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Objective To investigate the effect of high concentration of Caerulomycin A(Cae A) on HK2 in renal tubular epithelial cells and to explore the role of cytoplasmic nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3) in this process. Methods The effect of different concentrations of Cae A on the viability of HK2 cells was determined by MTT; the expression of kidney injury molecule(KIM-1) and NLRP3 was detected by real-time quantitative PCR, Western blot and immunofluorescence, while the effect of Cae A on the mRNA expression of IL-1β, IL-18, IL-33, MCP-1, TNF-α was also measured by real-time quantitative PCR. HK2 cells were divided into control group, high concentration of Cae A group and high concentration of Cae A plus NLRP3 inhibitor CY-09 group, and the expression of KIM-1 and NLRP3 protein was detected by Western blot. Results The results of MTT showed that high concentration of Cae A could inhibit HK2 cell viability. Real-time quantitative PCR, Western blot and immunofluorescence assays showed that high concentration of Cae A upregulated the expression of KIM-1 and NLRP3, as well as the mRNA levels of IL-1β, IL-18, IL-33, MCP-1, TNF-α, while CY-09 could down-regulate the expression of NLRP3 and KIM-1. Conclusion High concentration of Cae A significantly inhibited the viability of HK2 cells and induced damage and inflammatory response to HK2 with some nephrotoxicity that might be achievedviaNLRP3 pathway.

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Objective To investigate the biological role of aqueous-soluble components of sporoderm-removed Ganoderma lucidum spore powder(A-GSP) in head and neck squamous cell carcinoma(HNSCC). Methods The effects of A-GSP on the proliferation and metastasis of head and neck squamous cell carcinoma cells SCC15 and SCC25 were investigated using MTS, plate cloning, scratch healing experiments, and chamber penetration experiments. The effect of A-GSP on the apoptosis ability and cell cycle distribution of head and neck squamous cell carcinoma cells was detected using a flow cytometry assay kit. Western blot was used to detect the expression changes of epithelial mesenchymal transformation(EMT) molecules and cell cycle proteins after A-GSP treatment. Results After the action of A-GSP, the proliferation ability of head and neck squamous cell carcinoma cells decreased(F=62.72, P<0.000 1), the metastasis ability decreased(F=95.7, P<0.001), the apoptosis ability increased(F=87.65, P<0.001), and the G2/M phase stagnated(F=15.31, P<0.01). In addition, A-GSP treatment resulted in upregulation of epithelial cell markers E-cadherin, downregulation of mesenchymal cell markers Vimentin, N-cadherin, Snail, Slug, downregulation of cell cycle kinase CDK1, and upregulation of P21 and P27. However, it did not cause changes in the levels of apoptosis pathway proteins Caspase and Bax. Conclusion A-GSP inhibits the proliferation and metastasis of head and neck squamous cell carcinoma cells by inducing G2/M phase arrest and inhibiting EMT.

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Objective To explore the role of Argonaute(Ago) gene and RNA-Dependent RNA Polymerase(RDRP) gene of Aspergillus flavus in the growth and development about the RNAi mechanism. Methods A. flavus Ago1, Ago2, RDRP1, RDRP3 gene mutant strains were constructed by homologous recombination. The growth and development of the mutant strains were observed on potato dextrose agar(PDA)+ uracil uridine(UU) medium inoculated with 3 μl 106CFU/mL spores. 200,400 μg cell wall pressure agent conidored(CR), 0.8 mol/L,1.6 mol/L osmotic pressure agent NaCl, 2 mmol/L,4 mmol/L oxidative pressure agent hydrogen peroxide(H2O2) and 0.01%,0.02% genomic damage agent methyl mesylate(MMS) were added to the Yeast extract Glucose Minimum(YGM)+UU medium to analyze the stress response of the mutant strains. Results A. flavus mutant strains about ΔAgo1, ΔAgo2, ΔRDRP1, ΔRDRP3 were successfully constructed and its growth and development were normal. The ΔAgo1 and ΔAgo2 strains reduced the stress effects on cell wall and osmotic pressure compared to the control. Ago1 gene deletion reduced the effect of H2O2, and conversely RDRP3 gene deletion increased the inhibition of H2O2. The Ago2 and RDRP1 strains reduced the effect on genetic damage agent. In addition, ΔRDRP1 increased the effect of osmotic stress. Conclusion The Ago1, Ago2, RDRP1 and RDRP3 genes of A. flavus are not involved in the regulation of growth rate and asexual reproduction and can participate in the regulating of the host stress response to the environment.

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Objective The purpose of this study was to investigate the effects of plasma exosome-derived miR-29b-3p in myocardial ischemia-reperfusion injury(MIRI) rats on hypoxia/reoxygenation(H/R) cardiomyocyte after sevoflurane(SEV) postconditioning through targeting IGF1. Methods The GEO database was used to screen differentially expressed miRNAs in MIRI, and cardiomyocytes were treated with H/R to construct a MIRI cell model. The expression of miR-29b-3p and IGF1 in the MIRI cell model post-treated with SEV was intervened, and then the survival rate of cardiomyocytes was detected by MTT, apoptosis was detected by flow cytometry, and inflammatory factors(IL-1β and TNF-α) in cardiomyocytes in each group were detected by ELISA. Results Compared with Normal group, the expression of miR-29b-3p in plasma exosomes of MIRI rats was enhanced(P<0.05), and the target-binding relationship between miR-29b-3p and IGF1 was confirmed(P<0.05). After SEV post-treatment, the expression of miR-29b-3p in H/R-stimulated cardiomyocytes decreased, while the expression of IGF1 increased(both P<0.05). Overexpression of miR-29b-3p in plasma exosomes could significantly inhibit the survival rate of H/R cells after SEV treatment, aggravate apoptosis and inflammatory response, while knockdown of miR-29b-3p showed a opposite effects(all P<0.05). The rescue experimental data showed that overexpression of IGF1 could partially reverse the effects of overexpression of miR-29b-3p on H/R cell injury after SEV treatment(all P<0.05). Conclusion Plasma exosome-derived miR-29b-3p promotes H/R cardiomyocyte injury after SEV treatment by targeting IGF1.

