Regulation and mechanism of mannose-binding lectin on autophagy during the differentiation of 3T3-L1 adipocytes / 中华微生物学和免疫学杂志

Objective:To investigate the regulatory effects and mechanism of mannose-binding lectin(MBL) on autophagy during the differentiation of 3T3-L1 adipocytes, and provide the feasibility for targeting autophagy to prevent obesity and related pathological conditions in natural immunity.Methods:3T3-L1 preadipocytes were cultured in vitro and induced to differentiation. Cell differentiation and lipid accumulation were analyzed by oil red O staining and CCK-8 was used to detect the effect of different concentrations of MBL (0, 1, 5, 10 μg/ml) on cell proliferation ability at different differentiation stages. Western blot was used to analyze the expression of MBL(10 μg/ml) on the key autophagy factors LC3B, Beclin1 and p62 protein at different stages of differentiation, and the changes of lipid droplet accumulation under the intervention of MBL were observed by oil red O staining. The protein and mRNA expression of autophagy key factors under the intervention of different concentrations of MBL were detected by Western blot and qRT-PCR. And autophagy flow analysis based on autophagic degradation was used to further illustrate the autophagic activity. The expression and phosphorylation of adenosine monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR) signaling molecules were analyzed by Western blot. Results:The results of oil red O staining showed that 3T3-L1 preadipocytes could achieve complete differentiation after 10 days of induction. CCK-8 showed that the concentration of MBL (1-10 μg/ml) in the experimental group had no effect on cell proliferation at different differentiation stages. During the differentiation of 3T3-L1 preadipocytes, Western blot and qRT-PCR showed that the expression of autophagy-related proteins and mRNA levels was enhanced in the MBL treated group, and presented a concentration-dependent relationship. Oil red O staining showed that the lipid droplets in adipocytes at different stages of differentiation are reduced to varying degrees under the intervention of MBL. Fluorescence microscopy results further confirmed that MBL enhanced the autophagy activity of adipocytes by increasing the synthesis of autophagosomes. Moreover, under the intervention of MBL, the phosphorylation level of AMPK was significantly up-regulated, while the phosphorylation level of mTOR was significantly down-regulated, also showing a concentration-dependent relationship.Conclusions:MBL accelerates the autophagy process during the differentiation of 3T3-L1 adipocytes through AMPK/mTOR signaling pathway, reduces lipid accumulation, providing a possible functional pathway for the treatment of obesity and related metabolic diseases.

Regulation and mechanism of mannan-binding lectin (MBL) on adipogenic differentiation of 3T3-L1 preadipocytes / 中华微生物学和免疫学杂志

Objective:To investigate the regulatory effects and mechanism of mannan-binding lectin (MBL) on adipogenic differentiation of 3T3-L1 preadipocytes.Methods:3T3-L1 preadipocytes were induced to differentiate into adipocytes in vitro, and stimulated with different concentrations of MBL (0, 1, 10, 20 μg/ml). Firstly, changes in cell proliferation ability were detected by CCK-8. Then lipid accumulation was analyzed by Oil red O staining and intracellular triglyceride content determination. Further, the expression of adipogenic differentiation-related factors PPARγ and C/EBPα at protein and mRNA levels were detected by Western blot and qRT-PCR, respectively. Finally, Western blot was used to analyze the expression and phosphorylation of Akt, a signal molecule related to adipogenic differentiation. Results:MBL at the concentrations of 0, 1, 10 and 20 μg/ml had no effect on the proliferation of 3T3-L1 preadipocytes. The level of triglyceride in MBL treatment groups decreased in a dose-dependent manner on 3 d after 3T3-L1 preadipocyte differentiation. Results of Oil red O staining showed that the number of lipid droplets in MBL treatment groups reduced significantly, and the absorbance values also decreased significantly in a concentration-dependent manner. Western blot and qRT-PCR results showed that the expression of PPARγ and C/EBPα at both protein and mRNA levels in MBL treatment groups decreased significantly in a dose-dependent manner, and the phosphorylation level of Akt was significantly down-regulated as well.Conclusions:MBL regulates the adipogenic differentiation of 3T3-L1 preadipocytes via Akt signaling pathway.

