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Objective Cas9-RNP biomimetic nanoparticles cas9-RNP@MMs were prepared by encapsulating the Cas9 Ribonucleoprotein complex(RNP) using mouse macrophage membranes, with the aim of utilizing this biomimetic nanoparticle to deliver the Cas9-RNP complex for gene editing, and further study the endocytosis of Cas9-RNP@MMs and its gene editing effect in mouse macrophage RAW264.7in vitro, providing evidence for the development of low-toxicity biomimetic nanoparticle carriers that inhibitNLRP3 therapeutic targets. Methods The purified mouse macrophage membrane was mixed with the prepared cas9-RNP mixture, and after ultrasound, the CAS9-RNP@MMS was obtained by liposome extrusion instrument; The particle size of Cas9-RNP@MMs was measured by nanoparticle tracking analysis, and the particle morphology of Cas9-RNP@MMs was observed under transmission electron microscope. Laser confocal Fluorescence microscope imaging was used to analyze the endocytosis Cas9-RNP@MMs.The Biocompatibility of Cas9-RNP@MMs was measured by MTT assay. The expression ofNLRP3 was detected by qPCR and Western blot to verify the knockdown effect of Cas9-RNP@MMs onNLRP3 gene. Results The average particle diameter of Cas9-RNP@MMs prepared from macrophages was about 216 nm. Under laser confocal fluorescence microscope, the Cas9-RNP@MMs could be successfully endocysed by Raw246.7 cell. MTT assay indicated that the Cas9-RNP@MMs-treated mouse macrophage RAW246. 7 had good biocompatibility. qPCR and Western blot showed that twoNLRP3-specific guide RNA were mediated by Cas9-RNP@MMs, with good effect of knockdownNLRP3 gene expression. Conclusion Nano-scale vesicles Cas9-RNP@MMs loaded with Cas9-RNP complexes were successfully prepared by biomimetic nanoparticles. Cas9-RNP@MMs have good biocompatibility and can be efficiently endocytosed by RAW246.7 cells. Cas9-RNP@MMs containingNLRP3-specific sgRNA can specifically knock downNLRP3 gene expression.

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Objective To obtain chimeric antigen receptor macrophages(CAR-M) targeting HER2 stably transfected. Methods CAR lentivirus vector targeting HER2 was constructed and infected with human monocytic leukemia cell line(THP-1). CAR THP-1 cells with green fluorescent labeling were selected by sorting flow cytometry and continued to be culturedin vitro. The CAR THP-1 cells targeting HER2 were co-cultured with the endometrial cancer cell line Ishikawa with negative and positive HER2 expression, and their targeted phagocytosis of CAR-M to HER2 positive tumor cells was detected by imaging flow cytometry, and the targeted phagocytosis efficiency of CAR-M to HER2 positive tumor cells was detected by flow cytometry. Results CAR lentivirus infection with THP-1 cells was less efficient; After co-culture with cancer cells, flow cytometry and imaging flow cytometry showed that CAR THP-1 cells had enhanced phagocytosis of HER2 positive Ishikawa cells compared with the empty body group(P<0.01). Conclusion In this experiment, CAR THP-1 cell line targeting HER2 was established by constructing CAR lentivirus vector and transfecting THP-1 cells, and it was proved that CAR THP-1 could phagocytize HER2 positive Ishikawa cells through specific targeting.

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Objective To investigate the molecular mechanisms underlying the feedback regulation of single immunoglobin interleukin-1 related receptor(SIGIRR) expression by the nuclear factor kappa-κB(NF-κB) in human renal tubular epithelial cells(HKC). Methods The pLNCX2-G418-SIGIRR overexpression vector was constructed by molecular cloning, and the SIGIRR overexpression cells and control cells were constructed by infecting HKC cells after packaging with PT67 cells. Using IL-1β induction, Western blot verified that overexpression of SIGIRR inhibited NF-κB activation. After using NF-κB blocker and interfering with NF-κB activity, immunofluorescence assay verified that activated NF-κB regulated SIGIRR expression. Online tools predicted the presence of NF-κB binding sites in the SIGIRR promoter region. The SIGIRR promoter sequence containing the binding site was obtained from within human genomic DNA by molecular cloning, ligated to the luciferase vector pGL3-Luc, constructed pGL3-Luc-SIGIRR, and mutated the binding site. The luciferase reporter gene assay and chromatin immunoprecipitation technique(ChIP) were used to jointly verify that activated NF-κB could bind to the SIGIRR promoter region to regulate SIGIRR gene expression. Results The results showed that the constructed pLNCX2-G418-SIGIRR retroviral vector was verified by enzymatic digestion and sequencing to be identical to the coding sequence of the SIGIRR gene for comparison, the recombinant and control vectors were transferred into HKC cells after viral packaging, and the HKC/SIGIRR experimental and HKC/Co control cell lines were successfully constructed at the mRNA and protein levels of SIGIRR expression differences were statistically significant(P<0.05,P<0.001). Overexpression of SIGIRR cell groups reduced IL-1β-induced NF-κB activation compared to control cells(P<0.001). SIGIRR expression was downregulated after inhibition of NF-κB activation and interference with NF-κB expression. After extracting human genomic DNA, the SIGIRR target promoter sequence was obtained by molecular cloning method and linked to the vector, and the pGL3-Luc-SIGIRR luciferase vector was successfully constructed and targeted to mutate the vector, which was verified to be identical to the target sequence by digestion and sequencing. The luciferase reporter gene assay and CHIP assay confirmed that NF-κB could bind to SIGIRR promoter region and regulate SIGIRR expression. Conclusion It has been verified that SIGIRR can influence the activation of NF-κB in HKC cells, and activated NF-κB can bind to the promoter region of SIGIRR and regulate the gene expression changes of SIGIRR, forming a feedback system to control the over-activation of NF-κB.

