Effect of NLRP3/GSDMD/IL-1β pathway in post epileptic depressive behavior of developmental rats and its possible intervention approaches / 解放军医学杂志

Objective To investigate the effect of NLRP3/GSDMD/IL-1β pathway in post epileptic depressive behavior of developmental rats and its possible intervention approaches. Methods Sixty SD male rats aged 21 days were randomly divided into negative Control group, status epilepticus (SE) group, and SE+Dexamethasone (SE+DEX) group (20 each). The temporal lobe epilepsy (TLE) model was established by intraperitoneal injection of Lithium chloride-Pilocarpine (Licl-Pilocarpine) in SE group and SE+DEX group, the SE+DEX group was intraperitoneally injected with DEX [3 mg/(kg·d)] after the convulsion ceased for 7 consecutive days. Rats in SE group and Control group were given the same amount of normal saline at the same time point and by the same way. The post epileptic depressive behavior of rats was evaluated by sucrose partiality test (SPT), open field test (OPT) and forced swimming test (FST, n=8). Western blotting and RT-qPCR were used to detect the expression levels of protein and mRNA of NLRP3 and GSDMD in the hippocampus (n=3), and the expression level of IL-1β in the hippocampus was detected by ELISA (n=3). Results Compared with control group, the sucrose consumption rate decreased (68.50%±8.65% vs. 87.13%±3.31%), the time spent in the central area of the open field was reduced [(1.25±0.55) s vs. (4.73±2.57) s], the total movement distance decreased [(7.34±1.48) cm vs. (11.01±1.94) cm], while the immobility time (IMT) in water increased [(53.74±22.54) s vs. (27.18±11.86) s] in SE group; Compared with SE group, the sucrose consumption rate increased (78.8%±2.80% vs. 68.50%±8.65%), the time spent in the central area of the open field was prolonged [(4.00±2.35) s vs. (1.25±0.55) s], while the IMT was reduced [(22.85±7.85) s vs. (53.74±22.54) s] in SE+DEX group. All the differences were statistically significant (P<0.05). The expression levels of protein and mRNA of NLRP3 and GSDMD, as well as the IL-1β content, were significantly higher in SE group than in Control group (P<0.05); While the expression level of mRNA of NLRP3 was lower in SE+DEX group than in SE group (1.40±0.66 vs. 2.70±0.22, P<0.05), but no obviously difference between the two groups on the expression level of NLRP3 protein, The expression levels of GSDMD protein and mRNA were lower in SE+DEX group than in SE group (0.38±0.11 vs. 0.72±0.03, 1.14±0.44 vs. 1.80±0.08), the IL-1β content was also lower than that in SE group [(299.44±119.35) pg/ml vs. (571.37±18.48) pg/ml] with statistically significant difference (P<0.05). Conclusions NLRP3/GSDMD/IL-1β may be involved in the occurrence and development of epilepsy with depression. Short course intervention with dexamethasone may improve epileptic related depressive behavior in developmental rats by down-regulating GSDMD/IL-1β expression.

Effect of advanced maternal age on development of hippocampal neural stem cells in offspring rats / 中国当代儿科杂志

OBJECTIVE@#To study the effect of advanced maternal age (AMA) on the development of hippocampal neural stem cells in offspring rats.@*METHODS@#Ten 3-month-old and ten 12-month-old female Sprague-Dawley rats were housed individually with 3-month-old male rats (1:1, n=20), whose offspring rats were assigned to a control group and an AMA group. A total of 40 rats were randomly selected from each group. Immunofluorescence assay and Western blot were used to localize and determine the levels of protein expression of Nestin and doublecortin (DCX) on day 7 as well as neuronal nuclear antigen (NeuN) and glial fibrillary acidic protein (GFAP) on day 28 (n=8 for each marker). Immunofluorescence assay was also used to localize the hippocampal expression of polysialylated isoforms of neural cell adhesion molecule (PSA-NCAM) on day 14 (n=8 for each marker).@*RESULTS@#According to the Western blot results, the AMA group had significantly lower protein expression of DCX than the control group (P0.05). According to the results of immunofluorescence assay, the AMA group had significantly lower protein expression of Nestin, DCX, and PSA-NCAM in the hippocampal dentate gyrus (DG) region than the control group (P0.05). The AMA group had significantly higher expression of NeuN in the hippocampal CA1 region than the control group (P0.05). The AMA group had significantly lower expression of GFAP in the hippocampal CA1, CA3, and DG regions than the control group (P<0.05).@*CONCLUSIONS@#AMA may cause inhibition of proliferation, survival, and migration of hippocampal neural stem cells. AMA may also affect their differentiation into neurons and astrocytes, which will eventually lead to developmental disorders of hippocampal neural stem cells in offspring rats.

