The incomplete degradation of tumour cells by macrophages(Mφ)is a contributing factor to tumour progression and metastasis,and the degradation function of Mφ is mediated through phagosomes and lysosomes.In our preliminary experiments,we found that overactivation of NADPH oxidase 2(NOX2)reduced the ability of Mφ to degrade engulfed tumour cells.Above this,we screened out liquiritin from Glycyrrhiza uralensis Fisch,which can significantly inhibit NOX2 activity and inhibit tumours,to elucidate that suppressing NOX2 can enhance the ability of Mφ to degrade tumour cells.We found that the tumour environment could activate the NOX2 activity in Mφ phagosomes,causing Mφ to produce excessive reactive oxygen species(ROS),thus prohibiting the formation of phagolysosomes before degradation.Conversely,inhibiting NOX2 in Mφ by liquiritin can reduce ROS and promote phagosome-lysosome fusion,therefore improving the enzymatic degradation of tumour cells after phagocytosis,and subse-quently promote T cell activity by presenting antigens.We further confirmed that liquiritin down-regulated the expression of the NOX2 specific membrane component protein gp91 phox,blocking its binding to the NOX2 cytoplasmic component proteins p67 phox and p47 phox,thereby inhibiting the activity of NOX2.This study elucidates the specific mechanism by which Mφ cannot degrade tumour cells after phagocytosis,and indicates that liquiritin can promote the ability of Mφ to degrade tumour cells by suppressing NOX2.

Objective:To explore whether LBP3 exerts anti-tumor effects by promoting production of short-chain fatty acids(SCFAs)by intestinal microbiota and regulating function of polymorphonuclear myeloid-derived suppressor cells(PMN-MDSC).Methods:A subcutaneous H22 liver cancer model was employed to assess anti-tumor activity of LBP3 and its regulatory effects on PMN-MDSC.Pseudo-sterile tumor-bearing mouse model was used to investigate role of intestinal microbiota in tumor suppression of LBP3.Fecal microbiota transplantation(FMT)was conducted to explore immune regulatory role of LBP3-modulated flora.Serum SCFAs levels in tumor-bearing mice were quantified using liquid chromatography-mass spectrometry,and effect of SCFAs butyrate on arginase 1(Arg-1)expression was evaluated in vitro.Results:Both low-dose(125 mg/kg)and high-dose(250 mg/kg)LBP3 signifi-cantly inhibited tumor growth in H22 tumor-bearing mice,also led to a marked reduction in proportion of PMN-MDSC in both spleen and tumor,a reduced proportion of Treg in lymphoid tissues,a decrease in Arg-1 level within tumor,infiltration of CD8+T cells into tumor was significantly enhanced.However,these effects of LBP3 were did not observed in pseudo-sterile mice,while the above changes could be reproduced after fecal supernatant transplantation in high-dose LBP3 treatment group,suggesting a crucial role for gut microbiota.Furthermore,co-expression of Ly6G and SCFA receptor GPR43 in tumor was also observed.LBP3 treatment resulted in increased levels of SCFAs,particularly butyrate,in both blood and tumor tissues.In vitro,butyrate was shown to inhibit Arg-1 expression in MSC-2 cells,further supporting hypothesis that SCFAs mediate immune-modulatory effects of LBP3.Conclusion:LBP3 exerts its anti-tumor effects by promoting SCFA production,which subsequently inhibits function of PMN-MDSC.This highlights LBP3's potential as an immunomodulatory agent in cancer therapy.