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Objective To the effects and potential mechanisms of ST3GAL5 on biological behaviors of Bladder Urothelial Carcinoma(BLCA). Methods Differentially expressed genes related to bladder cancer were identified using microarray analysis. Suitable bladder cancer cell lines were then screened. In vitro experimental measurements, including CCK8, EdU, colony formation assays, transwell migration, flow cytometry apoptosis experiments, scratch assay, were used to evaluate the effects of ST3GAL5 on biological behaviors of BLCA. ST3GAL5 gene Kyoto Encyclopedia of Genes and Genomes(KEGG), gene set enrichment analysis(GSEA) were analyzed using The Cancer Genome Atlas(TCGA) database.Finally, Western blot technology was used to verify the classical proliferation and metastasis related pathway factors. Results The combination of bioinformatics analyses and experimental measurements demonstrate that ST3GAL5 expression is aberrantly down-regulated in human cell lines of BLCA. Through Cancer Cell Line Encyclopedia(CCLE) database, HT-1376 cell lines were successfully screened for vitro test. Upregulation of ST3GAL5 was found to suppress the malignant biological behaviour of bladder cancer. GSEA enrichment analyses exhibited that ST3GAL5 and its co-expressed genes inhibited cell proliferation, invasion and metastasis of bladder urothelial carcinoma by activation of the PPAR pathway and inhibition of the PI3K/AKT pathway. The results of Western blot experiments verified that the key proteins of the PPAR signaling pathway showed a significant increase and the key proteins of the PI3K/AKT signaling pathway showed a significant decrease(P<0.05) after ST3GAL5 overexpression in bladder cancer. Conclusion ST3GAL5 gene might act as an oncogenic suppressor gene in bladder cancer, possibly inhibit the proliferation, invasion and metastasis of bladder cancer cells by activating the PPAR signaling pathway and inhibiting related molecules in the PI3K/AKT signaling pathway.

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Objective To investigate the effects of D-allose on the restoration of neurological function, Galectin-3(Gal-3), adenosine monophosphate-activated protein kinase/mammalian target of rapamycin(AMPK/mTOR) and the expression of some inflammatory factors in ischemia-reperfusion injury(CIRI) mice. Methods A total of 50 male mice were randomly divided into control group(Con group), sham group(Sham group), cerebral ischemia-reperfusion injury group(MCAO group), cerebral ischemia-reperfusion injury+D-alolose group(MCAO+D-allose group) and cerebral ischemia-reperfusion injury+modified citrus pectin group(MCAO+MCP group). The middle cerebral artery occlusion/reperfusion(MCAO/R) model(reperfusion after 2 hours of MCA ischemia) was established by thread embolism. After successful modeling, the neurological function of mice was evaluated Longa score and rotated rod walking. TTC staining was used to observe the volume of cerebral infarction foci. The expression levels of Gal-3 and autophagy-related molecules were detected by Western blot and RT-PCR. Immunofluorescence was applied to detect the distribution of Gal-3 in brain tissue, and TNF-α, IL-8 secretion was detected with ELISA KIT. Results Compared with Con group and Sham group, the MCAO model represented significant increase in the Longa neurofunction score(P<0.01), cerebral infarction volume(P<0.01), Gal-3 expression and manifasted enhanced autophagy(P<0.01). After treatment with D-allose, it could significantly improve neurological dysfunction, reduce cerebral infarction volume(P<0.01), reduce the expression of Gal-3(P<0.01), inhibit AMPK phosphorylation, promote mTOR phosphorylation, and inhibit autophagy(P<0.01). The use of the Gal-3 inhibitor MCP alone could also achieve the effect of inhibiting autophagy. Conclusion D-allose can effectively promote the recovery of neurological function and reduce the volume of infarct foci in CIRI mice. The mechanism may involve inhibiting excessive cell autophagy by downregulating the expression of Gal-3, and reducing the release of inflammatory factors such as TNF-α and IL-8, thereby exerting neuroprotective effects.