Mannan-binding lectin regulates Treg/Th17 cell balance in mice with Candida albicans infection / 中华微生物学和免疫学杂志

Objective To investigate the regulation of Treg/Th17 axis by mannan-binding lectin (MBL) in mice with Candida albicans ( C. albicans ) infection. -ethods MBL gene-knockdown (MBL-/-) C57BL/6 mice and wild-type (WT) C57BL/6 mice were divided into four groups. C. albicans strains (2×107 CFU) were injected intraperitoneally into the mice of infection groups. Paraffin sections of mouse liver and kidney tissues were prepared on 3 d. Histopathological changes were observed with hematox-ylin and eosin ( HE) and Periodic acid-Schiff ( PAS) staining. Flow cytometry was performed to analyze Th17 and Treg cells in mice on 7 d. The levels of IL-10 and IL-17A were measured by ELISA. CD4+T cells were obtained from spleen cells by magnetic sorting. Expression of Foxp3 and RORγt at mRNA and protein levels were detected by qRT-PCR and Western blot, respectively. Results The mouse model of C. albicans infection was established successfully. After infection, the MBL-/- mice had higher percentages of Th17 cells, but lower percentages of Treg cells than the WT mice. ELISA results showed that compared with the WT mice with C. albicans infection, the MBL-/- mice had significantly increased IL-17A and decreased IL-10 after infection. Moreover, the expression of RORγt at both mRNA and protein levels was up-regula-ted, while that of Foxp3 was down-regulated in the MBL-/- mice than in the WT mice following infection. Conclusions MBL regulates the immune balance of Treg/Th17 cells in mice infected with C. albicans through promoting the differentiation of CD4+ T cells into Treg cell subsets and inhibiting the differentiation into Th17 cell subsets.

Correlative analysis of three sperm indexes and IVT-ET outcome after semen treatment / 重庆医学

Objective To investigate the correlation between three sperm indexes and the outcome of in vitro fertilization and embryo transfer(IVT-ET) before and after semen treatment.Methods The semen derived from 298 male patients treated with IVT-ET from the Affiliated Hospital of Southwest Medical University was retrospectively analyzed.Routine analysis of semen before treatment was performed according to the WHO standard.Vitrolife density gradient centrifugation combined with upstream method was used to analyze sperm parameters after sperm analysis,and the observation and statistics were performed after fertilization and sequential culture to day 3.Results The total viability of spermatozoa before and after semen treatment,the percentage of normal spermatozoa,sperm density,and other differences were statistically significant (P＜0.05);The normal sperm morphology rate was positively correlated with fertilization rate(r=0.487,P＜0.01) and cleavage rate(r=0.250,P＜0.05);The sperm vitality of post-processed semen was positively related to the fertilization rate (r =0.249,P＜0.05,);and significantly cleavage rate (r =0.272,P＜0.05);After treatment,sperm density was positively correlated with 2 PN(r=0.609,P＜0.01) and multiple PN fertilization rates(r=0.243,P＜0.05).Conclusion Our experimental results indicate that the parameters of post-processed sperm treated at the day when the oocytes were collected may be more helpful in predicting the fertilization rate and cleavage rate of IVF-ET.Therefore,the IVF-ET treatment protocols should be chose based on the normal sperm morphology rate and sperm vitality after semen treatment.

Effects of mannan-binding lectin on differentiation of Th17 cells / 中华微生物学和免疫学杂志

Objective To investigate the effects of mannan-binding lectin ( MBL) on the differen-tiation of Th17 cells (T helper cell 17, Th17). Methods CD4+T cells were separated in vitro from fresh human cord blood by MACS ( magnetic-activated cell sorting ) separator. Anti-CD3 monoclonal antibody ( McAb) and anti-CD28 McAb were used to stimulate CD4+T cells with IL-6 and TGF-β1 as inducers. Then, these cells were treated with ( MBL group) or without ( control group) different concentrations of MBL. Percentages of Th17 cells in different groups were detected by fluorescence-activated cell sorting( FACS) after 72 hours of culturing. Quantitative real-time PCR ( Q-PCR) was used to analyze the expres-sion of RORγt ( retinoid-related orphan receptor-γ-t) at mRNA level in both control and MBL groups. En-zyme-linked immunosorbent assay (ELISA) was performed to detect the levels of IL-17A in supernatants of cell culture from different groups. FACS was used to detect the percentages of Th17 cells in MBL-/-and wild type ( WT) mice. Results MBL could significantly reduce the percentage of Th17 cells after 72 hours of culturing as compared with the control group. Moreover, MBL could significantly down-regulate the expres-sion of RORγt at mRNA level and decrease the expression of IL-17A. Results of animal experiments showed that the percentages of CD4+RORγt+Th17 cells in MBL-/- mice were significantly higher than those in WT mice. Conclusion MBL can inhibit the differentiation of CD4+T cells to Th17 cells, which is induced by IL-6 and TGF-β1.

