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ObjectiveTo investigate the effect of Yishen Tongluo prescription （YSTLP） on apoptosis of renal tubular epithelial cells and explore the mechanism based on endoplasmic reticulum stress pathway of protein kinase R-like endoplasmic reticulum kinase （PERK）/activating transcription factor 4 （ATF4）/transcription factor C/EBP homologous protein （CHOP）. MethodThe db/db mice were randomly divided into model group， valsartan group （10 mg·kg-1）， and low， middle， high-dose YSTLP groups （1， 2.5， 5 g·kg-1）. Samples were collected after eight weeks of drug intervention. In addition， db/m mice in the same litter served as the control group. Human renal tubular epithelial cells （HK-2） were cultured in vitro and divided into the control group， advanced glycated end-product （AGE） group， and AGE + low， middle， and high-dose YSTLP groups （100， 200， 400 mg·L-1）. TdT-mediated dUTP nick end labeling （TUNEL） staining was used to detect the apoptosis rate of HK-2 cells. Methylthiazolyldiphenyl-tetrazolium bromide （MTT） assay was conducted to detect the viability of HK-2 cells. Calcium fluorescence probe staining and luciferase reporter gene method were adopted to detect the luciferase activity of folded protein response element （UPRE） and endoplasmic reticulum stress. Immunohistochemical （IHC） analysis was carried out to measure the protein expressions of phosphorylated PKR （p-PERK）， CHOP， and ATF4. Real-time polymerase chain reaction （Real-time PCR） was used to measure the mRNA expression levels of CHOP and X-box binding protein 1 （XBP1） in mouse kidney and HK-2 cells. Western blot was used to detect the protein expression level of p-PERK， PERK， CHOP， ATF4， B-cell lymphoma-2 （Bcl-2）， Bcl-2 associated X protein （Bax）， and cleaved Caspase-3 in mouse kidney and HK-2 cells. ResultIn the cellular assay， HK-2 cell viability was significantly reduced， and the apoptosis rate was elevated in the AGE group compared with the control group （P<0.01）. The mRNA and protein expression levels of apoptosis-related factor Bcl-2 were significantly reduced （P<0.01）， and those of Bax were significantly increased （P<0.01）. The protein expression level of cleaved Caspase-3 was significantly increased （P<0.01）. Compared with the AGE group， YSTLP administration treatment resulted in elevated cell viability and reduced apoptosis rate （P<0.01）. The mRNA and protein expression levels of Bcl-2 were significantly elevated in a time- and dose-dependent manner （P<0.01）， and those of Bax were significantly reduced in a time- and dose-dependent manner. The protein expression level of cleaved Caspase-3 was significantly reduced in a time- and dose-dependent manner （P<0.01）. The intracellular Ca2+ imbalance and UPRE luciferase fluorescence intensity were increased in the AGE group compared with the control group （P<0.01）. The mRNA levels of endoplasmic reticulum stress-related factors CHOP and XBP1 were significantly increased （P<0.01）， and the protein expression levels of p-PERK， CHOP， and ATF4 were significantly increased （P<0.05）. Compared with the AGE group， YSTLP effectively improved intracellular Ca2+ imbalance in HK-2 cells and decreased UPRE luciferase fluorescence intensity in a dose-dependent manner （P<0.01）. It reduced the mRNA levels of endoplasmic reticulum stress-related factors CHOP and XBP1 （P<0.01） and the protein expression levels of intracellular p-PERK， CHOP， and ATF4 in a dose- and time-dependent manner （P<0.01）. In animal experiments， the protein expression level of Bcl-2 was significantly reduced（P<0.01）， and that of cleaved Caspase-3 and Bax was significantly increased in the model group compared with the control group （P<0.05）. The protein expression level of Bcl-2 was dose-dependently elevated， and that of cleaved Caspase-3 and Bax was dose-dependently decreased in the YSTLP groups compared with the model group （P<0.01）. Compared with the control group， the mRNA expression levels of CHOP and XBP1 were significantly elevated in the model group （P<0.05， P<0.01）， and the protein expression levels of p-PERK， CHOP， and ATF4 were significantly increased （P<0.05）. Compared with the model group， YSTLP significantly decreased the mRNA expression levels of CHOP and XBP1 （P<0.01） and the protein expression levels of p-PERK， CHOP， and ATF4 （P<0.01）. ConclusionYSTLP can effectively inhibit endoplasmic reticulum stress and improve apoptosis of renal tubular epithelial cells， and its mechanism may be related to the regulation of the PERK/AFT4/CHOP pathway.