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Objective To detect the low-affinity penicillin-binding protein drug resistance, pbp4 gene, and multi-locus sequence typing(MLST) of clinically isolated E. faecalis. Methods 78 clinical isolates of E. faecalis were collected, and their drug resistance was detected by automated instruments; the mutation of pbp4 gene mutation was analyzed by PCR amplification and MLST. Results 78 strains of E. faecalis were highly resistant to ciprofloxacin, levofloxacin, rifampicin, erythromycin, tetracycline and high concentration of gentamicin, and were resistant to penicillin and gentamicin. The ampicillin resistance rate was 10.3%, and no strains were found to be resistant to nitrofurantoin, vancomycin, teicoplanin and linezolid; 8 strains of 78 E. faecalis had amplified TEM genes, and all of them were resistant to penicillin and ampicillin resistance, with a positive rate of 10.3%; the allelic profiles and sequence types of 78 strains of E. faecalis which were divided into 16 sequence types, of which ST179 and ST16 were the most, with 21 and 21 strains, respectively. 20 strains, accounting for 26.9% and 25.6%, the rest were ST6 type 8 strains(10.3%), ST4 type 7 strains(9.0%), ST585 type 6 strains(7.7%), ST480 type 4 strains(5.1%), ST28 strains 3 strains(3.8%) of the ST type were detected, and only 1 strain was detected for the other ST types. The analysis of the relationship between ST types and drug resistance showed that E. faecalis with the same ST type had similar drug resistance profiles. Conclusion The resistance mechanism of E. faecalis to β-lactam antibiotics is mainly caused by the production of β-lactamase mediated by TEM gene, which is not necessarily related to the mutation of pbp4 gene. The isolates of E. faecalis are mainly CC16(including ST16 and ST179) clones and drug resistance is serious. It is necessary to guide clinical medication and strengthen nosocomial infection monitoring according to its characteristics.

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Objective To explore whether the long-chain noncoding RNA associated with genomic instability in colon cancer can predict clinical prognosis and therapeutic.Methods The R package " limma" was used for differential analysis,and the prognostic risk model was constructed by univariate Cox analysis and multivariate Cox proportional risk regression analysis.The difference in prognosis was evaluated by Kaplan-Meier method,and the difference was significant by log-rank test.The efficiency of the prognostic model was evaluated using a time-dependent area under the subject operating characteristic curve(AUC).The R package "pRRophetic" was used to predict the sensitivity of patients to anticancer drugs.R software package rms was used to build a line graph,and the consistency index of the line graph was calculated.Real-time quantitative PCR was used to detect the difference in the expression levels of prognostic protective factors.Results A total of 22 LncRNAs associated with genomic instability in patients with prognosis were obtained,2 were protective factors for prognosis in patients with colon cancer,and 20 were risk factors for prognosis.A prognosis model composed of LncRNAs associated with genomic instability was constructed,and patients with high risk scores had lower AUCs and shorter median survival.The five-year survival AUC predicted by the model was 0.823 in the training set,0.722 in the validation set,and0.759 in the overall TCGA colon cancer patient population.Patients with low risk scores had lower half inhibitory concentration(IC50) of cisplatin and higher sensitivity(P<0.000 1).The expression of prognostic protective factors in colon cancer tissues was significantly lower than that in adjacent colon cancer tissues.Conclusion A prognostic risk model composed of 8 LncRNAs associated with genomic instability was constructed and verified.In addition,the model can also predict cisplatin drug sensitivity.A histogram was constructed combining the tumor stage and the prognosis model.The predictive ability of this graph for five-year survival of colon cancer patients is better than that of traditional histopathological features and prognostic models constructed by predecessors.

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Objective To explore the differences in the effects of psoriatic serum and a mixture of five proinflammatory cytokines on keratinocyte inflammation and proliferation. Methods The human immortalized keratinocyte cell line(HaCaT cells) was used to cultivate with serum of healthy human serum, psoriasis patients and M5 factors, and the cell proliferation was monitored by CCK-8. RT-qPCR was used to detect the mRNA expression levels of inflammatory factors interleukin 1β/8/21/23(IL-1β/8/21/23), proliferation markers keratin 6/16(K6/K16) and nucleus related antigen 67(Ki67) in HaCaT cells in each group. Results Healthy human serum, psoriasis serum and M5 factors could effectively promote the growth of HaCaT cells, and psoriasis serum and M5 factors could promote cell growth more than healthy human serum. Similar to M5 factors, serum from patients with psoriasis significantly promoted the mRNA levels of inflammatory factors IL-1β, IL-8, IL-23 and proliferation markers K6 and Ki67 in cells. Different from M5 factors, the serum of psoriasis patients had enhanced the mRNA level of inflammatory factor IL-21, but weakened the promotion of proliferation marker K16. Conclusion The serum microenvironment of patients with psoriasis can promote the proliferation and inflammatory response of keratinocytes higher than that of healthy people, and is similar to M5 factors, which can be used to build a keratinocyte model of psoriasis.

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Objective To observe the effect of hypoxia on pituitary adrenal axis in rats and the therapeutic effect of Gynostemma pentaphyllum saponins(GPs)intervention. Methods 50 male SD rats were randomly divided into normoxia control group, hypoxia control group, and hypoxia + GPs low, medium, and high dose intervention groups, with 10 rats in each group. Rats in the normoxia group were kept in a normoxia animal room for 28 days, while rats in the hypoxia group and drug intervention group were kept in a low-pressure oxygen chamber for 28 days. Among them, hypoxia+GPs low, medium and high dose groups of rats were gavaged daily with GPs of 40 mg/kg, 80 mg/kg, and 160 mg/kg, respectively. After 28 days, rats were killed in batches, their body weight and pituitary weight were measured, and pituitary index was calculated; HE staining method was used to observe the morphology and structure of rat pituitary gland; immunohistochemical and Western blot methods were used to observe the expression level of pituitary adrenocorticotropin(ACTH)protein in rats; ELISA method was used to observe serum ACTH and corticosterone(CORT)concentrations in rats. Results Hypoxia had no significant effect on pituitary gland morphology and quality in rats. Hypoxia might induce the expression of rat pituitary ACTH protein and an increase in serum ACTH and CORT concentrations, while GPs might reduce the expression of rat pituitary ACTH protein and serum ACTH and CORT concentrations induced by hypoxia. Conclusion GPs have a significant effect on the pituitary adrenal axis induced by hypoxia in rats, and there is a dose-effect relationship between GPs and effect.

