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Objective To buildSERPING1-knockout porcine fetal fibroblasts(PFFs) based on CRISPR/Cas9 technology and provide cell experimental materials for the construction of hereditary angioedema models. Methods Firstly, protein structure prediction software was used to analyze the amino acid homology between human and pigSERPING1. Secondly, the eighth exon was selected as the knockout target by screening the effective coding region of the pigSERPING1gene. A single guide RNA targeting pigSERPING1was designed, synthesized and linked to pX330 vector containing Cas9 endonuclease. G418 was used to obtain the positive monoclonal cells after transfection into PFFs. Finally, the circumstance of CRISPR/Cas9 mediated knockout was assessed by the T7 EN1 enzyme digestion assay and the genotypes of monoclonal cells were identified by sequencing analysis. Results Bioinformatic analysis revealed that theSERPING1protein of human and pig had high homology, amino acid sequence identity reached 65.87%. A vector targetingSERPING1was constructed successfully and transfected into cells. Monoclonal cells with knockoutSERPING1gene were obtained through drug screening. Sequencing confirmed the mutant genotype. Conclusion The human and pigSERPING1sequences and their protein structures are highly homologous, and it is suitable for the construction of disease model. CRISPR/Cas9 expression vectors were constructed to achieve theSERPING1gene targeting in PFFs.SERPING1-knockout monoclonal cells were obtained, which could contribute to the construction of theSERPING1knockout miniature pig model.

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Objective To explore the effect and mechanism of chrysin and chloroquine(CQ) on the proliferation and apoptosis of ovarian cancer cells. Methods Ovarian cancer specimens and normal ovarian specimens were selected after surgical resection and immunohistochemical experimental methods was used to detect the expression level of autophagy-related protein LC3 in ovarian cancer and normal ovarian tissues. The CCK-8 method was used to detect the proliferation of ovarian cancer cell SKOV3. Western blot and immunofluorescence were used to detect the expression of LC3 in SKOV3 cells. An electron microscope was used to detect the number of autophagosomes in SKOV3 cells. Flow cytometry was used to detect SKOV3 cell apoptosis. Results The expression level of LC3 in ovarian cancer was higher than that in normal ovarian tissues. SKOV3 cells were treated with chrysin at concentrations of 0, 20, 40, 60, 80, 100 μmol/L for 24 hours. As the concentration increased, cell proliferation decreased significantly. Compared with the control group, 40 μmol/L, chrysin significantly up-regulated the expression of LC3 after 24 hours(P<0.05). Compared with the control group, after 24 hours of SKOV3 cells treated with chrysanthemum, more autophagosomes were observed under transmission electron microscope(P<0.05), but no autophagosome was observed in the control group. When chrysin and chloroquine were used in combination for 24 hours, the expression of LC3 protein in the combined treatment group was higher than that in the chrysin and CQ treatment group(P<0.05). Chrysin could obviously induce apoptosis of SKOV3 cells, and the group of Chrysin+CQ showed the highest proportion of apoptosis(P<0.05). Conclusion Chrysin can induce autophagy to inhibit the proliferation of ovarian cancer cells and promote apoptosis.

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Objective To investigate the effect of blocking the B7/CD28 pathway on rheumatoid arthritis mice and its possible mechanism. Methods 40 DBA/1 mice were randomly divided into normal control group, model group, CTLA4-Ig low-dose group and cytotoxic T lymphocyte antigen-4 immunolobulin(CTLA4-Ig) high-dose group. Except for the normal control group, the other three groups were prepared for rheumatoid arthritis models. At 0, 5 and 10 days after the second immunization, the CTLA4-Ig low-dose group and CTLA4-Ig high-dose group were intraperitoneally injected with 50 mg/kg and 100 mg/kg CTLA4-Ig, respectively, the normal control group and the model group were injected with the same amount of saline. Vernier calipers were used to measure the thickness of the ipsilateral hindfoot of the mouse for arthritis score; the serum levels of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interleukin-6(IL-6) and interleukin-17(IL-17) were detected by ELISA, images were acquired and bone tissue morphometric parameters were measured through Micro-CT scanning, HE staining observed the pathological changes of the knee joint, TRAP staining detected osteoclasts in knee joint tissues, real-time fluorescent quantitative PCR and Western blot detected osteoprotegerin(OPG), receptor activator of NF-κB(RANK), receptor activator of NF-κB ligand(RANKL) mRNA and protein expression, immunohistochemical staining detected nuclear factor κB(NF-κB) expression. Western blot was used to detect the expression of related proteins in the NF-κB signaling pathway. Results Compared with the model group, after high-dose CTLA4-Ig treatment, RA mouse foot swelling and arthritis scores were reduced, the levels of TNF-α, IL-1β, IL-6 and IL-17 in serum were reduced, the knee joint damage was significantly reduced, bone mineral density(BMD), trabecular thickness(Tb.Th), bonevolume(BV) and bone volume fraction(BV/TV) increased, the number of TRAP positive cells decreased, the relative expression of OPG mRNA and protein in the tissue was up-regulated, while the relative expression of RANK, RANKL mRNA and protein were down-regulated. At the same time, the relative expression of p-p65 and p-IκBα protein decreased, the positive expression area of NF-κB decreased, and the difference were statistically significant(P<0.01); After CTLA4-Ig low-dose treatment, the serum levels of TNF-α, IL-1β, IL-6 and IL-17 in RA mice were significantly lower than those in the model group, and the area of NF-κB positive expression in the tissue was reduced, the difference were statistically significant(P<0.01). Conclusion CTLA4-Ig to block the B7/CD28 pathway has a significant therapeutic effect on mouse rheumatoid arthritis, which can inhibit the inflammatory response, reduce bone destruction and prevent the activation of the NF-κB signaling pathway.