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Objective To observe the interventional effects of human amniotic mesenchymal stem cells(hAMSCs) transfected with angiotensin converting enzyme 2(ACE2) gene on monocrotaline-induced pulmonary arterial hypertension(PAH) in rats. Methods (1) HAMSCs were transfected with lentivirus carrying ACE2 gene, and the expression of ACE2, insulin-like growth factor(IGF-1), granulocyte chemoattractant protein-2(GCP-2) gene, cell migration and tube formation ability was detected before and after transfection.(2) Male, healthy, 6-week-old SD rats were randomly divided into normal Control group(Control group), pulmonary hypertension group(PAH group), human amniotic mesenchymal stem cell treatment group(hAMSCs group) and ACE2-transfected human amniotic mesenchymal stem cell treatment group(ACE2-hAMSCs group). The mean pulmonary arterial pressure(mPAP) and right ventricular hypertrophy index(RVHI) were measured 2 weeks after the model was established. The lumen size of pulmonary arterioles and the thickness of vessel wall were observed by HE staining. The mRNA expression levels of GCP-2, ACE2, α-actin and angiotensin Ⅱ(Ang Ⅱ) in lung tissue were determined by QPCR. Results In vitrocell experiments showed that the cell migration ability and tube formation ability of ACE2-hamscs were higher than those of pure hAMSCs(P<0.05). Ace2-hamscs up-regulated the mRNA expressions of ACE2 and pro-angiogenic factors IGF-1 and GCP-2(P<0.01). After 2 weeks of intervention, the mPAP and cardiac RV/LV+S of the PAH group significantly increased compared with the normal control group(P<0.01), and the mPAP and cardiac RV/LV+S of the ACE2-hAMSCs group significantly decreased compared with the PAH and hAMSCs groups(P<0.01). Compared with PAH group, the mRNA expression level of ACE2 significantly increased in the lung tissue of ACE2-hamscs group. Conclusion ACE2-hAMSCs can significantly reduce the mean pulmonary arterial pressure of monocrotaline(MCT)-induced PAH rat model, and significantly improve pulmonary vascular and right ventricular remodeling. The therapeutic effect of ACE2-hamscs is significant in hAMSCs group. It is considered that ACE2-hAMSCs may not only promote the paracrine effect of hAMSCs, The expression of pro-vascular growth factors GCP2 and IGF-1 can repair the injured pulmonary vascular endothelial cells and reduce the high pressure of pulmonary artery. Moreover, the high expression of ACE2 in hAMSCs is related to the regulation of RAS.

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Objective To investigate the effect and mechanism of all-transretinoic acid(ATRA) on lipopolysaccharide(LPS)-induced acute myocardial injury in C57BL/6 mice. Methods Male mice of C57BL/6 strain were randomly divided into normal group, model group, ATRA group, and ferrostatin-1 group. Mice in the ATRA group were injected intraperitoneally with ATRA 15mg/(kg·d), ferrostatin-1 group received ferrostatin-1 2 mg/(kg · d), the normal group and the model group were given solvent. After one week of continuous administration, the model group, ATRA group, and ferrostatin-1 group were intraperitoneally injected with LPS 6 mg/kg. All mice were sacrificed after 6 hours. The contents of malondialdehyde(MDA) and glutathione(GSH) in serum of mice were detected. qPCR was used to detect mRNA levels of interleukin-6(IL-6) and tumor necrosis factor-alpha(TNF-α) in heart tissue.Hematoxylin-eosin(HE) staining was used to observe the changes of heart tissue in mice. Transmission electron microscopy(TEM) was used to observe the structure of mouse myocardial mitochondria. Western blot was used to detect the expression of ferroptosis markers glutathione peroxidase 4(GPX4), ferritin heavy chain 1(FTH1), Solute carrier family 7 member 11(SLC7A11), acyl-CoA synthetase long-chain family member 4(ACSL4) and related regulatory proteins, Nuclear factor erythroid 2-related factor 2(NRF2), kelch-like ECH-associated protein 1(KEAP1). Results Compared with the normal group, the MDA content in the serum of the model group increased and the GSH content decreased, the above changes were reversed in the ATRA group as well as in the ferrostatin-1 group. Compared with normal group, the mRNA levels of IL-6 and TNF-α in the heart tissue of model group increased steeply, the above changes were relieved in the ATRA group and the ferrostatin-1 group. There was no significant difference in HE staining of myocardial tissue among the groups of mice. Compared with the normal group, myocardial mitochondria in the model group showed the phenomenon of cristae reduction or disappearance under TEM, while myocardial mitochondrial injury was alleviated in the ATRA group and the ferrostatin-1 group. Western blot showed that GPX4, FTH1, SLC7A11, and NRF2 expression were reduced in the myocardial tissue of mice in the model group compared with the normal group, ACSL4 and KEAP1 expression increased. The above changes were reversed in the ATRA group as well as in the ferrostatin-1 group. Conclusion ATRA alleviates lipopolysaccharide-induced acute myocardial injury in mice by inhibiting ferroptosis.

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Objective To investigate the expression of Ca2+-independent phospholipase A2β(iPLA2β) in human renal tubular epithelial cells(HK-2) induced by high glucose(HG), the relationship between iPLA2β and ferroptosis and the protective mechanism of HG treated HK-2 cells. Methods The HK-2 cells were treated with 30 mmol/L glucose, the overexpression model was constructed by transfection of iPLA2β plasmid.Ferrostatin-1(Fer-1)(an inhibitor of ferroptosis)and erastin(an activator of ferroptosis) were used as controls. After 36 hours of intervention, the kit detected the levels of superoxide(SOD), malonaldehyde(MDA) and iron in HK-2 cells. DCF immunofluorescence was used to detect intracellular reactive oxygen species(ROS). The expression of ACSL4, GPX4, LPCAT3, TFR1 in HK-2 cells were measured by Western blot. Results The expression of iPLA2β downregulated in HG-induced injury of HK-2 cells. The levels of ROS and MDA in HK-2 cells increased, while the levels of GSH and SOD decreased. The expression of ACSL4, LPCAT3 and TFR1 decreased, and the expression of GPX4 increased in HK-2 cells. However, these indexes were improved after Fer-1 intervention. iPLA2β overexpression could reduce the injury of HK-2 cellsviaattenuation of KIM-1. Further research revealed that iPLA2β overexpression inhibited oxidative stress and ferroptosis in HK-2 cells injury induced by high glucose. Meanwhile, the improvement effect of iPLA2β on HG-induced HK-2 cells damage could be eliminated by erastin. Conclusion iPLA2β prevents HG-induced injury of HK-2 cellsviaregulating ferroptosis.