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Objective Based on metabolomics and transcriptomics analysis, to explore the molecular mechanism of spleen inflammation induced by high altitude hypoxia in mice through NOD-like receptor signaling pathway. Methods C57BL/6 mice were raised at an altitude of 400 m and 4 200 m respectively, with five mice in each group, and spleen tissues were collected after 30 days. Differential metabolites and differentially expressed genes in key pathways were screened by metabolomics and transcriptome analysis and correlation KEGG enrichment analysis, and the related network interaction diagram of differential metabolites and differentially expressed genes in key pathways was constructed and verified by RT-qPCR. Results Metabolomics analysis showed that 133 differential metabolites were screened from in the plain spleen control group(PSC group) and the plateau spleen test group(HST group), 95 of which were up-regulated while 38 of which were down-regulated. KEGG enrichment analysis showed that they were mainly involved in NOD-like receptor signaling pathway, HIF-1 signaling pathway, cholesterol metabolism and other metabolic pathways. The results of transcriptome analysis showed that a total of 4213 differentially expressed genes were identified in PSC group and HST group, including 1947 up-regulated genes and 2266 down-regulated genes. KEGG was enriched in 173 signaling pathways, including NOD-like receptor signaling pathway, MAPK signaling pathway, NF-κB signaling pathway and other pathways. Comprehensive analysis showed that the differential metabolites and differentially expressed genes were obviously enriched in NOD-like receptor signaling pathway. Therefore, the correlation network interaction map was constructed for the differential metabolites ATP and differentially expressed genes in NOD-like receptor signaling pathway. RT-qPCR results showed that compared with PSC group, the expression levels of DEGs related to NOD1 and NOD2(CHUK, TAB3, MAPK8) in the signaling pathway of NOD-like receptor and NLRP1-CASP1 pathway(NLRP1b, CASP1) in HST group were significantly enhanced. The mRNA expression levels of downstream inflammatory factors IL-6, IL-1 β, IL-18, INF-γ and TNF-α were up-regulated and differentially expressed. Conclusion Based on the combined analysis of metabolomics and transcriptomics, it was found that hypoxia stimulation at high altitude may affect the NOD-like receptor signaling pathway in vivo, and the differential metabolite ATP is positively correlated with the differential key genes in the pathway. ATP mediates the release of downstream inflammatory factors by activating NOD1, NOD2 pathways and NLRP1 inflammable-CASP1 pathways. Inflammatory response occurred in spleen tissue of mice.

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Objective To investigate the effects of Lin28 overexpression on the proliferation and osteogenic differentiation of human dental pulp stem cells(hDPSCs) through mTOR signaling pathway. Methods After transfecting lentiviral vectors of Lin28 gene in hDPSCs, the relative expression of Lin28 was detected by Real-time PCR. CCK-8 assay was applied to detect the effect on cell proliferation. qRT-PCR was used to research the expression levels of alkaline phosphatase(ALP), osteopontin(OPN) and osteocalcin(OCN). Western blot assay was processed to investigate the effects on the relative expression levels of ALP and OPN proteins. Alizarin red staining was utilized to detect the mineralized nodules. Results Compared with the control group, the cell proliferation of transfection group was promoted(P<0.05); The mRNA and protein expression levels of ALP, OPN and OCN in transfection group were significantly lower than those in control group(P<0.05), the expression level of ALP apparently decreased after the addition of mTOR inhibitor rapamycin(P<0.05); Alizarin red staining showed that the size and number of mineralized nodules formed in transfection group were markedly declined compared with empty carrier group(P<0.05). Conclusion Overexpression of Lin28 can inhibit the osteogenic differentiation of hDPSCs through suppress mTOR signaling pathway.

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Objectives To investigate the effect of sciatic nerve electrical stimulation(SNES) on motor function recovery in rats with incomplete spinal cord injury(SCI) and its possible mechanism. Methods The incomplete SCI model was constructed by modified Allen′s method. Forty-five Sprague-Dawley(SD) rats were randomly divided into three groups: Sham, SCI, and SNES. Electrical stimulation parameters were 1 ms pulse width and 100 Hz, with 20 mins each time, once a day for 21 days. The motor function was assessed by Basso-Beattie-Bresnahan(BBB) locomotor function scale, and the action potential conduction was detected by electrophysiology. Hematoxylin-eosin(HE) staining was used to observe the pathological changes in the spinal cord and the average cross-sectional area(CSA) of biceps femoris muscle fibers. The number of brain-derived neurotrophic factor(BDNF) and tropomyosin-related kinase B(TrkB) positive cells, relative mRNA and protein expression were analyzed by immunohistochemistry, reverse transcription polymerase chain reaction(RT PCR) and Western blot separately. Results On 21 d, the BBB score and average amplitude of action potential of SNES group were higher than those of SCI group, and the difference was statistically significant(P<0.05). Compared with SCI group, the pathological injury of spinal cord tissue in SNES group was significantly improved, and the average CSA of biceps femoris muscles had a statistical difference(P<0.05). The number of BDNF and TrkB positive cells in SNES group was higher than that in SCI group, and there were statistical differences(P<0.05). The relative mRNA and protein expressions of BDNF and TrkB in SNES group were higher than those in SCI group, with statistical differences(P<0.05). Conclusion These results show that SNES contributes to alleviating spinal cord tissue injury, promoting the recovery of motor function and delaying the atrophy of muscles below the injury level. The possible mechanism is related to the increased expression of BDNF-TrkB proteins.