The incomplete degradation of tumour cells by macrophages (Mϕ) is a contributing factor to tumour progression and metastasis, and the degradation function of Mϕ is mediated through phagosomes and lysosomes. In our preliminary experiments, we found that overactivation of NADPH oxidase 2 (NOX2) reduced the ability of Mϕ to degrade engulfed tumour cells. Above this, we screened out liquiritin from Glycyrrhiza uralensis Fisch, which can significantly inhibit NOX2 activity and inhibit tumours, to elucidate that suppressing NOX2 can enhance the ability of Mϕ to degrade tumour cells. We found that the tumour environment could activate the NOX2 activity in Mϕ phagosomes, causing Mϕ to produce excessive reactive oxygen species (ROS), thus prohibiting the formation of phagolysosomes before degradation. Conversely, inhibiting NOX2 in Mϕ by liquiritin can reduce ROS and promote phagosome-lysosome fusion, therefore improving the enzymatic degradation of tumour cells after phagocytosis, and subsequently promote T cell activity by presenting antigens. We further confirmed that liquiritin down-regulated the expression of the NOX2 specific membrane component protein gp91 phox, blocking its binding to the NOX2 cytoplasmic component proteins p67 phox and p47 phox, thereby inhibiting the activity of NOX2. This study elucidates the specific mechanism by which Mϕ cannot degrade tumour cells after phagocytosis, and indicates that liquiritin can promote the ability of Mϕ to degrade tumour cells by suppressing NOX2.

Objective To investigate the ameliorative effect and the related mechanism of rotating magnetic fields(RMF)on sevoflurane(SEV,a commonly used inhalational anesthetics in clinics)-induced cognitive impairments in elderly rats.Methods The cognitive functions of 24 elderly male SD rats which were allocated into 3 experimental groups(Control group,SEV group,and SEV+RMF group;n=8 per group)were assessedviawater maze testing,and the damage and repair of hippocampal neurons were detected using ELISA and Western blotting assays.Water maze testing was also conducted on additional 24 elderly male SD rats which were randomized into 3 groups(SEV group,SEV+RMF group,and SEV+autophagy inhibitor+RMF group;n=8 per group)for assessing their cognitive functions.Results Compared with SEV group,RMF intervention significantly reduced the escape latency and swimming distance in water maze test(P<0.01),while increasing the number of platform crossovers(P<0.001).RMF downregulated the serum levels of tumor necrosis factor-α,interleukin-1β,and interleukin-6 in SEV-treated rats(P<0.001),and led to remarkable reductions in both serum neuron-specific enolase and β-amyloid protein levels(P<0.001).Moreover,RMF upregulated the expressions of nerve growth factor and brain-derived neurotrophic factor in hippocampal neurons(P<0.001),suppressed the expression of apoptotic protein caspase-3 and autophagy related protein p62 in the hippocampus,and upregulate the expression of autophagy related protein Beclin-1(P<0.001).However,the administration of autophagy inhibitor 3-MA abolished the ameliorative effects of RMF on SEV-induced cognitive impairments in elderly rats(P<0.001).Conclusion RMF can effectively improves SEV-induced cognitive dysfunction in elderly rats,and this neuroprotective effect is mediated by the autophagy activation in hippocampal neurons.

Objective To investigate the ameliorative effect and the related mechanism of rotating magnetic fields(RMF)on sevoflurane(SEV,a commonly used inhalational anesthetics in clinics)-induced cognitive impairments in elderly rats.Methods The cognitive functions of 24 elderly male SD rats which were allocated into 3 experimental groups(Control group,SEV group,and SEV+RMF group;n=8 per group)were assessedviawater maze testing,and the damage and repair of hippocampal neurons were detected using ELISA and Western blotting assays.Water maze testing was also conducted on additional 24 elderly male SD rats which were randomized into 3 groups(SEV group,SEV+RMF group,and SEV+autophagy inhibitor+RMF group;n=8 per group)for assessing their cognitive functions.Results Compared with SEV group,RMF intervention significantly reduced the escape latency and swimming distance in water maze test(P<0.01),while increasing the number of platform crossovers(P<0.001).RMF downregulated the serum levels of tumor necrosis factor-α,interleukin-1β,and interleukin-6 in SEV-treated rats(P<0.001),and led to remarkable reductions in both serum neuron-specific enolase and β-amyloid protein levels(P<0.001).Moreover,RMF upregulated the expressions of nerve growth factor and brain-derived neurotrophic factor in hippocampal neurons(P<0.001),suppressed the expression of apoptotic protein caspase-3 and autophagy related protein p62 in the hippocampus,and upregulate the expression of autophagy related protein Beclin-1(P<0.001).However,the administration of autophagy inhibitor 3-MA abolished the ameliorative effects of RMF on SEV-induced cognitive impairments in elderly rats(P<0.001).Conclusion RMF can effectively improves SEV-induced cognitive dysfunction in elderly rats,and this neuroprotective effect is mediated by the autophagy activation in hippocampal neurons.