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Objective To observe the dynamic expression of recombinant lysyl oxidase like protein 1(LOXL1) in the lysine oxidase family in the liver of C57BL/6 mice infected withSchistosoma japonicumand explore its role in hepatic fibrosis. Methods Mice were infected subcutaneously with cercariae ofS.japonicum,and sacrificed with euthanasia in 6,9 and 12 weeks after infection. The sera and liver tissues were collected. The levels of liver fibrosis in mice was dynamically evaluated by HE and Sirius red staining, and the serum transaminases were detected. The dynamic expression levels of collagen type Ⅰ( Col1), LOXL1 and α-smooth muscle actin(α-SMA) in liver tissues were determined respectively by Western blot and qPCR. Finally, the dynamic levels of soluble and insoluble collagens were detected. Results The result of HE and Sirius red staining showed that hepatic fibrosis levels increased at 6 weeks, peaked at 9 week, and decreased at 12 week in response toS.japonicum infection. Western blot and q-PCR showed that the expression levels of LOXL1,Col1α1,Col3α1 and α-SMA was significantly up regulated and reached maximum at the 9th week in response toS.japonicuminfection. Soluble collagen protein levels reached maximum at the 9th week. and decreased at 12 week, however insoluble collagen protein levels continued to increase. Conclusion There may be a correlation between LOXL1 and fiber cross-linking in the process of hepatic fibrosis inS.japonicum, and it plays a role in promoting hepatic fibrosis.

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Objective To explore the possible mechanism of glycyrrhetinic acid on inhibiting malignant biological behaviors of melanoma by Wnt/β-catenin pathways. Methods The melanoma cells B16-F10 were selected as the research objects. The concentration gradient tests(0, 1, 2, 4 μmol/L) were conducted by MTT. The cells given cisplatin intervention was enrolled as positive controls. The cells invasion and migration were detected by Transwell chamber assay. The expression levels of Wnt/β-catenin pathway proteins, invasion and migration related proteins(MMP-2, MMP-9) in B16-F10 cells were detected by Wester blot. The xenograft models of nude mice were constructed, and they were divided into control group(without drugs treatment) and glycyrrhetinic acid group(40 mg/kg). The growth of tumor tissues, and expression levels of Wnt/β-catenin pathway proteins, invasion and migration related proteins were observed. Results MTT results showed that glycyrrhetic acid could inhibit the proliferation of B16-F10 cells in a concentration-dependent manner. The inhibition effect of glycyrrhetic acid(≥2 μmol/L) was significant on the proliferation of B16-F10 cells(P<0.05). The results of Transwell chamber assay showed that compared with control group, invasion and migration abilities of B16-F10 cells were significantly reduced after treatment with glycyrrhetinic acid(2, 4 μmol/L)(P<0.05). Wester blot results showed that compared with those without glycyrrhetinic acid treatment, expression levels of MMP-9, MMP-2, Wnt1 and β-catenin protein in B16-F10 cells significantly decreased after treatment with glycyrrhetinic acid(2, 4 μmol/L)(P<0.05). The results of tumor-bearing assay showed that compared with control group, weight and volume of tumors significantly decreased in glycyrrhetinic acid group, and expression levels of Wnt1, β-catenin, MMP-9 and MMP-2 proteins also significantly decreased(P<0.05). Conclusion Glycyrrhetinic acid can significantly inhibit the malignant biological behaviors of melanomain vitroandvivo. And its mechanism may be related to inhibiting the activation of Wnt/β-catenin signaling pathways.

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Objective To investigate the effects CXCL1 derived from TAMs on the progression of HCC by CXCL1 in tumor microenvironment(TME). Methods PMA induced human monocyte(THP-1) to obtain undifferentiated macrophages(M0) and then co-cultured with Huh 7 HCC cells. The biomarkers of macrophage phenotype and mRNA level of HCC were analysed by qRT-PCR. Colony formation assay, cell viability assay and transwell assay were performed to evaluate the biological alteration of HCC cells in TME. Epithelial-mesenchymal transition(EMT) molecular genetics were detected using western blot. The levels of CXCL1 in TME were measured by ELISA assay. Results High CXCL1 expression in HCC patients predicted poor prognosis. Abundant macrophages were found in CXCL1 high expression HCC tissues. In macrophages and HCC co-cultured model, significantly increasing CXCL1 expression was detected in both two cells and the biomarkers of M2 macrophages, CD163 and CD206 were elevated. The growth and migration of HCC cells were promoted. Conclusion Co-culture of macrophages with HCC cells induces macrophages pro-tumor phenotype and CXCL1 secretion which promotes the progression of HCC.

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Objective To explore the effects of long non-coding RNA(LncRNA) MIR22HG on proliferation, apoptosis and inflammatory response of rheumatoid arthritis(RA) fibroblast-like synoviocytes(FLSs) and its molecular mechanism. Methods Synovial tissue samples were collected from 37 RA patients and 30 joint trauma patients in our hospital, and the expression levels of MIR22HG and miR-22-5p in synovial tissue were detected by qRT-PCR. RA-FLSs in human was isolated, cultured and identified in vitro. MIR22HG siRNA interference plasmid (si-MIR22HG) and its negative control plasmid(si-NC),miR-22-5p inhibitor and its negative control(inhibitorNC) were transfected into RA-FLSs respectively or simultaneously. The expression levels of MIR22HG and miR-22-5p were detected by qRT-PCR. CCK-8 was used to detect the proliferation activity of cells in various groups.AnnexinⅤ-FITC/PI was used to detect the apoptosis rates of cells in various groups. ELISA was used to detect the levels of TNF-α,IL-1β and IL-6 in the supernatant of cells in various groups. Western blot was used to detect the protein expression levels of Bcl-2,Bax and Cleaved caspase-3 of cells in various groups. The targeting relationship between MIR22HG and miR-22-5p was verified by dual luciferase reporter gene assay. Results Compared with joint trauma patients,the expression level of MIR22HG in synovial tissues of RA patients increased(P<0. 05),while the expression level of miR-22-5p decreased(P<0. 05). Interference with MIR22HG inhibited the proliferation activity of RA-FLSs,decreased the levels of TNF-α,IL-1β and IL-6 in cell supernatant and the protein expression level of Bcl-2 in cells(P<0. 05),and increased the apoptosis rate,the expression level of miR-22-5p and the protein expression levels of Bax and Cleaved casepase-3(P<0. 05). However,inhibition of miR-22-5p expression reversed the effects of MIR22HG gene silencing on proliferation,apoptosis and inflammation of RA-FLSs(P<0. 05). Dual luciferase reporting assay showed that miR-22-5p was a potential downstream miRNA target of MIR22HG. Conclusion MIR22HG is highly expressed in synovial tissues of RA patients,and it may promote the proliferation and the inflammatory response of RA-FLSs and inhibit cell apoptosis by down regulating the expression of miR-22-5p.