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Objective To study the protective effect and mechanism of iPSC-MSCs on cartilage matrix in knee osteoarthritis(KOA) patients in vitro. Methods Cartilage tissues removed from KOA patients with joint replacement surgery were collected and subjected to tissue and cellular experiments, respectively. Cartilage tissue was cut into small pieces and randomly divided into a control group, an IL-1β(10 ng/ml) induction group, and iPSC-MSCs groups. Except for the control group, cartilage tissues from each group were stimulated with IL-1β(10 ng/ml) for 96 h and then co-cultured with different amounts of iPSC-MSCs(1×104, 1×105, 1×106) cells for 72 h. For in tissues, the pathological changes of isolated cartilage tissues were examined by HE staining. The levels of ADAMTS-4, ADAMTS-5, and type II collagen expression were analyzed by immunohistochemistry, while the levels of MMP13, IL-6, and IL-10 in culture supernatants were detected by ELISA kits. The 2 to 5 generations of chondrocytes, which were extracted from cartilage tissue of KOA patients, were stimulated with IL-1β(10 ng/ml) for 48 h and then co-cultured with different concentrations of iPSC-MSCs(1×104, 1×105, 1×106) cells for 72 h. Immunofluorescence and Western blot detected the expression of RUNX2, ADAMTS-4, and ADAMTS-5 in chondrocytes. Results Comparison with the control group, in the IL-1β-induced group, the levels of RUNX2, ADAMTS-4, and ADAMTS-5 increased, the level of type II collagen decreased, the levels of MMP-13 and IL-6 in the culture supernatant increased(P<0.05), and the level of IL-10 decreased(P<0.05); Compared with the IL-1β-induced group, co-culture of different numbers of iPSC-MSCs reduced the levels of MMP-13 and IL-6 in the supernatant, decreased the expression of RUNX2, ADAMTS-4, and ADAMTS-5, promoted type II collagen expression and elevated IL-10 levels. Conclusion iPSC-MSCs inhibited ADAMTS-4 and ADAMTS-5 expression in vitro, reduced cartilage extracellular matrix degradation, and played a role in articular cartilage protection.

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Objective To explore the regulatory role of microRNA-455-3p(miR-455-3p) in lymphangiogenesis of rat silicosis model,and to investigate the effect of miR-455-3p targeted regulation of vascular endothelial growth factor C(VEGF-C) on the tubular structure formation of human lymphatic endothelial cells(HLECs).Methods The rats were randomly divided into the silicosis model group and the normal control group.The silicosis model group were injected with silicon dioxide(SiO2) dust suspension,and the control group was injected with the same amount of normal saline.HE,Masson and immunohistochemistry staining were used to observe the pathological changes and lymphangiogenesis of lung tissue.The expression levels of miR-455-3p and VEGF-C in lung tissues of rats were detected by Quantitative real-time PCR(RT-qPCR) and Western blot;The miR-455-3p inhibitors and negative controls(NC) were transfected into HLECs,and the expression levels of miR-455-3p and VEGF-C in cells were detected by RT-qPCR and Western blot.The migration ability of HLECs was detected by scratch test,the ability of tubular structure formation was detected by matrigel tube formation test,and dual luciferase experiments were used to verify the targeting relationship between miR-455-3p and VEGF-C.Results Compared with the normal control group,in the silicosis model group,a large number of inflammatory cells gathered and collagen gradually deposited in the pulmonary interstitium,and there was lymphatic hyperplasia in the lung.The expression of miR-455-3p in the lung tissue was lower than that in the control group,and the expression of VEGF-C was higher than that in the control group;After transfection with HLECs,compared with the NC group,the expression of miR-455-3p in the cells of the Inhibitors group decreased,the expression of VEGF-C increased,and the ability of cell migration and tubular structure formation increased(P<0.05);VEGF-C was confirmed as a target gene of miR-455-3p by the dual luciferase experiments.Conclusion miR-455-3p can affect the tubular structure formation ability of HLECs and regulate lymphangiogenesis by targeting the expression of VEGF-C.

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Objective To analyze the effect of miR-148b-3p on the proliferation of keloid derived fibroblasts. Methods The expression levels of miR-148b-3p and SPARC in human keloid derived fibroblasts(HKF) and normal human fibroblasts(NFS) were analyzed by real time PCR. The expression level of SPARC protein was detected by Western blot. The effects of miR-148b-3p and SPARC on HKF proliferation were analyzed by CCK-8 method and plate clone formation. The target gene of miR-148b-3p was predicted using online analysis software, and then the luciferase reporter plasmid was constructed. The targeted binding site of miR-148b-3p and the target gene was analyzed by luciferase reporter gene method. Results miR-148b-3p was low expressed in HKF and SPARC was high expressed in HKF. Transfection of miR-148b-3p in HKF cells could down regulate the expression of SPARC and inhibit cell proliferation. Online analysis software predicted that miR-148b-3p could target the 3′-UTR binding SPARC; The results of dual luciferase reporter gene further confirmed that miR-148b-3p could target the 3′-UTR of SPARC. Transfection of SPARC eukaryotic expression plasmid into HKF transfected with miR-148b-3p could counteract the effect of miR-148b-3p and restore cell proliferation. Conclusion miR-148b-3p can inhibit the proliferation of HKF by targeting the 3′-UTR of SPARC and inhibiting its expression.