Objective:To explore whether LBP3 exerts anti-tumor effects by promoting production of short-chain fatty acids(SCFAs)by intestinal microbiota and regulating function of polymorphonuclear myeloid-derived suppressor cells(PMN-MDSC).Methods:A subcutaneous H22 liver cancer model was employed to assess anti-tumor activity of LBP3 and its regulatory effects on PMN-MDSC.Pseudo-sterile tumor-bearing mouse model was used to investigate role of intestinal microbiota in tumor suppression of LBP3.Fecal microbiota transplantation(FMT)was conducted to explore immune regulatory role of LBP3-modulated flora.Serum SCFAs levels in tumor-bearing mice were quantified using liquid chromatography-mass spectrometry,and effect of SCFAs butyrate on arginase 1(Arg-1)expression was evaluated in vitro.Results:Both low-dose(125 mg/kg)and high-dose(250 mg/kg)LBP3 signifi-cantly inhibited tumor growth in H22 tumor-bearing mice,also led to a marked reduction in proportion of PMN-MDSC in both spleen and tumor,a reduced proportion of Treg in lymphoid tissues,a decrease in Arg-1 level within tumor,infiltration of CD8+T cells into tumor was significantly enhanced.However,these effects of LBP3 were did not observed in pseudo-sterile mice,while the above changes could be reproduced after fecal supernatant transplantation in high-dose LBP3 treatment group,suggesting a crucial role for gut microbiota.Furthermore,co-expression of Ly6G and SCFA receptor GPR43 in tumor was also observed.LBP3 treatment resulted in increased levels of SCFAs,particularly butyrate,in both blood and tumor tissues.In vitro,butyrate was shown to inhibit Arg-1 expression in MSC-2 cells,further supporting hypothesis that SCFAs mediate immune-modulatory effects of LBP3.Conclusion:LBP3 exerts its anti-tumor effects by promoting SCFA production,which subsequently inhibits function of PMN-MDSC.This highlights LBP3's potential as an immunomodulatory agent in cancer therapy.

Objective To observe the effects of sevoflurane inhalation anesthesia and spinal canal anesthesia on cogni-tive function in patients with urinary surgery in the elderly. Methods Researched 68 patients that undergoing elective urinary surgery in elderly patients, and then randomly divided into the sevoflurane inhalation anesthesia group (group A) and spinal canal anesthesia group (group B). The two groups were recorded time of anesthesia operation, surgical blood loss and transfusion volume, low blood pressure and the number of hypoxemia occurred, at last used the simple mental state examination (MMSE) to evaluate two groups of patients with 1 d before anesthesia and postoperative 1 d, 3 d, 7 d cognitive function. Results Two groups of anesthesia operation in time, surgical blood loss and transfusion volume, low blood pressure and the number of hypoxemia occurred had no significant differences(P>0.05). Two groups of postoper-ative 1 d, 3 d MMSE score were significantly lower than before operation, and the group A was significantly lower than group B, the differences were statistically significant(P <0.05);MMSE of A group ofter treatment for 7days were signifi-cantly lower than before the operation, but there was no significant difference compared with controls of groupA ofter treatment for 7 days (P>0.05); The incidence of POCD postoperative 1 d and 3 d in groupA were significantly higher than that of group B, with significant difference (P<0.05), and the incidence of POCD difference of two groups of post-operative 7 d was not significant (P >0.05). Conclusion Sevoflurane inhalation anesthesia is spinal canal anesthesia cognitive dysfunction occurred more often in patients with elderly urinary surgery.