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Objective To investigate the protective effect of cordycepin(Cor) on cardiac function in aged rats and its mechanism. Methods The aged rats were divided into Model group, Cor administration group(Cor 5, 10, 20mg/kg),and young rats were used as Control group( Control). The levels of heart rate( HR),left ventricular ejection fraction( LVEF) and fractional shortening( FS) were detected by echocardiography. The levels of creatine kinase MB( CK-MB),myohemoglobin( Mb),cardiac troponin Ⅰ( cTnI),superoxide dismutase( SOD),malondialdehyde( MDA) and lactate dehydrogenase( LDH) were detected by ELISA. The pathological morphology of heart tissue was observed by HE staining. The apoptosis of myocardial cells was observed by TUNEL staining.Western blot detected the protein expression of cysteine aspartic protease 9( Caspase 9),cleaved cysteine aspartic protease 9( cleaved Caspase 9),cysteine aspartic protease 3( Caspase 3),cleaved cysteine aspartic protease 3( cleaved Caspase 3),nuclear factor E2 related factor 2( Nrf2),phosphorylation nuclear factor E2 related factor 2( p-Nrf2),quinone oxidoreductase 1( NQO1),heme oxygenase 1( HO-1). Results Compared with the elderly model group,HR,LVEF,FS increased,CK-MB,Mb,cTnI levels decreased,MDA,LDH content decreased,SOD activity increased,cleaved Caspase 9/Caspase 9,cleaved Caspase 3/Caspase 3 expression decreased,The expressions of Nrf2,NQO1 and HO-1 were increased,and the pathological morphology of cardiac tissue and myocardial apoptosis were improved in Cor groups. Conclusion Cor can improve cardiac function of elderly rats,relieve oxidative stress response,inhibit myocardial cell apoptosis and have a certain protective effect on cardiac tissue injury in rats,and the mechanism may be related to the promotion of Nrf2/HO-1 pathway related protein expression.

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Objective A prognostic risk model for lung adenocarcinoma patients was established based on the cancer genome atlas(TCGA)database to explore the prognostic performance of autophagy related gene risk model for lung adenocarcinoma patients and its correlation with immune microenvironment. Methods Clinical information and transcriptome data of lung adenocarcinoma patients were downloaded and extracted from TCGA database, and 232 autophagy-related genes were screened from the human autophagy database. cox regression analysis was used to screen out four autophagy genes independently associated with prognosis. The prognostic prediction model of lung adenocarcinoma was constructed by risk score, and the performance of prediction model was evaluated by ROC curve. The relationship between risk scores and tumor immune microenvironment was explored using ESTIMATE(estimation of stromal and immune cells in malignant tumour tissues using expression data) and CIBERSORT algorithms. Results Thirty differentially expressed autophagy-related genes were identified in lung adenocarcinoma, of which four autophagy genes(BIRC5,ERO1A,ITGB4,NLRC4) could predict the prognosis of the patients. Grouped by risk score, the Kaplan-Meier analysis demonstrated that the survival rate of high-risk group was significantly lower than that of low-risk group(P<0.000 1). The ROC curve proved the accuracy of the model in predicting the prognosis of lung adenocarcinoma(AUC=0.757). The ESTIMATE and CIBERSORT analyses revealed that the risk scoring model was associated with multiple immune cells and immune infiltrates in the tumor microenvironment. Conclusion Compared with clinical data, the autophagy gene prognostic risk model can better predict the prognosis of patients with lung adenocarcinoma. In the high-risk group, CD4+memory quiescent cells can improve prognosis in lung adenocarcinoma patients.

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Objective To investigate the role of connexin 43(Cx43) in acute sympathetic stress induced by phenylephrine(PE) overactivation of α1-adrenergic receptor(α1-AR) in cardiomyocytes. Methods Cardiomyocytes of H9 C2 rats were randomly divided into control group, PE alone treatment group, Gap26(Cx43 specific inhibitor) intervention group and Gap26 alone treatment group. PE alone treatment group was treated with 50 μmol/L PE for 15 min. The Gap26 intervention group was pretreated with 0.5 μmol/L Gap26 for 30 min, and then treated with 50 μmol/L PE for 15 min. The protein and mRNA expression levels of Cx43, NLRP3 inflammasome, interleukin-1β(IL-1β), Caspase-1, interleukin-18(IL-18) were detected by Western blot and qRT-PCR. The expression and co-location of Cx43 in cardiomyocytes were observed by immunofluorescence assay, and the expression of inflammatory cytokines IL-1β and IL-18 in cardiomyocytes was detected by ELISA. Results Compared with control group, the protein and mRNA levels of Cx43, NLRP3, Caspase-1 and IL-18 in PE group increased. Compared with PE alone treatment group, the protein and mRNA levels of Cx43, NLRP3, Caspase-1 and IL-18 decreased after Gap26 intervention, but they were still higher than those of control group. Similarly, immunofluorescence showed that Cx43 protein expression increased in PE alone group, while Cx43 expression was down-regulated in Gap26 intervention group compared with PE alone group. ELISA results showed that the expression of IL-1β and IL-18 was significantly up-regulated in PE alone group, but down-regulated in Gap26 intervention group. Conclusion Cx43 is involved in α1-AR activation induced cardiac acute sympathetic stress by regulating NLRP3 inflammasome.

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Objective To elucidate the molecular mechanism of receptor for advanced glycation end products(RAGE) inhibitor FPS-ZM1 in the treatment of depression in db/db mice. Methods 24 male db/db mice aged 6-8 weeks were randomly divided into type 2 diabetic group(db/db group), diabetic treatment group [db/db+FPS, FPS-ZM1 1.0 mg/(kg·d) was administered intraperitoneally] and diabetic solvent control group(db/db+Oil, mice were injected intraperitoneally corn oil with the same volume). The other 8 male db/m mice aged the same weeks were used as normal control. After administrating drugs for 12 weeks, the depression-like behavior of mice was detected by tail suspension and forced swimming test, the apoptosis of neurons in mice hippocampus was detected by TUNEL staining, and the expression of RAGE, nod-like receptor protein 3(NLRP3), interleukin-1β(IL-1β) and cysteinyl aspartate specific proteinase-1(Caspase-1) in mice hippocampus was detected by Western blot. Results In behavioral experiments db/db mice showed depressive symptoms, which could be improved by FPS-ZM1. Compared with db/m mice, the apoptosis rate of hippocampal neurons in db/db mice was significantly higher(P<0.01), which could also be significantly reduced by FPS-ZM1(P<0.01). The expressions of RAGE, NLRP3, IL-1β and Caspase-1 in the hippocampus of db/db mice were significantly higher than those of db/m mice(P<0.01) and FPS-ZM1 could still inhibit the expression of these proteins. Conclusion FPS-ZM1 can reduce the apoptosis of hippocampal neurons by inhibiting RAGE and its downstream NLRP3 signal pathway, and eventually improve the depressive symptoms of diabetic mice.