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ObjectiveTo explore the protective effect and mechanism of Zingiberis Rhizoma Recens alcohol extract on lipopolysaccharide （LPS）-induced acute lung injury in mice. MethodBalb/c mice were randomly divided into normal group， model group， dexamethasone group， and low- and high-dose Zingiberis Rhizoma Recens groups. Mice in the normal group were instilled with normal saline through the nose， and the other groups were instilled with normal saline containing LPS （50 μg）. After 30 minutes of modeling， the dexamethasone group was gavaged with 5 mg·kg-1 of dexamethasone acetate solution， the low- and high-dose Zingiberis Rhizoma Recens groups were gavaged with different doses of （7， 14 g·kg-1） of Zingiberis Rhizoma Recens alcohol extract， and the normal group and the model group were gavaged with the same volume of water. After 24 hours of modeling， the total number of white blood cells in bronchoalceolar lavage fluid （BALF） was detected by cell counter， and the levels of the inflammatory factors including tumor necrosis factor （TNF）-α， interleukin （IL）-1β， IL-6， and superoxide dismutase （SOD）， and myeloperoxidase （MPO） was detected by enzyme-linked immunosorbent assay （ELISA）. Haematoxylin-eosin （HE） staining method was used to observe the pathological changes of lung tissue in each group， and the Western blot was used to detect the protein expression of nuclear transcription factor （NF）-κB p65， phosphorylation （p）-NF-κB p65， and Toll-like receptor 4 （TLR4） in lung tissue. ResultCompared with the normal group， the white blood cell count in BALF and the levels of TNF-α， IL-1β， IL-6， and MPO in the model group was increased （P<0.01）， and the level of SOD was decreased （P<0.05）. Pathological damage of lung tissue was obvious， and the protein expression of NF-κB p65， p-NF-κB p65， and TLR4 in lung tissue was increased （P<0.01）. Compared with the model group， the white blood cell count in BALF and the levels of TNF-α， IL-1β， IL-6， and MPO in the treatment group was decreased （P<0.05，P<0.01）， and the level of SOD was increased （P<0.05，P<0.01）. Pathological damage of lung tissue was alleviated， and the protein expression of NF-κB p65， p-NF-κB p65， and TLR4 in lung tissue was decreased （P<0.01）. ConclusionZingiberis Rhizoma Recens alcohol extract may play a protective role in LPS-induced acute lung injury in mice by inhibiting the TLR4/NF-κB signaling pathway.
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Reflux esophagitis is an inflammatory disease of esophageal mucosa damage caused by the reflux of gastric contents into the esophagus. Its incidence is on the rise， and it has become an important precancerous disease of esophageal cancer. Studies have shown that the continuous inflammatory response stimulates the esophageal mucosa， causing abnormal proliferation of esophageal epithelial cells and damage to esophageal mucosal tissue， which eventually leads to the occurrence of heterogeneous hyperplasia and even carcinogenesis. The nuclear transcription factor-kappa B （NF-κB） signaling pathway is one of the most classical inflammatory and cancer signaling pathways. It has been found that abnormal activation of the NF-κB signaling pathway is crucial to the development and prognosis of reflux esophagitis and esophageal cancer. It is widely involved in the proliferation， autophagy， apoptosis， and inflammatory response of esophageal epithelial cells and tumor cells， accelerating the transformation of reflux esophagitis to esophageal cancer and making it a potential target for the treatment of reflux esophagitis and esophageal cancer. Currently， there is no specific treatment for reflux esophagitis and esophageal cancer， and large side effects often appear. Therefore， finding a promising and safe drug remains a top priority. In recent years， traditional Chinese medicine scholars have conducted a lot of research on NF-κB signaling pathway， and the results indicate that NF-κB signaling pathway is an important potential target for traditional Chinese medicine to prevent and treat reflux esophagitis and esophageal cancer， but there is a lack of comprehensive and systematic elaboration. Therefore， this paper summarized the relevant studies in recent years， analyzed the relationship among NF-κB signaling pathway， reflux esophagitis， esophageal cancer， and transformation from inflammation to cancer， and reviewed the research literature on the regulation of the NF-κB signaling pathway in traditional Chinese medicine to prevent and treat reflux esophagitis and esophageal cancer， so as to provide new ideas for the prevention and treatment of reflux esophagitis and esophageal cancer.
