[Mechanism of Effects of Genistein on the Reproductive System of Prepubertal Male Rats].

OBJECTIVE: To study the effects of genistein (GEN) on reproductive system in prepubertal male rats. METHODS: Thirty SPF-rated male SD rats were randomly divided into control group (Con group), low-dose group (G1 group) and high-dose group (G2 group), with 10 rats in each group. Corn oil, 150 mg/kg and 300 mg/kg GEN dissolved in corn oil of equal volume were respectively administered every day and weighed the next day. After 6 weeks, the rats were sacrificed, and the testis, epididymis and prostate were dissected, and organ coefficients were calculated. Histopathological changes of testis was observed. The number of sperm was counted and the rate of sperm malformation was calculated. The concentrations of serum testosterone and estradiol were detected by radioimmunoassay. The protein phosphatase 2, regulatory subunit B, gamma (PPP2R2C) protein expression in testicular tissue was detected by immunofluorescence assay. The mRNA and protein expression levels of PPP2R2C and cyclin dependent protein kinases 2 (CDK2) in rat testis were detected by real-time quantitative fluorescence PCR (RT-qPCR) and Western blot, respectively. The protein phosphatase 2A (PP2A) activity in testicular tissue was detected by immunoprecipitation. RESULTS: There were no statistically significant differences in body mass, sperm number, serum estradiol and PP2A enzyme activity among the groups ( P>0.05). The pathological structure of testicular in G2 group was disordered. Sperm abnormality rate in G1 and G2 groups was higher than that in Con group ( P<0.05). Serum testosterone concentration in G2 group was lower than that in Con group ( P<0.05). The expression of PPP2R2C and CDK2 in G2 group was higher than that in Con group ( P<0.05), but the protein level was lower than that in Con group ( P<0.05). PPP2R2C protein was expressed in testicular tissue in each group. CONCLUSION: Long-term exposure to high dose (300 mg/kg) GEN during prepuberty may cause adverse effects on reproductive function in adult male rats. Further investigation is needed to determine whether PPP2R2C-PP2A-CDK2 phosphorylation pathway affects reproductive system in rats.

[The Thyroid Hormone Receptor Mediated Luciferase Reporter Gene Assays Screening for EDCs].

OBJECTIVE: This study in order to use report gene assay based on the thyroid hormone receptor (TR) α/ß from human origin for screening endocrine disruptors chemicals (EDCs), evaluating the thyroid hormone activity of Bisphenol (BPA), 1-Naphthaleny methyl carbamate and 1-naphthol (1-NAP). METHODS: Using Rhesus monkey kidney cells (LLC-MK2) as transfection cell to establish the gene report assay system based on pGL-3-promega and pGL4.27 of TRα/ß through the method of transient transfection. Using T3 and T4 as positive subjects to evaluation the effectiveness of two detection systems and detect the thyroid hormone activity of BPA, 1-Naphthaleny methyl carbamate, 1-NAP. RESULTS: The TRß LLC-MK2 report gene assay based on pGL3-promega, the minimum detectable limit of T3 is 1.216×10-11 mol/L, the largest induction multiple was shown at 7.482×10-6 mol/L, the expression multiple of induced Lucifrerase was 5.98-fold that of the vehicle control, the EC50 was 3.327×10-8 mol/L; The minimum detectable limit of T4 was 1.622×10-8 mol/L, the largest induction Luc expression was 3.4-fold of vehicle control, the EC50 was 2.213×10-7 mol/L. The TRß LLC-MK2 report gene assay based on pGL4.27, the minimum detectable limit of T3 was 9.863×10-12 mol/L, the largest induction Luc expression as shown at 1.671×10-6 mol/L, resulting in 8.57-fold of vehicle control, the EC50 is 3.327×10-8 mol/L. The minimum detectable limit of T4 was 1.349×10-9 mol/L, the largest induction Luc expression was 4.6-fold of vehicle control, the EC50 is 4.074×10-7 mol/L. There was no thyroid hormone activity by using TRß report gene assay to evaluate BPA, 1-Naphthaleny methyl carbamate or 1-NAP, but 1-Naphthaleny methyl carbamate and 1-NAP have some degree receptor antagonism. CONCLUSIONS: The TRß LLC-MK2 report gene assay based on pGL3- promega and pGL4.27 show highly sensitive (pGL4.27 relatively higher), can be used to screen for EDCS and test chemical thyroid hormone activity effectively.

[Effects of Genistein on Whole Genome Expression Profiles of Mice Testes Tissue Cultured in vitro].

OBJECTIVE: To investigate the effects of Genistein (Gen) on the whole expression profiles of testes in mice by using the gene chip technology,and analyze the mechanism how Gen exerts testis on gene level. METHODS: The testicular tissues of suckling mice were cultured in vitro,and separated into control group and Gen group with the dose of 5 µmol/L. After 72 h of culture,we extracted the total RNA and purified it,then detected the gene expression by using Agilent gene array. RESULTS: Compared with the control,there were 84 genes expressed differently in Gen group,including 47 upregulated genes and 37 downregulated genes. We classified these genes as 14 categories in Gen group by GO ( P<0.05),including cell proliferation,cell death,the immune system and reproductive development. CONCLUSION: The whole gene chip test found that Gen can mainly affect the functions of testes by regulating the expression of gene related to cell development,proliferation and cell cycle function, which can influence the spermatogenesis and change the testis cell proliferation and apoptosis.