Effects of conbercept combined with PDGF receptor inhibitor on the proliferation, migration of RPE cells and VEGF expression in hypoxic conditions / 眼科新进展

Objective To investigate the effects of conbercept combined with platelet-derived growth factor (PDGF) receptor inhibitor on the proliferation and migration of human retinal pigment epithelial cells (ARPE-19),as well as mRNA and protein expression of vascular endothelial growth factor (VEGF).Methods ARPE-19 cells were cultured in in vitro and the cells in logarithmic growth phase were obtained and induced by different concentrations of CoCl2.Then cell proliferation and toxicity test kit (CCK-8) was used to detect the proliferation activity of ARPE-19 cells for screening the optimal concentration of CoCl2 to construct hypoxic model.The cultured ARPE-19 cells were divided into normal group,hypoxic group and hypoxia + conbercept group (different concentrations),and CCK-8 assay was applied to analyze the effects of conbercept with different concentrations on cell proliferation activity for screening the best concentration of conbercept.The ARPE-19 cells in logarithmic phase were divided into normal group,hypoxic group,hypoxia + PDGF receptor inhibitor group (different concentrations),and CCK-8 assay was performed to detect the effects of PDGF receptor inhibitor on cell proliferation with hypoxic damage activity for screening the best concentration of PDGF receptor inhibitor.The logarithmic growth phase cells were divided into normal group,hypoxic group,hypoxia + 20 mg · L-1 conbercept group,hypoxia + 200 μmol · L-1 PDGF receptor inhibitor group,hypoxia + conbercept + PDGF receptor inhibitor group (both optimal concentration),and then the cell proliferation in each group was detected by CCK-8 assay,and the migration was detected by transwell chambers.The level of VEGF protein in the supernatant of each group was detected by ELISA methods,and VEGF mRNA expression was measured by RT-PCR.Results CCK-8 assay results showed that the A value of ARPE-19 in the 100 μmol · L-1 CoCl2 group was the highest (1.063 ± 0.031),which was set to be the optimal concentration for preparation of hypoxic model.CCK-8 assay results showed the cell proliferation rate of 20 μg · mL-1 conbercept and 20 μmol · L-1 PDGF receptor inhibitor group was (94.58 ± 3.80) ％ and (96.72 ± 5.44) ％,respectively,which could achieve the most satisfying efficacy,thereby both concentrations were selected for follow-up experiments.The cell proliferation and migration ability and VEGF protein level decreased in the conbercept group,PDGF receptor inhibitor group and conbercept + PDGF receptor inhibitor group when compared with the hypoxic group,and the decrease in the combined group was the most significant.Moreover,VEGF mRNA expression in the conbercept group and combined group were decreased when compared with the hypoxic group.Conclusion Hypoxia can enhance cell proliferation and migration ability and induce the up-regulation of VEGC protein and mRNA expression.In addition,PDGF receptor inhibitor combined with conbercept has inhibitory effects on the migration and proliferation of ARPE-19 cells as well as VEGF protein and mRNA expression following hypoxia injury,which is superior to conbercept treatment alone.

Studies on causes of gynecology postoperative urinary retention and its acupuncture treatment / 中国针灸

Urinary retention is a frequent-encountered complication after gynaecological surgery. It affects the postoperative recovery and decreases the life quality of patients. In recent years, extensive researches on causes and treatments of postoperative urinary retention are carried out in clinic. And it is approved that acupuncture treatment, which includes body needling, moxibustion, combination of acupuncture and moxibustion, acupoint injection and medication plasters, has reliable effects and less side-effects. Acupuncture treatment on postoperative urinary retention keeps developing and innovating. And it is held to have better effect when compare with western medicine.

