before treatment. The differences in IL-10 expression level before and after treatment were not statistically significant in the Gold Bifid group and Changmei group (P>0.05). CONCLUSION: Golden Bifid combined with Changmei as microbial ecological agents has a positive efficacy on mild to moderate ulcerative colitis, which may be associated with the up-regulation of IL-10 expression.

Effects of PTTG down-regulation on proliferation and metastasis of the SCL-1 cutaneous squamous cell carcinoma cell line.

AIMS: To study effects of down-regulation of pituitary tumor-transforming gene (PTTG) on proliferation and metastasis ability of the SCL-1 cutaneous squamous cell carcinoma (CSCC) cell line and explore related mechanisms. METHODS: SCL-1 cells were divided into 3 groups (untreated, siRNA control and PTTG siRNA). Cell proliferation assays were performed using a CCK-8 kit and proliferation and metastasis ability were analyzed using Boyden chambers. In addition, expression of MMP-2 and MMP-9 was detected by r-time qPCR and Western blotting. RESULTS: Down-regulation of PTTG could markedly inhibit cell proliferation in SCL-1 cells, compared to untreated and control siRNA groups (P < 0.05). Real-time qPCR demonstrated that expression levels of PTTG, MMP-2 and MMP-9 in the PTTG siRNA group were 0.8%, 23.2% and 21.3% of untreated levels. Western blotting revealed that expression of PTTG, MMP-2 and MMP-9 proteins in the PTTG siRNA group was obviously down-regulated. The numbers of migrating cells (51.38 ± 4.71) in the PTTG siRNA group was obviously lower than that in untreated group (131.33 ± 6.12) and the control siRNA group (127.72 ± 5.20) (P < 0.05), suggesting that decrease of proliferation and metastasis ability mediated by PTTG knock-down may be closely correlated with down-regulation of MMP-2 and MMP-9 expression. CONCLUSION: Inhibition of PTTG expression may be a new target for therapy of CSCC.

Synergistic antitumor activity of reversine combined with aspirin in cervical carcinoma in vitro and in vivo.

A recent report showed that reversine treatment could induce murine myoblasts dedifferentiation into multipotent progenitor cells and inhibit proliferation of some tumors, and other reports showed that apoptosis of lung adenocarcinoma cells could be induced by aspirin. The aim of the present study was to evaluate the synergistic antitumor effects of reversine and aspirin on cervical cancer. The inhibition rate of reversine and aspirin on cervical cancer cell lines' (HeLa and U14) was determined by MTT method, cell cycle of HeLa and U14 cells was analyzed by FACS, mitochondrial membrane potential of HeLa and U14 was detected using a JC-1 kit. HeLa and U14 colony formation was analyzed by soft agar colony formation assay. The expression of caspase-3, Bcl-2/Bax, cyclin D1 and p21 was detected by qRT-PCR and Western Blotting. Moreover, tumor weight and tumor volume was assessed using a murine model of cervical cancer with U14 cells subcutaneously (s.c.) administered into the neck, separately or combined with drug administration via the intraperitoneal (i.p.) route. The inhibition rate of cells in the combination group (10 µmol/L reversine, 10 mmol/L aspirin) increased significantly in comparison to that when the drugs were used alone (P < 0.05); moreover, this combination could synergistically inhibit the proliferation of five cervical cancer cell lines (HeLa, U14, Siha, Caski and C33A). In the therapeutic mouse model, tumor weight and tumor volume of cervical cancer bearing mice was more reduced when compared with the control agents (P < 0.05) in tumor-bearing mice. The combination of reversine and aspirin exerts synergistic growth inhibition and apoptosis induction on cervical cancers cells.