Kangfuxinye Enema Combined with Mesalamine for Ulcerative Colitis: A Systematic Review and GRADE Approach.

OBJECTIVES: To critically appraise the efficacy and safety of Kangfuxinye enema combined with mesalamine for the ulcerative colitis (UC) patients and in addition to grade the quality of evidence by using the GRADE (grading of recommendations, assessment, development, and evaluation) approach. METHODS: A literature search was performed in the Cochrane Library, MEDLINE, EMBASE, CBM, CNKI, VIP, and WanFang Databases. The search restrictions were patients with UC and RCTs. Studies including other treatments except Kangfuxinye with mesalamine were excluded. RESULTS: Nineteen studies met the inclusion criteria. We found significant benefits of Kangfuxinye combined with mesalamine against mesalamine alone in improving response rate as well as reducing the recurrence rate and inflammation rate; meanwhile, the increase of the adverse events rate was not observed. Furthermore, the symptoms remission rate and the cure time were insignificant statistically. Additionally, GRADE results indicated that the quality of evidence regarding the above 6 outcomes was rated from very low to moderate quality. CONCLUSIONS: Although Kangfuxinye enema seems effective and safe for treating UC patients in this systematic review, Kangfuxinye enema combined with mesalamine was weakly recommended due to very low to moderate quality of available evidence by the GRADE approach.

[The Effects of All-trans Retinoic Acid on the Expression of Inflammatory Cytokines and Cartilage Damage Related Protease in Rats with Collagen Induced Arthritis].

OBJECTIVES: To investigate the effects of all-trans retinoic acid (ATRA) on arthritis and the expressions of inflammatory cytokines and cartilage damage related proteases of the collagen-induced arthritis model (CIA) rats in vivo. METHODS: The CIA model of rheumatoid arthritis was induced with C2 and incomplete Freund's adjuvant. The rats were randomly divided into control group, CIA model group and two ATRA dose groups (ATRA 0.50 mg/kg group and ATRA 1.00 mg/kg group). ATRA were given three times per week for six weeks in ATRA groups. Morphological changes, arthritis index (AI) scores, the semi-quantitative scores of pathology damage, the protein expressions of cartilage damage related proteases and the serum levels of TNF-α, IL-17A, IFN-γ, IL-4, IL-10 were observed. RESULTS: The AI scores of ATRA groups were similar to CIA model group ( P<0.05). Apparent morphological disorders in knee and ankle joints were observed in the CIA model group and ATRA 1.00 mg/kg group. The structure of knee joint was improved slightly in ATRA 0.50 mg/kg group. The serum levels of TNF-α, IFN-γ and IL-17A were decreased in both ATRA groups; ATRA also can increase the serum level of IL-4. Compared to CIA model group, the protein expressions of ADAMTS-4, MMP3, MMP1 were decreased in both ATRA groups ( P<0.05). CONCLUSIONS: ATRA, which was able to inhibit pro-inflammatory cytokines secretion, could correct the imbalance of Th1/Th2 and Th17/Treg. ATRA also can reduce the expressions of cartilage damage related proteases, which proved that ATRA may have a beneficial effect on rheumatoid arthritis.

[Prophylactic administration of all-trans retinoic acid alleviates inflammation in rats with collagen-induced arthritis].

OBJECTIVE: To investigate the effects of prophylactic administration of all-trans retinoic acid (ATRA) in relieving inflammation in a rat model of collagen-induced arthritis (CIA). METHODS: Female Wistar rats (6 to 8 weeks old) were randomly divided into normal control group, solvent control group, and prophylactic ATRA treatment (0.05, 0.5, and 5 mg/kg) groups. All the rats except for those in normal control group were subjected to subcutaneous injection of type II collagen and incomplete Freund adjuvant in the tails to induce CIA, followed by injection on the following day with saline, corn oil or different doses of ATRA 3 times a week. The arthritis index (AI) scores, histological scores, serum levels of TNF-α, IL-17A, and IL-10, and expressions of proteases related with cartilage damage were evaluated. RESULTS: On the 15th day after the primary immunization, the AI scores increased significantly in all but the normal control groups; the scores increased progressively in all the 3 ATRA groups but remained lower than that in the solvent control group, which was stable over time. The rats in the 3 ATRA groups showed obvious pathologies in the knee and ankle joints, but the semi-quantitative scores of pathology damage showed no significance among them. Compared with those in solvent control group, the serum IL-17A and TNF-α levels decreased, serum IL-10 level increased, and the expressions of ADAMT-4 and MMP-3 proteins decreased significantly in the knees in the 3 ATRA groups. CONCLUSION: ATRA can reduce the production of TNF-α and IL-17A and increase the production of IL-10 to alleviate the inflammation in rats with CIA. ATRA may delay the progression of RA by correcting the imbalance of Th1/Th2 and Th17/Treg.