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Objective To investigate the mechanism of resveratrol promoting fibronectin type Ⅲ domain-containing 5(FNDC5) degradation in skeletal muscle of male obese mice. Methods Six-week-old male C57BL/6 mice were randomly divided into three groups: standard control diet(SCD), high-fat diet(HFD) and high-fat diet treated with resveratrol(HFD+RES). HFD+RES group was intervened with resveratrolviagavage [400 mg/kg·d)] while fed HFD for 20 weeks. The body mass, serum TG, TC, LDL-C and HDL-C levels were detected. The pathological changes in skeletal muscle were detected by HE staining. The expression of FNDC5, SIRT1, SIRT2, LC3, p62, Beclin-1, ATG5, ATG7 was assessed by immunohistochemistry, RT-PCR and Western blot respectively. Results The body mass, serum TG, TC and LDL-C levels increased significantly, meanwhile HDL-C levels decreased in HFD group. Lipid deposition between skeletal muscle fibers were obvious in HFD group. The immunohistochemistry results showed that protein expression levels of SIRT1, SIRT2 and LC3 obviously decreased, while the protein levels of FNDC5 and p62 obviously increased. The expression levels of FNDC5 significantly increased, while the gene expression levels of SIRT1, SIRT2, LC3, Atg7 and Beclin-1 obviously decreased. All these responses were attenuated by treatment with RES. Conclusion RES has obvious effects of lipid-lowering and promoting FNDC5 degradation in skeletal muscle tissues, which may be related with SIRT1 and SIRT2-induced autophagy, thus resulting in degradation of FNDC5.

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Objective To investigate the effect of Arginine Vasopressin(AVP) on the median preoptic glutamatergic(MnPOVglut2) neurons and its mechanism. Methods Brain slices were prepared from male Vglut2-tdTomatomice. MnPOVglut2neurons expressing red fluorescent protein were located by using fluorescence microscope. Whole-cell patch clamp technique was used to observe the effect of AVP on the firing frequency of MnPOVglut2neurons, the effect of synaptic transmission blockers(STBs) on the AVP-induced change in the firing frequency of MnPOVglut2neurons, and the effect of AVP V1a receptor antagonist on the AVP-induced change in the firing frequency of MnPOVglut2neurons. Results The mean firing frequency of MnPOVglut2neurons increased during perfusion with artificial cerebrospinal fluid(ACSF) and AVP compared with that during perfusion with ACSF(P<0.01), indicating that AVP excited the MnPOVglut2neurons. The mean firing frequency of MnPOVglut2neurons still increased during perfusion with ACSF, STBs, and AVP compared with that during perfusion with ACSF and STBs(P<0.001); moreover, the magnitude of AVP-induced increase in firing frequency didn′t change significantly during perfusion with ACSF, STBs, and AVP compared with that during perfusion with ACSF and AVP(P>0.05), suggesting that AVP excited the MnPOVglut2neurons directly in a postsynaptic manner. The magnitude of AVP-induced increase in the firing frequency of MnPOVglut2neurons declined during perfusion with ACSF, STBs, AVP, and V1a receptor antagonist compared with that during perfusion with ACSF, STBs, and AVP(P<0.01), suggesting that AVP excited MnPOVglut2neurons directlyviaV1a receptor. Conclusion AVP can excite MnPOVglut2neuronsviaV1a receptor directly in a postsynaptic manner. This study reveals the molecular marker of MnPO neurons which AVP act on.

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Objective Investigate osteogenic differentiation of canine periodontal ligament stem cells(cPDLSCs)viaover-expression ephrinB2 in cPDLSCs. Methods cPDLSCs were isolated from the premolars and molars of Beagle. After transfected with EfnB2-GFP-Bsd and GFP-Bsd empty Vector, cPDLSCs were induced to osteogenic differentiation. Western blot was used to invest the expression of ephrinB2 protein. The effect of osteogenic differentiation of EfnB2-cPDLSCs and Vector-cPDLSCs were analyzed by RT-PCR, CCK-8, Alizarin-red S staining and ALP. Results There was no significant difference in cell proliferation between EfnB2-cPDLSCs and Vector-cPDLSCs. While EfnB2-cPDLSCs displayed an enhanced ALP activity and more prominent mineralized nodules compared with Vector-cPDLSCs. The odonto-/osteogenic genes in EfnB2-cPDLSCs were also highly enhanced. Conclusion The results of our study indicated that ephrinB2 gene-transfected cPDLSCs showed enhanced osteogenic differentiation.

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Objective To explore the effect and mechanism of lycorine on oxaliplatin(OXA) induced chemotherapy pain in mice. Methods 40 mice were randomly divided into 4 groups, 10 mice per group, which were respectively divided into control group, model group, administration group, and inhibitor group.A mouse model of chemotherapy induced pain was established by intraperitoneal injection of OXA for 5 consecutive days. Intrathecal administration of lycorine was performed. Behavioral changes and expression levels of inflammatory related proteins weredetected. Results Compared with control group, model group mice exhibited the increased number of spontaneous flinches, decreased mechanical nociceptive threshold, decreased movement distance and latency, and up-regulated expression levels of interleukin-1β(IL-1β), astrocytic marker glial fibrillary acidic protein(GFAP), cyclooxygenase-2(COX-2), NOD-like receptor protein 3(NLRP3), cysteinyl aspartate and specific proteinase 1(Caspase-1). Compared with model group, lycorine administration reduced the number of spontaneous flinches, increased mechanical nociceptive threshold, enhanced the movement distance and latency, bound and reduced COX-2 expression, down-regulated the expression levels of IL-1β, GFAP, NLRP3 and Caspase-1. Conclusion Lycorine reduces COX-2 expression, inhibits NLRP3 inflammasome activation, suppresses spinal inflammation, consequently alleviates pain behaviors and improved motor ability of mice.