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Objective To observe the effect of Methyltransferase like 3(METTL3) in cisplatin-induced acute kidney injury(AKI) in mice with hyperuricemia.Methods C57BL/6 mice were randomly divided into control group,hyperuricemia group,AKI group,and hyperuricemia+AKI group.The hyperuricemia model was established by injecting with gavage potassium oxyzinate and adenine for 21 days,and the AKI model was established by intraperitoneal injection of cisplatin for 3 days.Serum creatinine,urea nitrogen,blood uric acid,the pathological staining of HE and Masson were detected.The expression of alpha-smooth muscle actin(α-SMA),Kim-1 and METTL3 proteins were detected by Western blot.qPCR detected mRNA changes such as interleukin-6(IL-6),tumor necrosis factor(TNF-α),and IL-1β.The m6A assay detected overall methylation levels in mice.Results Compared with the AKI group alone,the serum creatinine,urea nitrogen and blood uric acid in the hyperuricemia+AKI group significantly increased.Pathological staining,Western blot,and qPCR also showed worsening of renal fibrosis and elevated METTL3 expression in the composite model group.The m6A test results showed that the overall methylation level of mice in the experimental group increased,and the composite model group was significantly higher than that in the AKI group.In vitro experiments,the METTL3-specific inhibitor STM2457 was given to effectively reduce the injury of renal tubular epithelial cells and reduce the level of inflammation and fibrosis.Conclusion Cisplatin can exacerbate renal inflammation and fibrosis caused by high uric acid;STM2457 has therapeutic effects in hyperuricemic nephropathy and cisplatin-induced acute kidney injury,and METTL3 may be a potential target for the treatment of related kidney injury.

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Objective To screen the RNA binding proteins interacting with NBV nonstructural protein σNS in host cells and to analyze their bioinformatics functions. Methods In this study, the eukaryotic expression vector pEF-HA-MB-S3 of NBV σ NS was constructed and transfected into HEK293T cells after verification. After RNase A treatment, the obtained protein lysate was enriched by immunoprecipitation to enrich σNS binding proteins, identified and analyzed by LC-MS/MS mass spectrometry, and the properties and specific functions of proteins were discovered with the help of related Bioinformatics tools. Results In this study, 32 candidate RNA binding proteins interacting with NBV σNS proteins were successfully screened, and the results of bioanalysis showed that these proteins were mainly located in cytoplasm and nucleus, and were mainly involved in biological processes such as cell metabolism, biological regulation, virus translation and transcription. Conclusion This study preliminarily analyzes the function of RNA binding proteins interacting with NBV σNS, which lays a foundation for further study on the mechanism of σNS protein in NBV life cycle.

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Objectives To investigate the expression and clinical significance of TRIM32 in Cutaneous Malignant Melanoma patients. Methods Real-time PCR was used to detect the expression of TRIM32 in human melanoma cell lines(A375,MV3) and human normal skin cell lines(HACAT). Clinical characteristics of 31 CMM patients were collected. The expression of TRIM32 in the CMM tissues and para-cancerous tissues was detected by immunohistochemistry(IHC). The correlation between TRIM32 expression and clinical characteristics was analyzed. Kaplan-Meier survival analysis and Cox univariate analysis model were used to analyze the prognostic factors. Results The expression of TRIM32 mRNA in A375 melanoma cells was significantly higher than that in HACAT normal skin cells(P<0.001),while the expression in MV3 cells was comparable to that in HACAT cells. The expression of TRIM32 protein in the CMM tissues, detected by IHC, was significantly higher than that in the para-cancerous tissues(P<0.001) and positively correlated with Breslow thickness(r=0.771,P<0.001) and Clark grade(r=0.635,P<0.001), but not with sex, ulcer, age and primary location of tumor(P>0.05). Also, TRIM32 expression level was significantly correlated with prognosis of CMM. Conclusion TRIM32 is highly expressed in A375 melanoma cells and CMM tissues. TRIM32 may be helpful for the prognosis of CMM patients.

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Objective To investigate the mechanism of sertoli/spermatogenic injury induced by cigarette smoke extract(CSE) based on ROS/NLRP3 signaling pathway.Methods The sertoli/spermatogenic of rat were cultured in vitro,and they were divided into control group,cigarette Smoke extract(CSE) group and CSE+NLRP3 inflammosome inhibitor MCC950 group.Cell viability was detected by CCK-8 assay;Lactate dehydrogenase(LDH) was used to detect cell membrane damage;reactive oxygen species(ROS) production was used to detect oxidative stress level;Hoechst/PI fluorescent staining was used to detect cell pyroptosis.Real-time quantitative PCR(RTqPCR) was used to determine the mRNA level of NLRP3,ASC,Caspase-1,GSDMD,IL-1β and IL-18.Western blot was used to determine the protein level of NLRP3,ASC,Caspase-1,GSDMD,IL-1β and IL-18.Results CCK-8 assay showed that cell viability decreased in CSE group(P<0.001),LDH leakage rate(P<0.001) and ROS(P<0.001) production increased.Compared to the CSE group,cell viability increased(P<0.001),LDH leakage rate(P<0.01) and ROS(P<0.001) production decreased in CSE+MCC950 group.The number of PI staining cells in CSE group was more than that in control group and CSE+MCC950 group,but staining cells in CSE+MCC950 group was less than that in CSE group.RT-qPCR and Western blot results showed that the expression levels of NLRP3,ASC,Caspase-1,GSDMD,IL-1β and IL-18 increased in CSE group but decreased in CSE+MCC950 group.Conclusion The oxidative stress and inflammation of sertoli/spermatogenic induced by cigarette smoke may be related to the activation of ROS/NLRP3 signaling pathway.