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Objective@#To study the effect of low concentrations of sodium fluoride on the osteogenic/odontogenic differentiation of human dental pulp cells (hDPCs) in vitro.@*Methods@#This study was reviewed and approved by the Ethics Committee. hDPCs were cultured using a modified tissue explant technique in vitro. The effects of different concentrations of sodium fluoride on the proliferation of hDPCs were measured by methylthiazol tetrazolium (MTT) assay. Appropriate concentrations were added to the osteogenic/odontogenic differentiation induction medium, and the cells were induced in vitro. Alizarin red S staining was used to detect the osteoblastic/odontogenic differentiation ability of the cells, and the mRNA expression of the key differentiation factors was detected by RT-qPCR. Moreover, the expression of key molecules of endoplasmic reticulum stress (ERS) was detected by RT-qPCR and Western blot. The data were analyzed with the SPSS 18.0 software package.@*Results@#Low concentration of NaF (0.1 mmol/L) could stimulate cell proliferation in vitro, while a high concentration (5-10 mmol/L) could inhibit cell proliferation (P<0.05). According to the literature and the experimental data, 0.1 mmol/L NaF was selected as the following experimental concentration. The levels of alizarin red S staining were increased after NaF induction of mixed osteogenic/odontogenic differentiation in vitro. The mRNA expression levels of key molecules for osteogenic/odontogenic differentiation, dentin sialophosphoprotein (DSPP), bone sialoprotein (BSP) and osteocalcin (OCN), were increased (P<0.05). The mRNA levels of ERS markers (splicing x-box binding protein-1 (sXBP1), glucose-regulated protein 78 (GRP78) and activating transcription Factor 4 (ATF4) were increased in NaF-treated cells. The protein expression levels of key ER stress molecules (phosphorylated RNA-activated protein kinase-like ER-resident kinase (p-PERK), phosphorylated eukaryotic initiation factor-2α (p-eIF2α) and ATF4) were higher in NaF-treated cells.@*Conclusion@#A low concentration of NaF promotes the osteogenic/odontogenic differentiation of hDPCs and increases the level of ER stress.
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OBJECTIVE To investigate the improvement effects of glycyrrhizin （GL） on Helicobacter pylori （HP）-associated gastritis in rats and its mechanism. METHODS HP-associated gastritis rat model was induced by inoculating with 1×109 cfu/mL HP. The model rats were randomly divided into model group， positive control group （HP standard quadruple group）， GL low-dose， medium-dose and high-dose groups （5， 20， 50 mg/kg）， with 12 rats in each group. Another 12 healthy rats were selected as normal control group. Except the normal control group and model group were given constant volume of normal saline intragastrically， the other groups were given corresponding drugs intragastrically， once a day， for 30 consecutive days. After administration， rats received 13C urea breath test， and delta-over-baseline （DOB） was recorded； the pathological and cellular morphological changes of gastric mucosa in rats were observed， and pathological scoring was performed； the levels of interleukin-8 （IL-8）， IL-1β， tumor necrosis factor-α （TNF-α）， reactive oxygen species （ROS） and malondialdehyde （MDA） were detected in gastric mucosa of rats； mRNA expressions of high mobility group box-1 protein （HMGB1） and nuclear factor-κ-B （NF-κB）， relative expressions of nitric oxide synthases （iNOS） and HMGB1， the phosphorylation level of NF- κBp65 were also detected in rats. RESULTS Compared with normal control group， the DOB value， histopathological score of gastric mucosa， the levels of IL-8， IL-1β， TNF-α， ROS and MDA， relative expressions of HMGB1 and NF- κB mRNA， relative expressions of iNOS and HMGB1 protein and the phosphorylation level of NF-κB p65 were all increased significantly in model group （P＜0.05）； the epithelial cells of gastric mucosa in rats were incomplete in structure and decreased in the number， with an increase in cell fragments and vacuoles， and significant cell pyknosis. Compared with model group， the changes of the above indexes in GL groups and positive control group were significantly reversed （P＜0.05）； the changes in the above indicators in the GL high-dose group were more significant than GL low-dose and medium-dose groups （P＜0.05）； the pathological changes of gastric mucosal cells in rats had all improved. CONCLUSIONS GL may inhibit inflammation and oxidative stress by inhibiting the activation of HMGB1/NF-κB pathway， thus relieving HP-induced gastric mucosal injury.