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Objective To investigate the expression, prognosis and biological role of hepatocyte nuclear factor 4A(HNF4A) in gastric cancer, and to study its effect on the proliferation of gastric cancer cells. Methods Tumor Immune Estimation Resource 2.0(TIMER2.0) and Gene Expression Profiling Interactive Analysis(GEPIA2) databases were used to analyze the relative expression levels of HNF4A in gastric cancer and normal tissue, KM Plotter was used to analyze the correlation between the expression level of HNF4A and the survival rate of gastric cancer patients, TISIDB database and R language(4.1.2) were used to analyze whether HNF4A was involved in the immune regulation process of gastric cancer. cBioPortal database was used to analyze the mutations of HNF4A in gastric cancer, GSEA 4.2 was used to analyze the functional enrichment of HNF4A, and LinkedOmics database was used to predict the genes that might be regulated by HNF4A. The relative expression of HNF4A in gastric cancer and adjacent tissues was detected by qRT-PCR, Western blot and immunohistochemistry(IHC). The proliferation and cell cycle of gastric cancer cells were analyzed by CCK-8, EdU, colony forming assay and flow cytometry. Results The expression of HNF4A increased in gastric cancer tissues(P<0.05), and the overall survival rate of gastric cancer patients with high HNF4A expression was worse(P<0.001). HNF4A was mainly missense mutated in gastric cancer. Immune cell infiltration showed that HNF4A was associated with B lymphocytes, CD8+T cells, neutrophils, macrophages and dendritic cells(allP<0.001). HNF4A was also associated with tumor mutation burden(r=0.28,P<0.0001) and microsatellite instability(r=0.13,P<0.01). After knockdown of HNF4A, cell proliferation ability was significantly inhibited, and cell cycle was arrested at G0/G1 phase. Conclusion HNF4A expression significantly increased in gastric cancer tissues, which is associated with poor prognosis, and may also be involved in immune regulation. Knockdown of HNF4A can inhibit the proliferation of gastric cancer cells.

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Objective To explore the mechanism of exosome-derived LncRNA ESCCAL-1 regulating the miR-874/ITGBL1 axis in the progression of colorectal cancer(CRC). Methods The differentially expressed genes in CRC were analyzed using the Gene Expression Omnibus(GEO) database. Expressions of LncRNA ESCCAL-1,miR-874 and ITGBL1 in CRC tissues and cell lines(SW480,SW620,HCT116 and HT29) and adjacent normal tissues and NCM460 cell lines were detected by qRT-PCR; 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazoliumbromide(MTT),clone formation and flow cytometry was used to detect cell proliferation,colony formation and apoptosis;dual luciferase reporter assays were used to verify the interaction between miR-874 and ESCCAL-1,ITGBL1; fluorescence in situ hybridization was used to determine the subcellular localization of LncRNAESCCAL-1. Exosomes were isolated from serum using the Exosome extraction kit. Results The expressions of ESCCAL-1 and ITGBL1 in CRC tissues and cell lines were higher than those in adjacent normal tissues and NCM460 cell lines,while the opposite was true for miR-874(P<0. 05). Knockdown of ESCCAL-1 can inhibit CRC cell proliferation and colony formation and promote apoptosis. There are specific binding sites for miR-874 and ESCCAL-1,and miR-874 inhibitor could partially reverse the effect of knockdown ESCCAL-1 in CRC(P<0. 05). ESCCAL-1 upregulates ITGBL1 by adsorbing miR-874. The serum levels of ESCCAL-1 and exo-ESCCAL-1 in CRC patients were higher than those in the control group. Serum exo-ESCCAL-1 may be a valuable diagnostic indicator for CRC treatment(P<0. 05).Conclusion ESCCAL-1 promotes CRC progression by regulating the miR-874/ITGBL1 axis,and ESCCAL-1 may be an effective molecular target for CRC therapy.

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Objective To investigate the oxidative stress injury of nano zinc oxide nanoparticles(ZnO NPs) on human myocardial cells(AC16), and to analyze the mechanism of ZnO NPs from the transcriptome level. Methods Dynamic light scattering(DLS) was used to characterize and detect ZnO NPs. After AC16 cells were exposed to ZnO NPs at different doses and at different times, the cell survival rate was determined by CCK-8 method. AC16 cells were divided into control group, ZnO NPs(50, 100, 200 μmol/L), after 6 h treatment, the mitochondrial membrane potential(MMP) and reactive oxygen species(ROS) were measured. AC16 cells were divided into control group, 50 μmol/L ZnO NPs group and 200 μmol/L ZnO NPs group. After 6 h exposure, total RNA was extracted by TRIzol for transcriptome analysis, and the differentially expressed genes were enriched by gene body(GO), Kyoto Encyclopedia of Genes and Genomes(KEGG). Results The results of DLS showed that the hydrodynamic diameter was(192.2±1.63) nm and the Zeta potential was(-23.26±1.05) mV. CCK-8 results showed that the survival rate of AC16 cells decreased with the increase of dose and time of exposure to ZnO NPs. Fluorescence quantification showed that with the increase of ZnO NPs exposure dose, MMP significantly decreased at 100 μmol/L ZnO NPs(P<0.05), and ROS significantly increased at 50 μmol/L ZnO NPs(P<0.05).Using the multifunctional microplate reader, it was observed that MMP and ROS were statistically significant at 100 and 50 μmol/L ZnO NPs, respectively, showing a decrease in MMP and an increase in ROS. Transcriptome analysis showed that 1 071 genes were enriched in the 50 μmol/L ZnO NPs group compared with the control group, including 561 up-regulated genes and 510 down-regulated genes. Compared with the control group, 7 164 genes were enriched in 200 μmol/L ZnO NPs group, including 4 098 up-regulated genes and 3 066 down-regulated genes. GO and KEGG analysis showed that the differential genes were mainly concentrated in ROS, antioxidant activity, mitochondrial cytochrome C release, apoptosis and other signaling pathways. Conclusion ZnO NPs can decrease the survival rate of AC16 cells and induce mitochondrial damage and oxidative stress, among which ROS-mediated oxidative stress and mitochondrial function changes are important toxic mechanisms of ZnO NPs induced AC16 cytotoxicity.