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Objective To investigate the mechanism of sertoli/spermatogenic injury induced by cigarette smoke extract(CSE) based on ROS/NLRP3 signaling pathway.Methods The sertoli/spermatogenic of rat were cultured in vitro,and they were divided into control group,cigarette Smoke extract(CSE) group and CSE+NLRP3 inflammosome inhibitor MCC950 group.Cell viability was detected by CCK-8 assay;Lactate dehydrogenase(LDH) was used to detect cell membrane damage;reactive oxygen species(ROS) production was used to detect oxidative stress level;Hoechst/PI fluorescent staining was used to detect cell pyroptosis.Real-time quantitative PCR(RTqPCR) was used to determine the mRNA level of NLRP3,ASC,Caspase-1,GSDMD,IL-1β and IL-18.Western blot was used to determine the protein level of NLRP3,ASC,Caspase-1,GSDMD,IL-1β and IL-18.Results CCK-8 assay showed that cell viability decreased in CSE group(P<0.001),LDH leakage rate(P<0.001) and ROS(P<0.001) production increased.Compared to the CSE group,cell viability increased(P<0.001),LDH leakage rate(P<0.01) and ROS(P<0.001) production decreased in CSE+MCC950 group.The number of PI staining cells in CSE group was more than that in control group and CSE+MCC950 group,but staining cells in CSE+MCC950 group was less than that in CSE group.RT-qPCR and Western blot results showed that the expression levels of NLRP3,ASC,Caspase-1,GSDMD,IL-1β and IL-18 increased in CSE group but decreased in CSE+MCC950 group.Conclusion The oxidative stress and inflammation of sertoli/spermatogenic induced by cigarette smoke may be related to the activation of ROS/NLRP3 signaling pathway.

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Objective To explore the microbial contamination status of household washing machines and evaluate the improvement effect of professional washing machine cleaning on microbial contamination.Methods Before and after cleaning,samples were collected asepticly from the outer wall of the inner cylinder,filter,sealing ring,detergent drawer box and washing water of 15 household washing machines,thus,a total of 150 samples were obtained.The total number of bacteria,fungi and coliform were detected to reflect the hygiene status of the washing machine.At the same time,Salmonella,Shigella and Candida albicans were isolated and cultured to reflect the contamination status of pathogenic bacteria.According to the detection and clearance rate of microorganisms,the cleaning effects of washing machine were comprehensively analyzed.Results Before washing,the total number of bacteria in the washing machine ranged from 1.25 × 103 to 1.19 × 106 CFU/cm2.After washing,the total number of bacteria in the washing machine ranged from 0.01 to 375 CFU/cm2.The bacterial clearance rates of the outer wall of the inner cylinder,the filter,the sealing ring,the washing water and the detergent drawer box were 99.93%,99.99%,99.99%,99.74% and 99.86%,respectively.Before washing,the total number of fungi in the washing machine ranged from 8.5 to 9.45 × 103CFU/cm2.After washing,the total number of fungi in the washing machine ranged from 0 to 49.22 CFU/cm2.The fungal clearance rates of the outer wall of the inner cylinder,filter screen,seal ring,washing water and detergent drawer box were 99.36%,99.74%,99.76%,99.51% and 99.64%,respectively.Before washing,the coliforms in the washing machine ranged from 0 to 4.6 × 104MPN/100 ml.After washing,the number of coliform bacteria was close to 0.Before washing,the average detection rate of Salmonella in the washing machine was 5.33%,the average detection rate of Shigella was 2.67%,and no Candida albicans was detected.After washing,Salmonella,Shigella and Candida albicans were not detected.Conclusion There are a large number of bacteria and fungi in the household washing machine before cleaning,and there are Salmonella and Shigella contamination.After professional cleaning,the hygiene condition of the washing machine is significantly improved.

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Objective Viral load is becoming more and more important in prevention of HIV/AIDS. Understanding the baseline of viral load before treatment in newly reported HIV/AIDS patients can provide a basis for AIDS prevention. Methods The viral load of newly reported HIV/AIDS patients in Jiangsu province in 2019 was detected by fluorescence quantitative PCR, the differences of viral load under different demographic characteristics were compared, and the factors of high viral load were analyzed. Results The mean viral load of 3098 newly reported HIV/AIDS patients in Jiangsu province was 4.33±1.05 log/mL, and the proportion of patients with viral load<5000 copies/mL was 20.85%(646/3 098). There were statistically significant differences in viral load among different genders(P<0.05), and male patients had higher viral load. The difference of viral load between HIV-infected and AIDS patients was statistically significant(P<0.05), and the viral load value of AIDS patients diagnosed with disease status was relatively high. Unmarried and married spouses were more likely to have higher viral load than divorced or widowed spouses. Higher baseline viral load level was associated with higher rates of virologic failure after antiviral therapy. Conclusion The overall viral load of HIV infected/AIDS patients is high, but there is a certain proportion of low viral load, suggesting that follow-up of low viral load should be strengthened in AIDS prevention and treatment. Strengthen interventions aimed at men and unmarried or married people with spouses to reduce the risk of transmission.