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Objective To explore the effect of miR-3188 on the proliferation, apoptosis, invasion and migration of gastric cancer cells and the underlying molecular mechanism. Methods The expression level of miR-3188 was examined in normal human gastric mucosal cells(GES-1) and human gastric cancer cell lines(HGC-27,MGC-803,BGC-823,MKN-45) by qRT-PCR. It was determined by bioinformatic analysis and dual luciferase reporter assay whether NRAGE was the target gene of miR-3188.miR-3188 mimic, miR-3188 inhibitor and negative control miR-NC were transfected into gastric cancer cell line HGC-27 respectively, and the expression of NRAGE on protein and mRNA levels was detected by Western blot and qRT-PCR. miR-3188 mimic, NRAGE overexpression plasmid(pc-NRAGE) and its negative control(miR-NC,pc-control) were separately or co-transfected into gastric cancer cell line HGC-27. CCK-8, flow cytometry and Transwell experiment were used to detect cell proliferation, apoptosis, invasion and migration in each group. The expressions of epithelial-mesenchymal transition(EMT), proliferation, apoptosis, invasion and migration related proteins [CyclinD 1,matrix metalloproteinase 9(MMP 9),Bcl-2,N-cadherin, Vimentin, E-cadherin] in each group were detected by Western blot. Results The expression of miR-3188 in human gastric cancer cell lines was lower than that of human normal gastric mucosal cells. Bioinformatics analysis and dual luciferase reporter assay confirmed that miR-3188 could target and bind to NRAGE 3′UTR.Western blot and qRT-PCR confirmed that miR-3188 negatively regulated the expression of NRAGE on the protein level in HGC-27 cells, but not on the mRNA level. Compared with the miR-NC group, Transfected miR-3188 mimic reduced the proliferation, invasion and migration ability of HGC-27 cells, and increased the apoptosis rate, and the co-transfection of pc-NRAGE could reverse the above effects. Compared with the miR-NC group, when miR-3188 was overexpressed in HGC-27 cell, the expression of cyclinD 1, MMP 9, Bcl-2, N-cadherin, and Vimentin was down regulated, while E-cadherin expression was up-regulated, and the above effects could be reversed by co-transfection with pc-NRAGE. Conclusion The miR-3188/NRAGE axis may play important roles in the progression of gastric cancer, and miR-3188 may be a potential therapeutic target for gastric cancer.

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Objective To investigate the anti-inflammatory effect of polysaccharide from atractylodes atractylodes(PAMK) on rheumatoid arthritis(RA) rats and its effect on Toll like receptor 4/nuclear factor kappa-B(TLR4/NF-κB) signaling pathway. Methods RA rat model was induced by type Ⅱ collagen, after successful modeling, the rats were divided into model group, positive drug group(Tripterygium wilfordii polyglycoside tablet), PAMK low-dose and high-dose groups, normal control group was also set, with 12 rats in each group. Tripterygium wilfordii polyglycoside tablets or Atractylodes macrocephala polysaccharide were administered intragastric administration, once a day for 4 weeks. The body weight, paw swelling rate and arthritis index of rats were measured; the thymus index and spleen index were calculated; HE staining was used to observe the histopathological changes of synovial membrane of ankle joint; The levels of interleukin-1β(IL-1β), interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) in serum and synovial tissue of ankle were determined by ELISA; The expression levels of total protein TLR4, MyD88, phosphorylated NF-κB p65(Ser536) and nuclear protein NF-κB p65 in synovial tissue of ankle were detected by Western blot. Results Compared with the normal control group, synovial hyperplasia and unclear layers were observed in the model group, body weight decreased(P<0.05), paw swelling rate, arthritis index, thymus index and spleen index increased(P<0.05), the levels of IL-1β, IL-6 and TNF-α in serum and synovial tissue of ankle, and the protein expressions of TLR4, MyD88, p-NF-κB p65 and NF-κB p65 in synovial tissue of ankle increased(P<0.05). Compared with model group, the histomathological changes of synovial of ankle joint in positive drug group and PAMK high-dose group were significantly improved, body weight increased(P<0.05), paw swelling rate, arthritis index, thymus index and spleen index decreased(P<0.05), the levels of IL-1β, IL-6 and TNF-α in serum and synovial tissue of ankle, and the protein expressions of TLR4, MyD88, p-NF-κB p65 and NF-κB p65 in synovial tissue of ankle decreased(P<0.05), however, there were no significant differences in the above indexes between PAMK low-dose group and model group(P>0.05). Conclusion PAMK can reduce the inflammatory response and improve RA, and the mechanism may be related to the inhibition of TLR4/NF-κB signaling pathway activation.

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Objective To construct and identify an overexpression recombinant adenovirus vector carrying the mouse PTG gene(NM＿016854), and to lay a foundation for in-depth study of the function of PTG. Methods The coding sequence of the mouse PTG gene was chemically synthesized, amplified by polymerase chain reaction(PCR), digested with restriction enzymes, and inserted into the GV314 vector(CMV-MCS-3 FLAG-SV40-EGFP) to obtain the recombinant shuttle plasmid pGV314-PTG. BamHⅠ/AgeⅠ double enzyme digestion was further carried out, and the product was transferred into linearized expression vector pDC315 to construct recombinant adenovirus Ad-PTG, which was transfected into HEK293 T cells and packaged into recombinant virus particles. After repeated amplification of several generations of HEK293 T cells, the recombinant adenovirus was purified and titer detected. Finally, PCR, Western blot and sequencing were used to verify the recombinant adenovirus. Results After PCR, Western blot and sequencing, the results showed that the pGV314-CMV-MCS-3 FLAG-SV40-EGFP-PTG overexpression adenovirus vector(Ad-PTG) was successfully constructed, and the virus titer measured by end-point dilution method was 4×1010PFU/ml, Western blot and RT-qPCR showed that the protein and mRNA expression levels of PTG increased significantly. Conclusion The recombinant adenovirus vector carrying mouse PTG gene is successfully constructed, and the expression of PTG gene in hepatocytes is effectively up regulated.