Effects of mannan-binding lectin on differentiation of Th17 cells / 中华微生物学和免疫学杂志

Objective To investigate the effects of mannan-binding lectin ( MBL) on the differen-tiation of Th17 cells (T helper cell 17, Th17). Methods CD4+T cells were separated in vitro from fresh human cord blood by MACS ( magnetic-activated cell sorting ) separator. Anti-CD3 monoclonal antibody ( McAb) and anti-CD28 McAb were used to stimulate CD4+T cells with IL-6 and TGF-β1 as inducers. Then, these cells were treated with ( MBL group) or without ( control group) different concentrations of MBL. Percentages of Th17 cells in different groups were detected by fluorescence-activated cell sorting( FACS) after 72 hours of culturing. Quantitative real-time PCR ( Q-PCR) was used to analyze the expres-sion of RORγt ( retinoid-related orphan receptor-γ-t) at mRNA level in both control and MBL groups. En-zyme-linked immunosorbent assay (ELISA) was performed to detect the levels of IL-17A in supernatants of cell culture from different groups. FACS was used to detect the percentages of Th17 cells in MBL-/-and wild type ( WT) mice. Results MBL could significantly reduce the percentage of Th17 cells after 72 hours of culturing as compared with the control group. Moreover, MBL could significantly down-regulate the expres-sion of RORγt at mRNA level and decrease the expression of IL-17A. Results of animal experiments showed that the percentages of CD4+RORγt+Th17 cells in MBL-/- mice were significantly higher than those in WT mice. Conclusion MBL can inhibit the differentiation of CD4+T cells to Th17 cells, which is induced by IL-6 and TGF-β1.

Analysis of microdeletions of azoospermia factor genes on Y chromosome in infertile males / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the location and characteristics of microdeletions of Y chromosome azoospermia factor (AZF) genes in infertile males with azoospermia and severe oligozoospermia in southern Sichuan.</p><p><b>METHODS</b>Multiplex PCR was used to detect 18 sequence tagged sites (STS) involved in Y chromosome AZF microdeletions among 224 infertile males (including 134 azoospermia cases and 90 severe oligozoospermia cases) and 70 healthy males.</p><p><b>RESULTS</b>Among the 224 infertile males, the overall frequency of microdeletions was 12.1% (27/224), and were 13.4% (18/134) in those with azoospermia and 10.0% (9/90) in those with severe oligozoospermia. The most frequent microdeletions have occurred in the AZFc region (51.9%). Compared with the 6 STS loci recommended by European Academy of Andrology and European Molecular Genetics Quality Network, 22.7% more deletions were detected based on the 18 STS loci selected from the AZF region.</p><p><b>CONCLUSION</b>Identification of Y chromosome microdeletions has a significant implication on the diagnosis of male infertility. The most frequent microdeletions have occurred in the AZFc region in southern Sichuan. To use more sequence tagged sites for the screening can improve the reliability and detection rate of Y chromosome microdeletions.</p>

The influence of processed total motile sperm count on clinical pregnancy rate of intrauterine insemination / 重庆医学

Objective To investigate the influence of processed total motile sperm (PTMS) count of husband on clinical pregnan-cy rate of intrauterine insemination(IUI) .Methods We retrospectively analyzed a total of 229 cycles of IUI among 131 patients in our hospital during the past three years .The cycles were divided into 5 groups according to the PTMS count :group A(0 .05) among five groups .Conclusion Ideal clinical pregnancy can be achieved when the PTMS count is between 3 × 106 and 5 × 106 .