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Objective To explore the application value of metagenomic second-generation sequencing(mNGS) in patients with suspected central nervous system(CNS) infection in the general intensive care unit(ICU), and to provide reference for rapid and accurate diagnosis of central nervous system infection patients in the general ICU. Methods The data of 82 patients who underwent cerebrospinal fluid mNGS examination in the general ICU of our hospital from 2018 to 2021 were collected. According to the inclusion criteria, 52 patients with suspected CNS infection, who had undergone routine biochemical and culture testing as well as mNGS on cerebrospinal fluid samples, were included in the final data analysis. The clinical diagnosis of CNS infection was taken as the "gold standard", and the application value of the two methods, traditional culture and mNGS, for clinical diagnosis were compared. Results Among the 52 patients, 32 were finally diagnosed with CNS infection, 24 of them were positive for mNGS in cerebrospinal fluid, and 5 were positive in culture of cerebrospinal fluid. The sensitivity and specificity of the two methods were 75.00% vs 15.63% and 55.00% vs 95.00%, The negative predictive values were 72.73% vs 83.33%, and 57.89% vs 41.00%, respectively; there was no significant difference in the detection rates between the two methods(P>0.05). Of the 32 patients with CNS infection, 14 had bacterial infection, 9 had viral infection, 2 had fungal infection and 7 had other pathogenic bacteria infection. The sensitivity of mNGS to detect pathogens of viral infection was 66.70%, the specificity was 95.30%, and the positive predictive value was 75.00%, and the negative predictive value was 93.20%, which was highly consistent with the gold standard of clinical final diagnosis(Kappa value=0.649, P<0.01). Conclusion Cerebrospinal fluid mNGS has a higher diagnostic accuracy for central viral infection compared to bacterial infection, and it is recommended to use mNGS for rapid screening of patients with suspected central virus infection.

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Objective To investigate the clinical application value of chemokine ligand(CCL)21 and its receptor chemokine(CCR)7 in patients with anti-neutrophil cytoplasmic antibody associated vasculitis.Methods The serum levels of CCL21 and its receptor CCR7 were measured by ELISA methods in 102 patients with AASV and 70healthy controls.At the same time,the correlation among the CCL21,CCR7,MPO,PR3 and general laboratory index of TNF-α,IL-6,BUN,CRE,C3,C4 ESR,CRP,BVAS in AASV patients was analyzed.Results The serum levels of the CCL21 and CCR7 were significantly higher in AASV patients than that in healthy controls [(276.95±57.34) vs(179.91±19.45);(85.85±18.77) vs(54.83±18.18) pg/ml](P<0.05).The serum levels of the CCL21 and CCR7 in MPO positive patients were higher than those in MPO negative patients [(294.94±58.15) vs(244.35±46.76);(90.64±18.38) vs(78.37±13.44) pg/ml](P<0.05).Moreover,The levels of the CCL21 and CCR7 were similar to those of TNF-α,IL-6,CRE,BUN,CRP,BVAS and MPO in AASV patients,the levels of the CCL21 and CCR7 in AASV patients were positively correlated.The serum levels of the CCL21 and CCR7 were found to be predictive of AASV activity with ROC.Conclusion The serum level of CCL21 and CCR7in AASV patients increased significantly,especially in MPO positive patients,and related to patients with renal function index and related inflammation index.Serum CCL21 and CCR7 are the influencing factors of BVAS in AASV patients,and have important significance in the diagnosis and judgment of AASV disease.

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Objective To establish a Nomogram clinical predictive model of liver failure(PHLF) after hepatectomy for hepatocellular carcinoma(HCC) based on ICG-R15. Methods Clinical data of 219 patients with hepatocellular carcinoma who underwent radical hepatectomy in the second affiliated Hospital of Anhui Medical University from February 2017 to March 2022 were analyzed retrospectively. The influencing factors of PHLF were screened by Logistics regression and the Nomogram model for predicting the occurrence of PHLF was established. Compared with the traditional model, its predictive efficiency was tested. Results In this study, ICG-R15(OR=1.07, P<0.05), PLT(OR=0.99, P<0.05), INR(OR=1.50, P<0.05), HBV-DNA>1000IU(OR=2.26, P<0.05), AFP>400ng/L(OR=2.60, P<0.05) and open surgery(OR=0.26, P<0.05) were the influencing factors of PHLF. A Nomogram prediction model was established based on these factors, and through ROC and DCA analysis, it was found that the prediction efficiency and clinical value of this model were better than those of the traditional model. Conclusion The Nomogram clinical predictive model of hepatocellular carcinoma(HCC) PHLF is successfully established based on ICG-R15.

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Objective To explore the effects of Er: YAG laser irrigation technology on smear layer removal and dentin microhardness. Methods A total of 102 ex vivo premolars were selected to amputate the crown, leaving a root length of 13 mm, after mechanical preparation, there were divided into experimental group(3% sodium hypochlorite, 3% NaOCl) and control group(0.9% sodium chloride solution, NS), and received syringe, ultrasound and Er: YAG laser terminal irrigation, which were specifically grouped into NaOCl+syringe, NaOCl+ultrasound, NaOCl+laser, NS+syringe, NS+ultrasound and NS+laser groups(n=17). Scanning electron microscopy(SEM) evaluated smear layer removal from the upper, middle and apical parts of the root. The Vickers microhardness tester measured the microhardness before and after irrigation. Results The NaOCl+laser group had the best effect on removing the root canal smear layer, and the amplitude of reducing the microhardness of dentin was the largest. There was no significant difference in the reduction of microhardness compared with the NaOCl+ultrasonic group(P>0.05). Conclusion Er: YAG laser-activated NaOCl solution improves the clearance of the smear layer and reduced the microhardness of dentin.