Semen quality and sperm ultrastructure in infertile men with varicocele / 中华男科学杂志

<p><b>OBJECTIVE</b>To examine and analyze semen quality and sperm ultrastructural characteristics of infertile patients with varicocele.</p><p><b>METHODS</b>This study included 118 infertile patients with varicocele (the VC group) and 76 normal semen donors (the control group). We obtained routine semen parameters, seminal plasma biochemical markers and the levels of reproductive hormones in the subjects, and observed the changes in sperm structure under the scanning electron microscope and transmission electron microscope.</p><p><b>RESULTS</b>Compared with the normal control, the VC patients showed significantly decreased sperm concentration, sperm progressive motility, sperm viability (P < 0.05), but no remarkable difference in semen volume and non-progressive motility (P > 0.05). The concentrations of zinc and alpha-glycoside enzyme in the seminal plasma were markedly reduced in the VC group in comparison with the controls (P < 0.05), but there was no significant difference in the level of fructose (P > 0.05), nor in such seminal plasma biochemical markers as FSH, LH, T and E2 between the two groups (P > 0.05). The percentage of morphologically normal sperm was dramatically lower in the VC than in the control group ([56.76 +/- 15.32]% vs [12.34 +/- 6.58]%, P < 0.05), and the sperm deformities were mostly in the head and neck, mainly tapering pin head accompanied by complex abnormal differentiation.</p><p><b>CONCLUSION</b>This study demonstrated that VC may lead to oligo-astheno-terato zoospermia, and hence male infertility, which may be attributed to the changes of seminal plasma microenvironment and sperm ultrastructure.</p>

Effect of testosterone on the expression of CMTM family of the male spermatogenesis suppression rats / 药学学报

This study is to investigate the influence and the expression of CMTM family of testosterone on spermatogenesis suppression in the male rats treated by gossypol and cyclophosphamide. Gossypol (50 mg kg(-1)) and cyclophosphamide (20 mg kg(-1)) were administered to male rats to induce spermatogenesis suppression. Testosterone propionate was administrated at the dose of 5 mg kg(-1) every other day for 6 times. Sperm was collected from the left caudal epididymis, the count and motility of sperm were analyzed by CASA. Morphological change of testis tissue was observed with HE staining. The expression of CMTM family was examined by Western blotting assay. Gossypol (50 mg kg(-1)) and cyclophosphamide (20 mg kg(-1)) decreased the count and motility of sperm, and the pathological change of testis tissue was also observed. But, testosterone (5 mg kg(-1)) had positive effect. Furthermore, CMTM4 down-expressed remarkably in the gossypol and cyclophosphamide treated rats, the expression of the CMTM4 was up-expressed after testosterone administration. On the contrary, the expression of CMTM2 increased significantly only in gossypol treated male rats, but not in cyclophosphamide treated male rats. The expression of CMTM2 was down-expressed after testosterone administration. However, no obvious change of CMTM2 was observed in cyclophosphamide treated rats. Testosterone did not influence the expression of CKLF1, CMTM3 and CMTM5, the CMTM6, CMTM7 and CMTM8 of CMTM family were not detected in testis tissue. These demonstrated that the spermatogenesis effect of testosterone (5 mg kg(-1)) was associated with the expression of CMTM family, and CMTM2 and CMTM4 may take part in the spermatogenesis process.