The role of mannan-binding lectin in inhibition of inflammatory responses induced by peptidoglycan and its mechanism in THP-1/CD14 cells / 中华微生物学和免疫学杂志

Objective To investigate the effects of mannan-binding lectin(MBL) on TNF-α production induced by peptidoglycan (PGN) and its mechanism in human THP-1/CD14 monocytes.Methods The THP-1/CD14 cells were stimulated for 24 h with PGN at the indicated ratios after pretreated with human natural MBL at concentrations ranging from 1 to 20 mg/L for 2 h.The content of TNF-α and IL-6 in culture supernatants were detected by ELISA,and the levels of TNF-α and IL-6 mRNA expressions in these cells were determined by RT-PCR.FACS was used to investigate the interaction of MBL with THP-1/CD14 cells and the impact of MBL on PGN binding to THP-1/CD14 cells.Western blot was used to detect PGN-induced NF-κB translocation in THP-1/CD14 cells.Results ELISA showed that secretion of TNF-α and IL-6 from THP-1/CD14 cells could be induced by PGN ;The productions of TNF-α and IL-6 by THP-1/CD14 cells induced with PGN were profoundly inhibited by MBL at higher concentrations (10-20 mg/L) but not MBL at lower concentrations (1 mg/L).RT-PCR analysis also indicated that the mRNA expressions of TNF-α and IL-6 in THP-1/CD14 cells were decreased by MBL at higher concentration,compared to the corresponding THP-1/CD14 cells stimulated with PGN only.FACS showed that the binding of MBL to THP-1/CD14 cells was evident in a Ca2+-dependent manner.PGN could competitively inhibit the binding of MBL to THP-1/CD14 cells.MBL could competitively inhibit the binding of PGN to THP-1/CD14 cells by binding to THP-1/CD14 cells directly.Similarly,MBL at higher concentration (20 mg/L) decreased the NF-κB translocation in THP-1/CD14 cells.Conclusion MBL may inhibit TNF-α and IL-6 production induced by PGN in THP-1/CD14 cells through NF-κB signaling pathways,suggesting that MBL can play some roles in the regulation of PGN-induced inflammatory response.

Mannan-binding lectin inhibits IL-8 and TNF-α production induced by C. albicans in THP1/CD14 cells / 中华微生物学和免疫学杂志

Objective To investigate the effects of mannan-binding lectin (MBL) on IL-8 and TNF-α production induced by Candida albicans ( C. albicans) in human THP1/CD14 monocytes. Methods The THP1/CD14 cells were stimulated for 24 h with heat-inactivated yeast form or hyphal form cells of C. albicans strain at the indicated ratios after pretreated with human natural MBL at concentrations ranging from 1 to 20 mg/L for 2 h. The content of IL-8 and TNF-α in culture supernatants were detected by ELISA,and the levels of IL-8 and TNF-α mRNA expressions in these cells were determined by RT-PCR. Western blot was used to detect C. albicans-induced NF-κB translocation in THP1/CDI4 cells. Results ELISA showed that secretion of IL-8 and TNF-α from THP1/CD14 cells could be induced by both yeast cells and hyphal cells. Hyphal cells proved to be much less efficient than yeast cells in stimulating production of IL-8and TNF-α by THP1/CD14 cells. The productions of IL-8 and TNF-α by THP1/CD14 cells induced with C.albicans were profoundly inhibited by MBL at higher concentrations ( 10-20 mg/L) but not MBL at lower concentrations ( 1 mg/L). RT-PCR analysis also indicated that the mRNA expressions of IL-8 and TNF-αt in THP1/CD14 cells were decreased to various extents by MBL at higher concentration, compared to the corresponding THP1/CD14 cells stimulated with C. albicans only. Similarly, MBL at higher concentration ( 20mg/L) decreased the NF-κB translocation in THP1/CD14 cells. Conclusion MBL may inhibit IL-8 and TNF-α production induced by dimorphism C. albicans in THP1/CD14 cells, suggesting that MBL can play some roles on the regulation of C. albicans immune response.