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Objective To investigate the effects of miR-203 a-3 p overexpression on pulmonary fibrosis after acute lung injury induced by lipopolysaccharide(LPS) in rats and its possible mechanism. Methods The rat model of pulmonary fibrosis after acute lung injury was established by multiple low-dose injections of LPS. The successfully modeled rats were randomly divided into model group, agomir-negative control group(agomir-NC group) and miR-203 a-3 p agomir group(agomir group), and another control group was set, with 15 rats in each group. One day before modeling and one week after modeling, miR-203 a-3 p agomir was injected by caudal vein for intervention. Two weeks after modeling, the wet/dry specific gravity(W/D) and hydroxyprolinic acid(Hyp) content of lung tissue were determined. The pathological changes and the degree of pulmonary fibrosis of lung tissue were observed by HE staining and Masson staining. The levels of interleukin-1β(IL-1β), interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) in broncholveolr lavage fluid(BALF) were determined by ELISA. The expression levels of miR-203 a-3 p and follicle-statin like 1(Fstl1) mRNA in the lung tissues of rats were detected by qRT-PCR. The protein expression level of Fstl1 in lung tissue of rats was detected by Western blot. The targeting relationship between miR-203 a-3 p and Fstl1 was detected by dual luciferase reporting system. Results Compared with control group, the degree of lung injury and pulmonary fibrosis in model group were relatively serious, the W/D value and Hyp content of lung tissue increased(P<0.05), the levels of IL-1β, IL-6 and TNF-α in BALF increased(P<0.05), the expression of miR-203 a-3 p in lung tissue decreased(P<0.05), while the mRNA and protein expression levels of Fstl1 increased(P<0.05). Compared with model group, the degree of lung injury and pulmonary fibrosis were improved in agomir group, the W/D value and Hyp content of lung tissue decreased(P<0.05), the levels of IL-1β, IL-6 and TNF-α in BALF decreased(P<0.05), the expression of miR-203 a-3 p in lung tissue increased(P<0.05), while the mRNA and protein expression levels of Fstl1 decreased(P<0.05). The luciferase reporter gene assay confirmed that Fstl1 was the target gene of miR-203 A-3 p. Conclusion Overexpression of miR-203 a-3 p improves pulmonary fibrosis after LPS-induced acute lung injury, and the mechanism may be related to the targeted down-regulation of Fstl1 expression.

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Objective To study the miR-1285 through Yes-associated protein 1(YAP1) on the proliferation, apoptosis and mechanism of chronic myelogenous leukemia K562 cells. Methods The plasmid of miR-1285 was constructed to make K562 cells overexpress miR-1285 and knock down miR-1285. They were divided into four groups: miR-1285 mimics, mimics control, miR-1285 inhibitor, and inhibitor control; qRT-PCR was used to quantitatively analyze the expression of miR-1285 of these four groups. The expression level of miR-1285 in the group was used to verify the transfection efficiency. The apoptosis and proliferation of the four groups were detected by CCK-8 and Annexin V-FITC; the changes of YAP and its downstream molecule epidermal growth factor receptor(EGFR) were detected by Western blot, and apoptosis related molecules BAX, Bcl-2 protein expression was detected. Results After knock down miR-1285, it can promote the proliferation of K562 cells and inhibit apoptosis. The expression of YAP increased, the expression of downstream molecule EGFR increased correspondingly, the expression of apoptosis-related molecule BAX decreased, and the expression of Bcl-2 increased; After expressing miR-1285, K562 cell proliferation decreased, apoptosis increased, YAP expression decreased, downstream molecule P-EGFR expression decreased, apoptosis molecule BAX expression increased, and Bcl-2 expression decreased. Conclusion miR-1285 can inhibit the proliferation and promote apoptosis of K562 cells of chronic myelogenous leukemia by inhibiting the expression of YAP. It is expected to become a targeted molecule for the treatment of chronic myelogenous leukemia.

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Objective To study the resistance characteristics, carbapenemase genotypes and the homology of carbapenem-resistantEscherichia coli(CREC). Methods 6 092Escherichia coliisolated from clinical specimens in The First Affiliated Hospital of Anhui Medical University were collected and 71 strains of CREC were selected. The identification and antimicrobial susceptibility test were carried out by Vitek-2 Compact. Confirmation of carbapenemase phenotype was performed by modified hodge test(MHT), modified carbapenem inactivation method(mCIM) and carbapenemase nordmann-poirel(Carba NP) test. Carbapenemase-encoding genes(blaKPC, blaNDM, blaVIM, blaIMP,etc.) were identified by PCR and positive amplification products were sequenced, and then analyzed by using BLAST programs. ERIC-PCR fingerprinting was used to determine the clonal relationship between the different strains. Results CREC strains were mainly distributed in intensive care unit(ICU) and burn department, and the source of specimens was mainly urine. The drug susceptibility results showed that the resistance rates of CREC to ciprofloxacin, levofloxacin and cotrimoxazole were all above 70%, and the resistance rates to amikacin and tobramycin were less than 50%. Among 71 CREC strains, the number of positive strains for MHT, mCIM and Carba NP were 45, 67 and 69, and the positive rates were 63.38%, 94.37% and 97.18%, respectively. Carbapenemase genes were detected in 43 CREC isolates, of which 34 strains(79.07%, 34/43) carried blaNDM, 9 strains(20.93%, 9/43) carried blaKPC-2. In addition, the rates of strains harbored blaNDM-1or blaNDM-5were 20.59%(7/34) and 79.41%(27/34), respectively. Other carbapenem genes such as blaIMP, blaVIMand blaOXA-48were not detected. According to the fingerprint of ERIC-PCR, CREC was divided into 19 genotypes A-S, and no dominant genotype was found. Conclusion Drug resistance rate of clinically isolated CREC in our hospital is high, showing multi-drug resistance. blaNDMis the main carbapenemase gene of CREC. The epidemic CREC in our hospital has high genetic diversity and the homology of CREC is dispersive.