Inhibitory effect of dexamethasone on myeloid differentiation factor 88 and tumor necrosis factor-alpha expressions in mouse peritoneal macrophages / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the inhibitory effect of dexamethasone (DEX) on myeloid differentiation factor 88 (MyD88) and tumor necrosis factor-alpha (TNF-alpha) expression in mouse peritoneal macrophages in innate immune response to Penicillium marneffei (PM).</p><p><b>METHODS</b>Mouse peritoneal macrophages were cultured in the presence of heat-inactivated yeast-phase PM with or without DEX, and the protein and mRNA expressions of MyD88 in the macrophages were detected using Western blotting and real-time PCR, respectively. TNF-alpha in the cell culture supernatant was measured with enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>DEX suppressed TNF-alpha production by the macrophages co-cultured with PM. The expressions of MyD88 were up-regulated by PM stimulation, whose effect was inhibited by the application of DEX.</p><p><b>CONCLUSION</b>The inhibitory effect of DEX on PM-induced proinflammatory responses of the macrophage is directly associated with the inhibition of MyD88 expression.</p>

Effect of Penicillium marneffei on TLR-2, TLR-4, and Dectin-1 expression and TNF-alpha production in macrophage / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effects of heat-killed Penicillium marneffei (PM) on the expressions of toll-like receptor-4 (TLR-4), toll-like receptor-2 (TLR-2) and dendritic cell associated C-type lectin-1 (Dectin-1)and the production of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). in mouse peritoneal macrophages.</p><p><b>METHODS</b>Mouse peritoneal macrophages were cultured in the presence of heat-killed yeast-phase PM for 24 h, and the average fluorescence intensity of TLR-2, TLR-4, and Dectin-1 in the macrophages was detected using flow cytometry. Fluorescent staining of the macrophages was performed to observe the fluorescence of TLR-2, TLR-4, and Dectin-1 with confocal microscopy. TNF-alpha mRNA in the cell culture supernatant was measured with real-time PCR, and TNF-alpha protein detected using enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>The average fluorescence intensity of TLR-2, TLR-4 and Dectin-1 in the macrophages was increased in response to a 24-h PM stimulation, and the stimulated macrophages produced large amounts of TNF-alpha.</p><p><b>CONCLUSION</b>PM up-regulates the expression of TLR-2, TLR-4 and Dectin-1 in mouse peritoneal macrophages, and their expressions are directly associated with macrophage activation.</p>

The Interaction Between Dendritic Cells and Penicillium marneffei Yeasts / 微生物学通报

To assay the influence of dendritic cells(DCs)on the function of anti-infective immunity to Penicillium marneffei.DCs were generated from peripheral blood mononuclear cells(PBMC)and pulsed with Penicillium marneffei yeasts.DCs morphology was observed by the inverted microscope and cell surface markers of DCs were analyzed by flow cytometry.The concentrations of IL-12p70 were detected by ELISA. Mixed lymphocyte reaction was performed to assay the proliferation of T cells.The mRNA of CCR7 and CXCR4 were detected by the Real-time PCR quantifications.The acquired DCs exhibited irregular appearance and numerous long dendrites under light microscope.DCs and Penicillium marneffei yeasts were co-cultured for 24 h,numerous yeasts were observed inside the cells;an enhanced expression of the cell sur-face markers CD86、CD83、HLA-DR and CD40 were demonstrated;the expression of CCR7 and CXCR4 mRNA were also increased;the improved proliferation of T cells were observed in the mixed lymphocyte reaction.Yeasts-pulsed DCs secreted more IL-12p70 than that of non-pulsed,but less than that of LPS-pulsed DCs.DCs can engulf the Penicillium marneffei yeasts.When pulsed with Penicillium marneffei yeasts,DCs improved their expression of the co-stimulatory molecules and chemokine receptor CCR7、CXCR4,enhanced their capacity to process antigen.DCs play an important role in host defense against Penicillium marneffei infection.But the low level of the IL-12p70 production may lead to deficiency in the cell-mediated immunity against Penicillium marneffei.

A Convenient Method to Select DNA Fragments of Cephalosporium acremonium with Promoter Function / 微生物学通报

A promoter-trap vector pGBT14 for selecting promoters of fungus gene was constructed with E. coli-yeast shuttling plasmid pGBT9. Using this vector, a0. 5-2. 0kb chromosomal DNA library of Cepholosporium acremonium was constructed, and twenty four DNA fragments with promoter function in Saccharomyces oerevisiae Y153 were selected from this DNA library. And the promoter function of these DNA fragments was analyzed.