Effect of angiotensin-(1-7) on proliferation and secretion of cultured rat's glomerular mesangial cells induced by transforming growth factor-β1 / 中国医师杂志

Objective To explore the effect of angiotensin-(1-7) [Ang-(1-7)] on proliferation and secretion in cultured rat's glo-merular mesangial cells(GMC) induced by transforming growth factor-β1 (TGF-β1). Methods GMC in logarithmic growth phase were in-cubated, and then were divided into 4 groups: control, Ang-(1-7), TGF-β1,and TGF-β1 + Ang-(1-7) group. Cell numbers of rat's GMC were detected by WST-1. Laminin(LN) and collagenlV (ColⅣ) mRNA expressions in GMC were analyzed by RT-PCR. The secretion of LN and ColⅣ in culture medium of rat's GMC was measured by radioimmunoassay. Results Ang-(1-7) significantly inhibited basal and TGF-β1-induced proliferation of cultured glomerular mesangial cells. Ang-(1-7) significantly down-regulated LN and Col Ⅳ mRNA expressions in glomerular mesangial cells and also inhibited the secretion of LN and ColⅣ induced by TGF-β1(P <0. 05). Conclusions Ang-(1-7) can inhibit basal and TGF-β1-induced proliferation, and inhibit ECM secretion of cultured rats glomerular mesangial cells.

Interventional effects of emodin on transforming growth factor-beta1/integrin-linked kinase signal way in interleukin-1beta-induced transdifferentiation of rat tubular epithelial-myofibroblasts / 中西医结合学报

To study the effects of transforming growth factor-beta1/integrin-linked kinase (TGF-beta1/ILK) signal way in interleukin-1beta (IL-1beta)-induced rat tubular epithelial-myofibroblast transdifferentiation (TEMT), and to investigate whether emodin inhibits IL-1beta-induced TEMT through the TGF-beta1/ILK signal way-dependent mechanism.

Angiotensin-(1-7) inhibit the expression of c-fos in angiotensin II-induced proliferative glomerular mesangial cells / 中国医师杂志

Objective To investigate the effect of angiotensin-(1-7) on the expression of cellular c-fos in angiotensin II -induced proliferative glomerular mesangial cells (GMC). Methods GMC were treated with angiotensin II and different dose of angiotensin-(1-7). GMC number were evaluated by crystal violet staining and the expression of c-foe were detected by western blot. Results Angiotensin-(1-7) inhibit angiotensin II -induced GMC proliferation as well as the expression c-foe in a concentration dependent manner. Conclusion c-fos is involved in the inhibiting effects of angiotensin-(1-7) on angiotensin II -induced GMC proliferation.

Influence of angiotensin-converting enzyme gene polymorphism on the anti-proteinuria efficacy of angiotensin-converting enzyme inhibitor in Han nationality of southern Sichuan province in China / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the association between angiotensin-converting enzyme (ACE) gene polymorphism and the reducing urinary protein efficacy of angiotensin-converting enzyme inhibitor (ACEI) in patients with primary chronic glomerulonephritis in Han nationality of southern Sichuan province.</p><p><b>METHODS</b>Ninety-nine primary glomerulonephritis patients with urinary protein were enrolled in this study. They were treated with benazepril for at least 3 months. The ACE gene insertion/deletion(I/D) polymorphisms in intron 16 were determined by PCR. A comparison of the reducing urinary protein efficacy of benazepril was made between the patients with different ACE genotypes.</p><p><b>RESULTS</b>Urinary protein excretion was significantly higher in patients with ACE DD genotype than that in patients with II genotype. After benazepril treatment for 3 months, the rates of urinary protein decline were observed. The rates of reduction of proteinuria in patients with DD genotype and ID genotype were obviously higher than that in patients with II genotype(P<0.05).</p><p><b>CONCLUSION</b>Benazepril could decline the rate of urinary protein excretion in patients with primary chronic glomerulonephritis, and significant relationship was observed between ACE gene polymorphism and the reducing urinary protein efficacy of ACEI.</p>

Inhibitory effect of phorbol 12-myristate 13-acetate and angiotensin-(1-7) on angiotensin Ⅱmediated proliferation and secretion of cultured rat glomerular mesangial cells / 医学研究生学报