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Objective To explore the effect and mechanism of breviscapine(Bre) on the formation of intracranial aneurysm(IA) and its influence on nuclear factor erythroid 2-related factor 2(Nrf2) pathway in a rat model. Methods The rat model of IA was established by elastase injection. The rats were randomly divided into Sham group, Model group and Bre group with 15 rats in each group. The Bre group was intraperitoneally injected with 50 mg/kg Bre every day, the Sham group and Model group were intraperitoneally injected with the same volume of normal saline for 3 weeks. During this period, the incidence of IA, survival rate and systolic blood pressure were recorded, immunofluorescence staining and qRT-PCR were employed to assay the expression of α-smooth muscle actin(α-SMA) and smooth muscle 22α(SM22α). Hydrogen peroxide(H2O2, 0.5 mmol/L) was used to treat rat vascular smooth muscle cells(VSMC) to induce oxidative damage, and then co-incubated with Bre(100 μmol/L) or/and ML385(Nrf2 inhibitor), Western blot, qRT-PCR, ELISA, DCFH-DA fluorescence staining and flow cytometry were used to detect the expression of Nrf2, contractile phenotype-related protein and inflammatory cytokine, reactive oxygen species(ROS) production and cell apoptosis rate, respectively. Results In IA rats, Bre treatment up-regulated the expression of nuclear Nrf2, improved the pathological changes of IA, reduced the incidence of IA, improved the survival rate, and lowered the systolic blood pressure. In VSMC treated with H2O2, Bre pretreatment increased the expression of Nrf2, antioxidant enzymes, α-SMA and SM22α, reduced the expression of matrix metalloproteinases(MMP)-2 and MMP-9, the production of ROS and cell apoptosis, and reduced inflammation in cerebral arteries cell infiltration and expression of pro-inflammatory cytokines. Inhibition of Nrf2 weakened the therapeutic effect of Bre pretreatment in H2O2treated VSMC. Conclusion Bre can effectively reduce the oxidative stress and inflammation in VSMC, thereby reducing the formation and rupture of IA in rat. The mechanism may be related to the activation of the Nrf2 pathway.

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Objective To investigate the role of LncRNA RP11-20 G6.3 and Toll-like receptor 4(TLR4)/nuclear factor kappa-B(NF-κB) signaling pathway in airway inflammation and remodeling in chronic obstructive pulmonary disease(COPD). Methods The wild-type(WT) and TLR4 knock out(KO) C57 BL/6 male mice were used to construct COPD model. The expression of TLR4/NF-κB signaling pathway protein was analyzed by Western blot; the levels of interleukin-1β(IL-1β), interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) in bronchoalveolar lavage fluid( BALF) were detected by enzyme-linked immunosorbent assay( ELISA). The differentially expressed LncRNAs were identified by RNA sequencing and functional enrichment analysis,and the competing endogenous RNAs( ceRNA) networks were predicted using RNAhybrid. Luciferase reporter was used to explore relationships between genes. Results In COPD model, compared with WT mice, the lung inflammation of TLR4-/-mice was significantly reduced,the expression of NF-κB related protein,the levels of inflammatory factors( IL-1β,IL-6 and TNF-α) in BALF and Masson trichromatic staining area significantly decreased in TLR4-/-mice. RNA sequencing and functional enrichment analysis confirmed that RP11-20G6. 3 was one of the differentially expressed LncRNA. RP11-20G6. 3 was up-regulated in lung tissue of COPD patients,and its expression was significantly correlated with FEV1( ρ = 0. 549,P = 0. 047). RP11-20G6. 3 plasmid intervention aggravated the inflammation of lung tissue in TLR4-/-COPD mice,and increased the Masson tricolor area around bronchi and the levels of inflammatory factors( IL-1β,IL-6 and TNF-α) in BALF. Luciferase reporter gene analysis showed that RP11-20G6. 3 acted as a ceRNA for miR-34c-5p in COPD,and sponges the latter to upregulate Col1a1. Conclusion TLR4/NF-κB relays the damage signals following COPD and activates the downstream RP11-20G6. 3/miR-34c-5p/Col1a1 ceRNA network which triggers airway inflammation and remodeling.

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Objective To study the antidepressant effects of extracts fromZanthoxylum bungeanumandZanthoxylum schinifoliumin mice exposed in chronic restraint stress. Methods 45 mice of SPF male ICR were randomly divided into five groups: normal group, model group, sertraline hydrochloride group,Zanthoxylum bungeanumextract group andZanthoxylum schinifoliumextract group, with 9 mice in each group. Except for the normal group, mice in all groups were chronically restrained to establish the depressive model. The normal group and model group were given olive oil by intragastric administration, and other groups were given sertraline hydrochloride(10 mg/kg),Zanthoxylum bungeanumextract(200 mg/kg) andZanthoxylum schinifoliumextract(200 mg/kg), respectively. The behaviors of mice in each group were evaluated by open-field test and forced swimming test. The levels of brain-derived neurotrophic factor(BDNF), 5-hydroxytryptophan(5-HT), glutathione(GSH) and nitric oxide(NO) in serum and brain were determined by ELISA. Results Compared with model group,Zanthoxylum bungeanumextract could reduce the percentage of immobility time in forced swimming test, increase the level of cortical BDNF, and improve the levels of 5-HT and GSH in cortex and serum, with statistical significance(P<0.05).Zanthoxylum schinifoliumextract could improve the behavior of mice in open field test and forced swimming test, increase the levels of cortex 5-HT and serum GSH, and decrease the contents of NO in serum and cortex. Conclusion The extracts ofZanthoxylum bungeanumandZanthoxylum schinifoliumcan improve the depression-like behaviors of mice exposed to chronic restraint stress.

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Objective To explore the mechanism of maternal vitamin D deficiency(VDD)-induced fetal intrauterine growth restriction( IUGR) during pregnancy. Methods Four weeks female CD-1 mice were divided into two groups: control mice( CTRL) and vitamin D deficiency mice( VDD). VDD mice were fed with diet with low concentration of vitamin D and mated with normal male mice. Several pregnant mice were killed on gestational sixteenth day,and the placentas,serum of maternal and fetal mice were collected. Proteins of placental key enzymes of steroid hormone synthesis and signaling pathways of oxidative stress were measured using Western blot. Serum estrogen,progesterone and 25-( OH)-D were measured via radioimmunoassay. The remaining pregnant mice were killed. Placentas and fetus were weighted,fetus height and placental diameter were calculated. The pregnancy outcomes were observed. Results Diet with low concentration of vitamin D were used and VDD mice model were established after five weeks. Maternal VDD during pregnancy decreased the weight of placenta and fetus,reduced the height of fetus and the diameter of placentas. The expression of placental aromatase cytochrome p450( CYP19) and the level of estrogen were increased in VDD pregnant mice. Besides,maternal VDD during pregnancy increased the expressions of placental heme oxygenase 1( HO-1),NADPH oxidase 4( NOX-4) and 3-nitrotyrosine( 3-NT).VDD promoted placental nuclear factor E2-related factor 2( Nrf-2) nuclear translocation. Conclusion Maternal vitamin D deficiency during pregnancy causes IUGR partially through evoking placental oxidative stress and promoting estrogen synthesis.