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Objective To observe the effect of angiotensin(1-7) [Ang(1-7)] on the apoptosis and angiogenesis of fibroblasts in the oral submucosal fibrosis(OSF), and to explore the effect preliminarily mechanism. Methods Fibroblasts were isolated and cultured from human buccal mucosal tissue, the cell morphology was observed by inverted microscope, and the expression of vimentin was detected by immunofluorescence staining; areca nut extract(ANE) was used to induce human fibroblasts to simulate the in vitro model of fibroblasts in OSF, the experimental groups included control group(normally cultured cells), ANE group(100 μg/ml ANE cultured cells for 48 hours), ANE+low-dose Ang(1-7) group(100 μg/ml ANE+10-7 mol/L Ang(1-7) cultured cells for 48 h), ANE+high-dose Ang(1-7) group(100 μg/ml ANE+10-5 mol/L Ang(1-7) cultured cells for 48 h), immunofluorescence staining detected the expression of α-smooth muscle actin(α-SMA), ELISA method detected the content of Collagen I and Collagen Ⅲ in the cell culture supernatant, MTT method detected cell proliferation activity, flow cytometry detected cell apoptosis, the tubule formation experiment detected the vascularization of human umbilical vein endothelial cell(HUVEC); After the mRFP-GFP-LC3 virus was transferred to the cells, the level of autophagy was detected by immunofluorescence staining,Western blot detected the expression of autophagy-related protein Beclin-1 and the ratio of LC3-Ⅱ/LC3-Ⅰ. Results The isolated and cultured cells were in a long spindle shape,and Vimentin was positively expressed,indicating that fibroblasts were successfully isolated; Compared with the ANE group,the fluorescence expression of α-SMA protein in ANE low dose Ang(1-7) group and ANE + high dose Ang(1-7) group significantly decreased,the contents of Collagen I and Collagen Ⅲ in the culture supernatant were reduced(P<0. 05),cell proliferation activity decreased(P<0. 05),and cell apoptosis rate increased(P<0. 05),the cell culture supernatants of the two groups inhibited the angiogenesis of HUVEC(P<0. 05),endophagosomes were reduced(P<0. 05),Beclin-1 protein expression was reduced(P<0. 05),and the ratio of LC3-Ⅱ/LC3-Ⅰ was down-regulated(P<0. 05); in addition,the effect of ANE + high-dose Ang(1-7) group was stronger than that of ANE + low-dose Ang(1-7) group(P<0. 05). Conclusion Ang(1-7) can inhibit the activation of fibroblasts induced by ANE,promote cell apoptosis,and reduce the angiogenesis of HUVEC,this mechanism may be related to the regulation of cell autophagy.

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Objective To study the effect of Musashi-2(MSI2) overexpression on the imbalance of proliferation and apoptosis of human ovarian granulosa cell line(KGN) induced by dihydrotestosterone(DHT). Methods The gene expression profiles of human ovarian granulosa cells(GCs) in primary culture were statistically analyzed to screen the differentially expressed genes. pcDNA3.1-MSI2 eukaryotic expression plasmid was constructed and transiently transfected into the KGN cells, and the overexpression effect of MSI2 was detected by Quantitative Real-time PCR(RT-qPCR) and Western blot. After overexpressing MSI2 in DHT induced KGN cells, MTT colorimetry and Edu staining were used to detect the proliferation of cells in each group, and flow cytometry(FCM) was further used to detect the apoptosis of cells in each group. Results The mRNA expression level of MSI2 gradually decreased during the primary culture of human ovarian GCs. And pcDNA3.1-MSI2 was successfully constructed and transfected into KGN cells to improve the mRNA and protein expression levels of MSI2. Then MTT, EdU and FCM results showed that compared with the blank group, DHT induction could significantly reduce the proliferation rate and increase the apoptosis rate of KGN cells(P<0.05). However, after MSI2 overexpression, the proliferation rate of KGN cells increased and the apoptosis rate decreased(P<0.05), which were close to the blank group. Conclusion Overexpression of MSI2 can effectively alleviate the imbalance of proliferation and apoptosis of KGN cells induced by DHT, indicating that MSI2 plays an important role in GCs growth and follicle development.

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Objective To explore the factors associated with the distance from the anal verge to rectal cancer(DAVtRC). Methods 102 patients with rectal cancer provided the clinicopathological data. Two groups of patients were created: low rectal cancer group(LRC) for those with DAVtRCs less than 7cm and mid/high rectal cancer group(M/HRC) for those with DAVtRCs greater than 7 cm. The two groups′ clinical and pathological differences were compared. Quantile regression was used to analyze the associations between DAVtRC and the clinicopathological factors. Results LRC had a lower median DAVtRC(5.00 cm [IQR: 4.00, 6.00 cm]) than that in M/HRC [10.00 cm(IQR: 10.00, 11.00 cm)](P<0.01), but had higher mean monocytes[(0.41±0.16)vs(0.34±0.11),P<0.05]. In the model with continuous and categorical variables at 95% quantile, DAVtRC was negatively associated with age(β=-0.204,P<0.01), TNM stage(Ⅲ+ⅣvsⅠ:β=-6.623,P<0.01), platelet(PLT,β=-0.024,P<0.01), vessel invasion(β=-1.544,P<0.01), but positively associated with BMI(β=0.278,P<0.01), tumor diameters(β=0.548,P<0.01), male(β=1.421,P<0.01), low and middle differentiations(β=4.727,P<0.01), T stage(T2vsT1:β=4.422,P<0.01), N stage(N1vsN0:β=6.670,P<0.01), nerve invasion(β=1.825,P<0.01), carcinoembryonic antigen(CEA,β=0.068,P<0.01), lymphocyte(L,β=3.068,P<0.01), platelet lymphocyte ratio(PLR,β=0.045,P<0.01), lymphocyte monocyte ratio(LMR,β=1.088,P<0.01).Quantile regression curves showed that the coefficients of age, body mass index(BMI), tumor diameter, PLT, PLR and LMR were different in different quantiles of DAVtRC. Conclusion DAVtRC is significantly associated with multiple clinicopathological factors in rectal cancer, but in different quantiles, the coefficients are different.