Objective To explore the optimal experiment conditions of CCK-8 and MTS for cell proliferation assays in human amniotic epithelial cells and to evaluate the cytotoxicity of these reagents. Methods Human amniotic epithelial cells (hAECs) in logarithm growth stages were prepared in different cell concentrations with DMEM/F12 and 10% FBS. The sensitivity and optimal wavelengths was determined based on the optical density (OD) measured at 450 nm and 492 nm. The optimal time was determined under the conditions of the same cell concentration and defined OD values. HAECs were treated with DMSO, CCK-8 and MTS for 1 h, 2 h, 3 h, and 4 h, respectively. 24 h later, cytotoxicity of the CCK-8 and MTS was evaluated by determination of cell proliferation and Trypan Blue staining. Results The optimal detection wavelength was 450 nm for CCK-8, and 492 nm for MTS. The sensitivity of CCK-8 was slightly lower then that of MTS. The optimal time for incubation hAECs with CCK-8 was 4 h within 1~4 h. The inhibitory on cell proliferation and cytotoxicity of CCK-8 were weaker then those of MTS. Conclusion CCK-8 is a convenient reagent with low cytotoxicity for detection of the proliferation of hAECs.

[Objective] To propose a criterion for making conclusions on paternity tests based on STR genotyping. [Method] To use binomial distribution formula to calculate minimal numbers of STR loci that must be tested for different scenarios in paternity testing. [ Results ] We proposed a set of criteria for making STR paternity testing conclusions. For triplet tests, concluded "paternity positive" for the following four cases when the cumulative paternity index (PI) was greater than 10 000: 1) no inconsistent STR locus was detected in 15 loci (PE > 0.571 4/locus) or 2) only one inconsistent STR locus was detected in 19 loci or 3) only two inconsistent STR loci were detected in 28 loci or 4) only three inconsistent STR loci were detected in 35 loci; otherwise, concluded "paternity negative" when at least four inconsistent STR loci had been detected. For single parent tests, concluded "paternity non-exclusive" for the following cases when the cumulative PI was greater than 10 000: 1) no inconsistent STR locus was detected in 18 loci (PE>0.411/locus) or 2) only one inconsistent STR locus was detected in 29 loci or 3) two inconsistent STR loci were detected in 41 loci; concluded "paternity negative" when three or more inconsistent loci were detected. [Conclusion] Our experience has proven that these criteria are robust in STR paternity testing.

OBJECTIVETo study transforming growth factor-beta1 (TGF-beta1) autoproduction in keloid fibroblasts and the regulation effect of blocking TGF-beta intracellular signaling on rhTGF-beta1 autoproduction.

METHODSKeloid fibroblasts cultured in vitro were treated with either rhTGF-beta1 (5 ng/ml) or recombinant adenovirus containing a truncated type II TGF-beta receptor gene (50 pfu/cell). Their effects of regulating gene expression of TGF-beta1 and its receptor I and II were observed with Northern blot.

RESULTSrhTGF-beta1 up-regulated the gene expression of TGF-beta1 and receptor I, but not receptor II. Over-expression of the truncated receptor II down-regulated the gene expression of TGF-beta1 and its receptor I, but not receptor II.

CONCLUSIONSTGF-beta1 autoproduction was observed in keloid fibroblasts. Over-expression of the truncated TGFbeta receptor II decreased TGF-beta1 autoproduction via blocking TGF-beta receptor signaling.

Activin Receptors, Type I ; biosynthesis ; pharmacology ; Cells, Cultured ; Down-Regulation ; Fibroblasts ; drug effects ; metabolism ; Gene Expression ; Humans ; Keloid ; metabolism ; Protein-Serine-Threonine Kinases ; RNA, Messenger ; genetics ; metabolism ; Receptors, Transforming Growth Factor beta ; biosynthesis ; metabolism ; Sensitivity and Specificity ; Signal Transduction ; Trans-Activators ; metabolism ; Up-Regulation