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Objective To investigate the effects of embryonic inflammation on the hippocampal synaptosomal-associated protein 25(SNAP-25) level and cognitive function in middle-aged. Methods During gestational days 15-17, the CD-1 maternal mice received a daily intraperitoneal injection of lipopolysaccharides(LPS, 50 μg/kg) or the equal volume of normal saline, and the corresponding offspring were regarded as LPS group and CON group respectively. At the age of young(3-month-old) and middle-aged(15-month-old), the spatial learning and memory ability was assessed using Morris water maze(MWM), and the expression of hippocampal SNAP-25 protein was detected by immunohistochemical method and Western blot. Results Compared with the 3-month CON group, the 15-month CON group had longer swimming distance(P<0.01), lower swimming distance percentage(P<0.01) in the target quadrant, and higher hippocampal subregions(CA1, CA3, DG) SNAP-25 levels(P<0.01). The same results were obtained in 15-month LPS group compared with 15-month CON group in learning and memory phase(P<0.05), and higher hippocampal subregions(CA1, DG) SNAP-25 levels(P<0.01). Pearson correlation analysis indicated that the hippocampal CA1 and CA3 subregions SNAP-25 level was positively correlated with the swimming distance, but negatively correlated with the percentage of swimming distance in the target quadrant. Conclusion Embryonic inflammation can accelerate the impairment of spatial learning and memory and the increase of hippocampal CA1 and CA3 subregions SNAP-25 protein in middle-aged CD-1 mice, and there may be a correlation between them.

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Objective To investigate the role of protein demethylase of lysine-specific demethylase 2 A(KDM2 A) in airway inflammation and remodeling in rats with bronchial asthma. Methods Intraperitoneal injection of chicken ovalbumin(OVA) sensitized aerosol to stimulate the asthma rat model. Eighteen 6-8 weeks old SPF SD female rats were randomly divided into 3 groups, namely the control group, the asthma group for 4 weeks, and the asthma group for 8 weeks. After the success of the asthma model, the infiltration of inflammatory cells in the trachea and perivascular of the lung tissue was observed by HE staining; Goblet cell proliferation and mucus secretion were observed by PAS staining; Masson staining was used to observe the fibrosis of airway wall; The proliferation of airway smooth muscle was observed by fluorescence immunohistochemistry; The protein expression level of KDM2 A was detected by Western blot. Results The inflammatory scores and inflammatory cell counts of rats in the asthma 4-week group and the asthma 8-week group were higher than those in the control group. There was a statistically significant difference in the above indicators between the asthma two groups(P<0.01). Compared with the control group, the proliferation of goblet cells and mucus secretion in the airway epithelium, the deposition of collagen fibers in the lung tissue and the proliferation of bronchial smooth muscle cells increased in the asthma groups(P<0.01). With the prolonged OVA sensitization time, airway inflammation and airway remodeling changes were more severe in the asthma 8-week group than those in the asthma 4-week group. The results of Western blot showed that the expression of KDM2 A in the lung tissues of the asthmatic 4-week group and the asthmatic 8-week group was higher than that of the control group, and the expression of KDM2 A in the asthma 8-week group was also higher than that of the asthma 4-week group. The differences were statistically significant(P<0.01). The expression of KDM2 A protein in lung tissue of rats in each group was positively correlated with airway inflammation score, total number of inflammatory cells, goblet cell mucus secretion score, lung tissue fibrosis ratio, and airway smooth muscle hyperplasia area(allP<0.01). Conclusion KDM2 A may play an important role in the pathogenesis of airway inflammation and airway remodeling in bronchial asthma.

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Objective To explore the effects of melatonin(MT) on the fertilization of in vitro matured oocytes in controlled ovarian hyperstimulation(COH) cycles and the development of subsequent embryos. Methods Imma-ture human oocytes from COH cycles were collected for in vitro maturation(IVM) culture and intracytoplasmic sperm injection( ICSI) insemination,and then the cleavage embryo and blastocyst culture medium supplemented with 0,10-11,10-9,10-7or 10-5mol/L MT were used in order to perform embryo culture in vitro,next the formed high-quality blastocysts were picked up and cryopreserved via vitrification,finally,array-CGH technology was used to detect aneuploidy of rewarmed high-quality blastocysts. Results The blastocyst rate of 10-9mol/L group was significantly higher than that of 10-11,10-7,10-5and 0 mol/L groups( P<0. 05,P<0. 01,P<0. 000 1,P<0. 01),respectively; the high-quality blastocyst rate of 10-9mol/L group was higher than that of other groups,but there were only significant differences compared with 10-5mol/L and 0 mol/L groups( P<0. 01,P<0. 05); the incidence of aneuploidy in MT group( 17. 6%) was lower than that in non-MT group( 33. 3%),but no significant difference was found. Conclusion The addition of MT to human embryo culture medium can promote the in vitro development of embryo,which is related to concentration,and 10-9mol/L is the optimal concentration.

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Objective To explore the molecular biological mechanism of curcumol in regulating ferroptosis and autophagy. Methods The Pharm Mapper database was used to screen out curcumol targets, various known related databases were used to establish a database of ferroptosis and autophagy related targets, and the String database was used to construct a protein-protein interaction network. The key targets were enriched and analyzed using DAVID database. Results 152 curcumol targets, 259 ferroptosis targets, and 796 autophagy targets were obtained; curcumol mainly regulated the ferroptosis process through PTGS2, ALB, MAPK1, MAPK8, and MAPK14 targets, and curcumol mainly through HSP90 AA1, MAPK1, MAPK8, ALB, NOS3 targets regulated the autophagy process, and curcumol mainly regulated the ferroptosis and autophagy process through ALB, MAPK1, MAPK8 targets. Conclusion Curcumol can exert pharmacological effects by regulating ferroptosis and autophagy.

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Objective To investigate the pathogenicity of rare damaging variants from ciliary pathway to human lumbosacral neural tube defects(NTDs). Methods The coding region sequences of 49 ciliary genes were amplified by AmpliSeq technique and sequenced by PGM sequencing platform for screening the rare damaging variants, and the parents without phenotype were used as controls to evaluate the pathogenicity of variants. Results The rare de novo mutation of GLI3 gene(c.C580 T,p.H194 Y) was detected in one patient.The rare complex heterozygous mutations of CRB2 gene(c.G1392 C,p.R464 S;c.T3448 C,p.C1150 R) was detected in another patient. Conclusion Rare damaging variants from ciliary pathway may be associated with the occurrence of human lumbosacral NTDs.