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Objective To quantitatively evaluate the left ventricular myocardial work index(MWI) of patients with end-stage renal disease(ESRD) with preserved ejection fraction using left ventricular pressure-strain loop(PSL), and group them according to left ventricular mass index(LVMI), in order to compare the effect of different LVMI levels on left ventricular MWI. Methods 81 patients with stage 5 chronic kidney disease, including 43 males and 38 females, were divided into left ventricular hypertrophy group(LVH group)(n=44) and non-left ventricular hypertrophy group(NLVH group)(n=37) according to LVMI, and 37 healthy people matched in age and sex with the ESRD groups were randomly selected as the control group. The general clinical data, conventional echocardiographic parameters, global longitudinal strain(GLS), and MWI namely global work index(GWI), global constructive work(GCW), global wasted work(GWW) and global work efficiency(GWE) of the three groups were compared. The correlation between MWI and various parameters were analyzed, and the independent predictors of MWI were analyzed too. Result The results showed that there were no significant differences in age, gender, height, weight and other general clinical data as well as left ventricular ejection fraction(LVEF) among the three groups. In two ESRD groups, GWW was higher than that in control group, GWE was lower than that in control group, and were more significant in LVH group(P<0.05). LVMI was positively correlated with GWW, and negatively correlated with GWE, respectively, with specific independent correlation. Conclusion GWW and GWE can detect early impairment of left ventricular systolic function in ESED patients, and myocardial function impairment in LVH group is more serious than that in NLVH group. LVMI is an independent positive factor of GWW and an independent negative factor of GWE.

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Objective To investigate the value of left ventricular global longitudinal strain(LVGLS) in predicting early chemotherapy related cardiotoxicity in breast cancer patients after chemotherapy. Methods Eighty-six breast cancer patients were enrolled in this study and received 6 cycles of doxorubicin +cyclophosphamide. Conventional echocardiography parameters were measured before chemotherapy(T0), immediately after chemotherapy(T1), 6 months(T2) and 12 months after chemotherapy(T3). LVGLS was obtained by 2D speck tracing imaging(2D-STI), and the relative change of LVGLS(△LVGLS) was calculated at T1. With the occurrence of cardiotoxicity as the clinical outcome, receiver operating characteristic(ROC) curve was plotted.The parameters were analyzed at T1 to evaluate the value of △LVGLS in predicting cardiotoxicity. Results (1) Although the change in LVEF was not different between T0 and T1(P>0.05), LVEF decreased significantly at T2 and T3(P<0.05).(2) Compared with T0, LVGLS decreased at T1, which was further damaged at T2 and T3(P<0.05).(3) Seven patients(8.1%) developed chemotherapy-related cardiotoxicity. LVGLS and △LVGLS were correlated with cardiotoxicity at T1 stage, and △LVGLS was more significant(r=0.64,P<0.001). ROC curve analysis showed that a △LVGLS>12% at T1 predicted cardiotoxicity, with sensitivity of 92.2%, specificity of 76%, and area under the curve was 0.88. Conclusion The LVGLS decreased earlier than LVEF in breast cancer patients after doxorubicin treatment. △LVGLS>12% is a good predictor of cardiotoxicity in patients with breast cancer.

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Objective To investigate risk factors related to coronary artery lesion(CAL) in children with Kawasaki disease. Methods Retrospective analysis was conducted on clinical data of 144 children with Kawasaki disease. The cases were divided into two groups according to whether they had CAL or not. There were 50 children in CAL group and 94 children in non-coronary artery lesion(NCAL) group. The clinical and laboratory indicators in the two groups were analyzed and compared, and the diagnostic efficacy of these indicators for Kawasaki disease combined with CAL was analyzed by drawing the receiver operating characteristic(ROC) curve. Results There was no statistical significant difference in age, gender, duration of fever, use time of immunoglobulin, erythrocyte sedimentation rate, C-reactive protein, serum amyloid A, N-terminal pro brain natriuretic peptide, white blood cell, hemoglobin and platelet between the two groups(P>0.05). In CAL group, the serum levels of 25-(OH)D3and albumin were lower, while the levels of alanine transaminase, interleukin-6 and procalcitonin were higher than those in NCAL group(P<0. 05). Multivariate logistic regression analysis showed that the levels of serum 25-(OH)D3(OR= 0. 984,95% CI: 0. 974 ～ 0. 995),albumin(OR = 0. 857,95% CI: 0. 771-0. 951),alanine transaminase(OR= 1. 011,95% CI: 1. 005-1. 017) and interleukin-6(OR = 1. 002,95% CI: 1. 000-1. 005) were significantly related with coronary artery lesion in children with kawasaki disease(P<0. 05). The levels of 25-(OH)D3,albumin,alanine transaminase and interleukin-6 in serum had diagnostic value for Kawasaki disease combined with CAL,and the area under the curve(AUC) were 0. 660,0. 652,0. 711,and 0. 700,respectively. The AUC of combined diagnosis of four indicators was 0. 816. Conclution Decrease of serum 25-(OH)D3and albumin levels,increase of serum interleukin-6 and alanine transaminase levels in children with Kawasaki disease are risk factors for CAL,combined detection on these multi-indicators have diagnostic value for Kawasaki disease combined with CAL.

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Objective To explore the value of vector angle of Lorenz plots(LPs) and the role of combining B-line slope in improving the diagnostic efficiency of arrhythmia. Methods LPs of 119 cases with ventricular premature contraction(VPC group), 97 cases with supraventricular premature contraction(SPC group), 52 cases with type II°I atrioventricular block(Ⅱ°Ⅰ group) and 54 cases with type Ⅱ°Ⅱ atrioventricular/sinoatrial block(Ⅱ°Ⅱ group) were retrospectively analyzed. The B-line slope and vector angle were measured, and the differences between groups were compared. Receiver operating characteristic curve was used to analyze the diagnostic efficacy of B-line slope, vector angle and their combination between groups, and MedCalc software was used for statistical comparison. The consistency of intra-observer and inter-observer measurements of B-line slope and vector angle was evaluated using Intra group correlation coefficient(ICC) and Bland-Altman plot. Results There were significant differences between VPC group and SPC group, and between Ⅱ° Ⅰ group and II°II group(P<0.05).The area under the curve(AUC) of B-line slope, vector angle, and the combination of the two in distinguishing ventricular and supraventricular premature contraction were 0.81,0.84 and 0.87 respectively, and the AUC in distinguishing type Ⅱ°Ⅰ and type Ⅱ°Ⅱ atrioventricular/sinoatrial block were 0.76, 0.78 and 0.80 respectively. The ICC of vector angle was better than B-line slope(Intra-observer 0.99vs0.98, Inter-observer 0.97vs0.96). Conclusion Vector angle can be used to identify the type of arrhythmia, and has good intraobserver and interobserver consistency. Its combination with B-line slope has the highest accuracy in diagnosing arrhythmia, providing a new reference for clinical diagnosis and treatment.