Objective: To explore the influence of angiotensin-(1-7) \[Ang-(1-7)\] and phorbol 12-myristate 13-acetate (PMA)on the proliferation and secretion of cultured rat glomerular mesangial cells (GMC) induced by angiotensinⅡ(AngⅡ). Methods: Ang-(1-7) and PMA was used in cultured rat glomerular mesangial cells induced by AngⅡ, synthesis of DNA and protein, change of cell number were observed for rat GMC proliferation. Secretion of PcⅢ and HA was measured by radioimmunoassay in culture medium of rat GMC. Results: Ang-(1-7) and PMA both inhibited the AngⅡinduced synthesis of DNA and protein, increase of cell number, and secretion of PcⅢ and HA in cultured rat GMC. Conclusion: Ang-(1-7) and PMA both can inhibit the AngⅡ induced proliferation and secretion of cultured rat GMC.

The Effect of Angiotensin-(1-7) on the Angiotensin II-induced Proliferation of Rat Glomerular Mesangial Cells / 中国医师杂志

Objective To explore the effect of angiotensin-(1-7) on the angiotensin-II(Ang II) induced proliferation of cultured rat's glomerular mesangial cells(GMC). Methods Ang-(1-7) was used in the cultured rat's GMC treated by AngⅡ, the synthesis of DNA and protein in GMC was measured by incorporation of thymidine and leucine, respectively, and the proliferation of GMC was measured by crystal violet staining. Results Ang-(1-7) inhibited the synthesis of DNA and protein of cultured GMC treated by AngⅡ in a dose-dependent manner. Ang-(1-7) also reduced the number of GMC treated by AngⅡ.The effects of Ang-(1-7) could not be blocked by both -AngⅡ,a specific AngⅡAT1 receptor antagonist, and AngⅡAT_2 receptor antagonist PD123319. Conclusion Ang-(1-7) could inhibit the proliferation of cultured rat's GMC induced by AngⅡ. The effects of Ang-(1-7) were not mediated by AngⅡAT1 and AT2 receptors.

Effect of ERK1/2/c-Fos signal pathway on angiotensin-(1-7) inhibiting proliferation of rat glomerular mesangial cell strain induced by angiotensin Ⅱ / 中国病理生理杂志

AIM:To investigate the effect of ERK1/2/c-Fos signal pathway during angiotensin-(1-7)inhibiting proliferation of rat glomerular mesangial cell strain(GMCS)induced by angiotensin Ⅱ.METHODS:Rat glomerular mesangial cells(GMC)were co-cultured with angiotensin Ⅱ and different doses of angiotensin-(1-7).The numbers of GMC were evaluated by crystal violet staining.The amounts of p-ERK1/2 and c-Fos expressions were detected by Western blotting.RESULTS:Angiotensin-(1-7)showed its inhibitory effects on GMC number increasing induced by angiotensin Ⅱ as well as the amounts of p-ERK1/2 and c-Fos expressions in a concentration dependent manner.CONCLUSION:ERK/c-Fos signal pathway is involved in the inhibitory effects of angiotensin-(1-7)on angiotensin Ⅱ-induced GMC proliferation.

Protective effect of glucocorticoid preconditioning on the myocardial ischemic and reperfused rabbit heart / 西安交通大学学报(医学版)

Objective To study the protective effect of glucocorticoid preconditioning on the myocardial ischemic and reperfused hearts.Methods Totally 18 rabbits were randomly divided into three groups: myocardial ischemia-reperfusion model(model),high-dose glucocorticoid given by one time group(high-dose group) and low-dose glucocorticoid given by several times group(low-dose group),with six rabbits in each group.Myocardial ischemia was induced by left anterior descending coronary artery ligation.ST segments were recorded by the BL-420 biological signal acquisition system.Plasma malondial dehyde(MDA) was examined before ischemia,at 15 min after ischemia and 30 min after reperfusion;ischemic heart muscles were prepared with cryotomy and stained histochemically.Succinic dehydrogenase activity was observed in the ischemic region.Results There was shorter time of ST-segment recovery in the high-dose group and the low-dose group than that in the model group.Serum level of MDA in the high-dose group was lower than in the low-dose group(P