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Objective To explore the prognostic factors of patients with cervical cancer and establish a nomogram to predict overall survival of patients with cervical cancer. Methods The information of age, pathological features, radiotherapy and chemotherapy of 513 patients with cervical cancer were collected. The Log-rank test and Cox regression models were used for analyzing overall survival of patients with cervical cancer. A nomogram was established based on the results of multivariate analysis. The C-index, calibration plots and receiver operating characteristic curves were used to evaluate the predictive ability and accuracy of the nomogram. Results A total of 456 patients were followed up in this study, 57 cases were lost to follow-up, the loss rate was 11.11%. As the results of univariate and multivariate analysis showed that the adenocarcinoma, poor differentiation, tumor size ≥4 cm, advanced stage, and no radiotherapy were independent risk factors for overall survival of cervical cancer. A nomogram for overall survival of cervical cancer was established based on multivariate analysis, and after validated, the C-index of the nomogram was 0.83. Meanwhile, the 1-, 3-, 5-year calibration plots of the nomogram matched the 45-degree line well. In addition, the 1-, 3-, 5-year AUC values of the nomogram were 0.836, 0.847 and 0.824, respectively. Conclusion Based on the results of multivariate analysis, the five prognostic factors of cervical cancer, including histological type, cell differentiation, tumor size, stage, and radiotherapy were used to establish a nomogram with good predictive ability and accuracy. The establishment of the overall survival nomogram of cervical cancer was useful for gynecological oncologists to make a more accurate assessment of the prognosis of patients with cervical cancer and guide the clinical individualized treatment.

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Objective To investigate the associations of the single nucleotide polymorphism(SNP) rs1803489, rs7617620 and rs13078528 in vestigial like family member 4(VGLL4) gene with the risk of non-cardia gastric cancer. Methods The case-control study design was used. 450 cases with non-cardia gastric cancer confirmed by pathology and 450 normal controls were collected in the Han population in Baotou; SNP rs1803489, rs7617620, and rs13078528 inVGLL4were genotyped using polymerase chain reaction(PCR)-restriction fragment length polymorphism(RFLP) technology. Results VGLL4gene SNP rs1803489 was associated with the risk of non-cardia gastric cancer in the Han population in Baotou. Compared with the carriers of GG genotype, the carriers of AG genotype had a higher risk of non-cardia gastric cancer(OR=1.511, 95%CI=1.095-2.085,P<0.05). SNP rs7617620 and rs13078528 were not associated with non-cardia gastric cancer(P>0.05). Conclusion SNP rs1803489 inVGLL4may have an effect on the risk of non-cardia gastric cancer in the Han population in Baotou. SNP rs7617620 and rs13078528 may not play a major role in the risk of non-cardia gastric cancer.

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Objective To establish an assessment model for predicting the aggressiveness of papillary thyroid microcarcinoma(PTMC) and to provide a theoretical basis for actively monitoring the same. Methods 264 PTMC patients were included from October 2017 to January 2021. All patients were confirmed by postoperative pathology. 154 cases collected from October 2017 to April 2019 were included in the model group while 110 cases collected from May 2019 to January 2021 were included in the validation group. We analyzed the clinical data, ultrasound characteristics, and BRAF V600 E gene status of 154 patients in the model group with confirmed PTMC based on pathological examination. Single factor regression was used to screen out risk factors for PTMC invasion, and these factors were then included in a multivariate logistic regression analysis to establish a risk prediction model; the established model was used to evaluate the diagnostic efficacy of 110 PTMC patients in the validation group. Results Multivariate analysis showed that male sex, age<45 years, microcalcification, tumor diameter>5 mm, suspected extraglandular invasion and lymph node metastasis on ultrasound, and BRAF V600 E gene mutation were all risk factors for PTMC invasion. A scoring model was established according to risk factors, and the higher the score, the higher the risk. In the 110-case verification group, the area under the predictive performance curve of the evaluation prediction model was 0.774(95%CI: 0.685-0.848), the cut-off value was 0.450 2, the sensitivity was 83.3%, and the specificity was 62.9%. The model therefore had good diagnostic efficacy. Conclusion Ultrasound combined with BRAF V600 E gene detection model can predict the aggressiveness of PTMC to a certain extent, which can provide reference for the selection of clinical treatment options.

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Objective To investigate the risk factors affecting the patency time of the125I seeds stent in malignant obstructive jaundice. Methods A retrospective analysis of 113 patients with malignant obstructive jaundice underwent biliary tract125I seeds stent implantation. The gender, age, obstruction site, type of125I seeds stent, primary tumor type, and postoperative response to treatment of tumor were enrolled for analysis to evaluate the related risk factors affecting the patency time of the stent. Results Univariate analysis showed that the location of biliary obstruction, the type of125I seeds stent, the type of primary tumor, and the type of primary tumor were the main factors affecting the patency time of the stent(P<0.001); Cox multivariate regression analysis showed biliary obstruction location, the type of125I seeds stent, and whether the primary tumor treated were independent factors that affected the patency time of the stent(P<0.001). Conclusion Multi-factor analysis shows that the location of biliary obstruction, the type of125I seeds stent, and the primary tumor are independent risk factors that affect the patency of the stent for malignant obstructive jaundice, which shows important markers for evaluating the prognosis of patients treated with this method.