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Objective To evaluate the value of multi-detector CT(MDCT) upper airway imaging in the diagnosis of obstructive sleep apnea hypopnea syndrome(OSAHS) and in determining the location of upper airway obstruction. Methods MDCT was used to scan the upper airways of 85 clinically confirmed adult patients with different degrees of OSAHS(73 males and 12 females) in calm breathing phase and forced inhalation phase and 60 normal adults(50 males and 10 females) in calm breathing phase to obtain nasal cavity,nasopharynx,palatopharynx and oglosopharynx volumes. Parapharyngeal fat volume was measured in OSAHS patients and normal subjects. In addition,three groups of clinical data related to OSAHS patients were recorded,including sleep apnea hypopnea index(AHI),body mass index(BMI) and lowest blood oxygen saturation(LaSO2). Finally,the measured data and clinical data of each group were statistically analyzed. Results The volume of nasopharynx and palatopharynx in the calm breathing group was significantly smaller than that in the control group,with statistical significance. Palatopharyngeal volume forced inspiratory phase was significantly smaller than calm breathing phase in the experimental group. The parapharyngeal fat volume in the experimental group was significantly higher than that in the control group. AHI was positively correlated with BMI and parapharyngeal fat volume. LaSO2was negatively correlated with AHI and BMI,respectively. Conclusion MDCT upper airway imaging has good clinical application value in the diagnosis,treatment and postoperative evaluation of OSAHS disease due to the significant anatomical difference between OSAHS patients and normal subjects.

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Objective To explore the value of T2WI and enhanced T1WI magnetic resonance imaging models in differentiating inverted papilloma and polyp of nasal cavity and paranasal sinus. Methods 54 cases of inverted papilloma(NIP) and 51 cases of polyp(NP) with complete T2WI and enhanced T1WI images confirmed by pathology were collected. ITK snap was used to outline all levels of the lesion. Pyromics was used to extract image omics features. Firstly, the mRMR) was used for feature extraction, and then rfe-SVM was used to remove the feature of minimum score and establish a prediction model. The sensitivity and specificity of ROC curve were used to evaluate the performance of the model, and were verified in the validation set. Results A total of 1 133 image omics features were extracted, and 30 features were retained after mrmr dimensionality reduction for the establishment of prediction models. The AUC value of T1WI enhanced prediction model was: training set 0.98, validation set 0.95, the sensitivity and specificity of training set were 89.7% and 100% respectively, and the sensitivity and specificity of validation set were 93.8% and 93.3% respectively. The AUC value of T2WI prediction model was: training set 0.95, validation set 0.91, the sensitivity and specificity of training set were 82.1% and 95.6% respectively, and the sensitivity and specificity of validation set were 93.8% and 84.2% respectively. Conclusion MRI based on T1WI enhancement prediction model and T2WI prediction model have certain value in differentiating inverted papilloma and polyp of nasal cavity and paranasal sinus.

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Objective To compare the diagnostic efficacy of three screening methods, including the epidemiological survey and the new gastric cancer scoring system proposed in the Expert Consensus Opinion on Early Gastric Cancer Screening Process in China(Shanghai, 2017), and the Kyoto classification score for screening gastric in early gastric cancer diagnosis. Methods Patients who underwent endoscopic submucosal dissection and were pathologically confirmed were included in the gastric cancer group, patients with non-gastric cancer in the same period were consecutively included in the control group. Questionnaires and serological tests were performed in all enrolled patients, and the endoscopic presentations was recorded according to the classification of Kyoto gastritis during gastroscopy. The risk stratification and the detection rates of gastric cancer were estimated by different screening methods, and the receiver operating characteristic curve(ROC curve) of subjects diagnosed with each screening method alone and in combination were plotted to compare the area under the ROC curve(AUC) and the diagnostic efficacy. Results Among all participants, 84 were patients with gastric cancer and 120 were normal people without gastric cancer. The Kyoto risk score had the highest diagnostic value(AUC=0.79) among the others, and its sensitivity and specificity were 72.6% and 79.1%, respectively, but the combined diagnosis of different methods did not significantly improve the diagnostic efficacy. The Kyoto risk score had a more discriminative value in the medium to high-risk group delineated by the new scoring system. Conclusion The Kyoto risk score has high clinical value in the diagnosis of early gastric cancer and can be used in conjunction with the new scoring system for early gastric cancer risk assessment.

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Objective To retrospectively analyze the efficacy of aerosol inhalation of N-acetylcysteine(NAC) solution combined with bronchoalveolar lavage in children withMycoplasma pneumoniaepneumonia(MPP). Methods A retrospective study was conducted on 120 children with MPP. According to different treatment methods, they were divided into combined treatment group(43 cases), atomization group(40 cases) and bronchoalveolar lavage group(37 cases). Among them, while receiving conventional treatment, children who were treated with aerosol inhalation of N-acetylcysteine alone were included in the atomization group, and those who were treated with bronchoalveolar lavage alone were included in the bronchoalveolar lavage group, and children treated with the combination of the above two treatments were included in the combined treatment group. The clinical efficacy, imaging recovery and incidence of adverse reactions were compared among all groups. Results Compared the total effective rate among the three groups, there was no significant difference between the atomization group and the bronchoalveolar lavage group(P>0.05), which were both lower than that of the combined treatment group(P<0.05); the difference in the improvement rate of lung imaging among the three groups was statistically significant(P<0.05), the combined treatment group was the highest, and the BAL group was higher than the atomization group; There was no significant difference in the duration of fever and pulmonary rales between the combined treatment group and the BAL group(P>0.05), which were shorter than those in the atomization group(P<0.05); There was no significant difference in duration of cough, shortness of breath and hospital stay between the atomization group and the BAL group(P>0.05), which were longer than those in the combined treatment group(P>0.05). There was no serious adverse reactions in the three groups. There was no significant difference in the three groups(P>0.05). Conclusion Compared with single treatment, N-acetylcysteine solution combined with bronchoalveolar lavage is more effective and has more advantages in the treatment ofMycoplasma pneumoniaepneumonia in children.