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Objective To investigate the change of serum microRNA-15 a(miR-15 a) expression level in patients with gestational diabetes mellitus and its relationship with maternal and infant adverse outcomes. Methods 143 cases of gestational diabetes mellitus patients were selected as the study group, and 124 healthy pregnant women were selected as the control group, and the expression levels of serum miR-15 a of the two groups were detected by real time fluorescent quantitative reverse transcription polymerase chain reaction(qRT-PCR). Follow up to delivery, the adverse maternal and infant outcomes of the two groups were observed, and the relationship between the expression level of serum miR-15 a and the adverse outcomes of maternal and infant in gestational diabetes mellitus was analyzed by multivariate Logistic regression analysis method. Results The expression level of serum miR-15 a in the study group was higher than that in the control group, and the difference was statistically significant(P<0.05). The incidence rates of abortion, premature rupture of membranes, polyhydramnios, postpartum hemorrhage, postpartum infection, premature infants, macrosomia, fetal distress, neonatal asphyxia, neonatal respiratory distress syndrome and neonatal hypoglycemia and the total incidence rates of maternal and perinatal adverse outcomes in the study group were higher than those in the control group(P<0.05), but there was no significant difference in the incidence rate of fetal death between the study group and the control group(P>0.05). The proportions of age>35 years old, pre-pregnancy body mass index ≥24.0 kg/m2, history of adverse pregnancy and childbirth and poor blood glucose control and the expression level of serum miR-15 a in poor maternal outcome group and poor perinatal outcome group were higher than those in good maternal outcome group and good perinatal outcome group(P<0.05), and multivariate Logistic regression analysis showed that the above indicators were the risk factors of adverse maternal and infant outcomes in patients with gestational diabetes mellitus(P<0.05). Conclusion The expression level of serum miR-15 a in gestational diabetes mellitus patients abnormally increases, and it can increase the risk of adverse maternal and infant outcomes, which is closely related to the occurrence of adverse maternal and infant outcomes. Besides, the age, pre-pregnancy body mass index, adverse pregnancy history, blood glucose control also have a certain impacts on the occurrence of adverse maternal and infant outcomes in patients with gestational diabetes mellitus.

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Objective To explore the predictive value of left ventricular spherical index(LVSI) in prognosis of patients with dilated cardiomyopathy(DCM). Methods The DCM patients from hospital were included. All DCM patients were treated with standard anti-heart failure drugs and followed up for six months. They were divided into a good prognosis group according to whether adverse cardiovascular events(MACE) occurred(n=77) and poor prognosis group(n=154). All patients′ age, course of disease, body mass index(BMI), blood pressure, N-terminal fragment of the BNP precursor(NT-proBNP), echocardiogram indicators and other clinical data were collected, and the drug consumption during the follow-up period and the occurrence of MACE events were recorded. Binomial logistic regression was used to analyze the risk factors of MACE events in DCM patients, and receiver operating characteristic(ROC) curves were used to analyze the predictive value of LVSI for MACE events in DCM patients. Results Compared with the good prognosis group, the patients in the poor prognosis group had a longer course of disease, faster heart rate, and increased NT-proBNP, left atrium diameter(LAD), left ventricular end diastolic diameter(LVEDd), and LVSI; systolic blood pressure and left ventricular ejection fraction(LVEF) decreased(P<0.001), and other indicators had no statistical difference. The results of binomial logistic regression analysis showed that the duration of heart failure(OR=1.187,95% CI:1.091-1.290, P<0.001), admission systolic blood pressure(OR=2.089, 95%CI:1.243-3.510,P=0.005), LVEDd(OR=1.241, 95%CI:1.080-1. 426,P = 0. 002),LVEF( OR = 0. 862,95% CI: 0. 771-0. 963,P = 0. 009),LVSI( OR = 1. 462,95% CI:1. 262-1. 693,P<0. 001),NT-proBNP( OR = 1. 001,95% CI: 1. 000-1. 001,P = 0. 002) were independent predictors of MACE in DCM patients. LVSI of 61. 95% was the best critical value for predicting the occurrence of MACE. At this time,the area under the ROC curve was 0. 821( 95% CI: 0. 769-0. 874,P<0. 001),the maximum Youden index was 0. 461,and the sensitivity: 0. 695,specificity: 0. 766. Conclusion LVSI,LVEDd,LVEF,heart failure course,admission systolic blood pressure,NT-proBNP are the prognostic factors of DCM patients. The left ventricular spherical index has a certain predictive effect on the prognosis of DCM patients.

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Objective To investigate the expression of miR-155 in peripheral blood of type 2 diabetes patients(T2 DM) associated with diabetic foot ulcer(DFU) and its relationship with the onset of DFU. Methods Sixty newly diagnosed T2 DM patients without DFU(T2 DM group), 112 T2 DM patients with DFU(DFU group), and 60 healthy controls with normal glucose tolerance(NC group) were included. MiR-155 levels were determined by quantitative real-time PCR, while clinical features and risk factors of DFU were explored. Results A significant decrease in the expression level of miR-155 in peripheral blood was observed in T2 DM group compared with NC group(P<0.05), and a markedly increased miR-155 expression level was noted in DFU group compared with T2 DM group(P<0.01). Moreover, there was a positive correlation between the expression level of miR-155 in peripheral blood and the course of foot ulcer and Wagner grade of foot ulcer(P=0.02,P=0.01) while a negative correlation in the expression level of miR-155 with healing rate of DFU after eight weeks(P=0.04). The multiple stepwise logistic regression analysis confirmed that a high expression of miR-155 was an independent risk factor for DFU(OR=3.98,P=0.002). Conclusion An increased expression of miR-155 in peripheral blood of T2 DM patients is an independent risk factor for DFU and is closely related to the prognosis of DFU.

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To study the antiviral effect ofZedoary TurmericOil Injection on novel coronavirus, SARS-CoV-2 viroid cell lines were preparedin vitroand treated with different concentrations of Zedoary Oil. The cell number and relative fluorescence value(RLU) were observed and measured, and the 50% effective inhibitory concentration(IC 50) was calculated. Four patients with Coronavirus Disease 2019 were clinically included, including 2 in the control group and 2 in the experimental group. The control group received conventional treatment, and the experimental group receivedZedoary TurmericOil Injection in addition to conventional treatment. The nucleic acid conversion rate, conversion time, pulmonary imaging changes, fever reduction time, clinical improvement time and adverse events of the patients were observed.In vitroexperiment, the relative fluorescence value decreased with increasing concentration ofZedoary TurmericOil, which was significantly different from that of the control group(P<0.05). The IC50 was 0.26 μg/ml.In vivostudy, the novel coronavirus nucleic acid in stool of case 1 in the test group turned negative in 3 days, the cough symptom of case 2 was significantly relieved, and there was obvious absorption in pulmonary imaging. The negative conversion time of novel coronavirus nucleic acid in the control group was 5 and 7 days respectively. No adverse events occurred in the experimental group.Zedoary TurmericOil had strong inhibitory effect on SARS-COV-2 virusin vitrowhich was dose-dependent.In vivotreatment of COVID-19,Zedoary TurmericOil Injection combined with conventional treatment can improve the cough caused by SARS-COV-2 infection, promote SARS-COV-2 to turn negative, promote absorption of lung lesions, and reduce lung injury, with no obvious adverse events.