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@#Objective To study the effect of ankyrin repeat domain 49(ANKRD49)on the migration of human lung adenocarcinoma cell line NCI-H1299 and its mechanism.Methods NCI-H1299 cells were infected with lentivirus vector carrying ANKRD49 gene and shRNA targeting ANKRD49 to construct the cell models stably overexpressing and knocking down ANKRD49. Meanwhile,the control cell models infected with empty lentivirus vector and lentivirus vector with scramble sequences were constructed respectively. The expression levels of ANKRD49 mRNA and protein were detected by real-time fluorescence quantitative PCR and Western blot. The effect of ANKRD49 on cell migration was measured by scratch test. The mRNA and protein levels of matrix metalloproteinase(MMP)-2/9 and tissue inhibitor of metalloproteinase(TIMP)-1/2 were detected by real-time fluorescence quantitative PCR and Western blot. The protein expression levels of p65,p-p65,IκBα and p-IκBα were detected by Western blot.Results The levels of ANKRD49 mRNA and protein in the ANKRD49 overexpression group were significantly higher than those in the control group(t = 70. 02 and 45. 68,respectively,each P < 0. 001). Compared with the control group,the migration ability of cells in the ANKRD49 overexpression group significantly increased at 24 h and 48 h(t = 5. 343 and 3. 282,P = 0. 005 9 and 0. 030 4,respectively);The mRNA transcription levels and protein expression levels of MMP-2 and MMP-9 significantly increased(t = 9. 304 and 6. 193,P =0. 000 7 and 0. 003 5,respectively),while the mRNA and protein expression of TIMP-1 and TIMP-2 decreased significantly(t = 3. 858 and 3. 517,P = 0. 018 2 and 0. 024 5,respectively),and the values of MMP-2/TIMP-1 and MMP-9/TIMP-2 significantly increased(t = 17. 7 and 9. 682,P < 0. 001 and < 0. 01,respectively);The expression of p-p65 and pIκBα significantly increased,the total protein levels of p65 and IκBα showed no obvious change,and the values of p-p65/p65 and p-IκBα/IκBα significantly increased(t = 3. 962 and 5. 370,P = 0. 016 7 and 0. 005 8,respectively). However,knocking down of ANKRD49 presented the opposite results.Conclusion ANKRD49 promotes the migration of NCI-H1299cells by enhan-cing the expression of MMP-2/9,the values of MMP-9/TIMP-1 and MMP-2/TIMP-2 via activating NF-κB/p65 signa-ling pathway.
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Objective To investigate the effects of chronic starvation stress on the proliferation and migration of colorectal cancer cells, as well as the underlying mechanisms. Methods By using prolonged serum starvation to simulate chronic starvation stress in tumor cells, we established enduring serum-deprived models of SW480 and DLD-1 cells and observed cellular morphological change. Effects of prolonged serum starvation on SW480 and DLD-1 proliferative and migratory capabilities were assessed using CCK-8 and Transwell assays. Differential gene-expression analysis on SW480 cultured with 1% FBS or 10% FBS medium was followed by GO and KEGG pathway assessments. Migration-related protein interactions were explored using String database and Metascape software, leading to 16 genes being selected for RT-qPCR validation. Protein levels of ITGB1 and key molecules in the relevant pathways were measured. Mobility changes in SW480 were observed through Transwell assay after ITGB1 knockdown or STAT3 inhibition. Results Prolonged serum starvation significantly inhibited the proliferation of SW480 and DLD-1 cells, and DLD-1 mobility, while enhanced SW480 migration. Transcriptome analysis revealed that prolonged serum deprivation caused the upregulation of 3016 genes, among which 283 were involved in cell migration. Metascape analysis identified the correlations among potential core genes ITGB1, CD44, TNS1, STAT3, etc. Prolonged serum deprivation increased the mRNA levels of VTN, TNS1, VEGFA, STAT3, and ITGB1 while also increasing the protein levels of ITGB1 and MMP2 and the phosphorylation levels of JAK2 and STAT3. Mobility reduction in prolonged serum-starved SW480 cells was achieved through ITGB1 knockdown or a STAT3 inhibitor. Conclusion Colorectal cancer cells can endure chronic starvation stress which enhances migration capability by upregulating ITGB1 expression.
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ObjectiveThe differential expression of microRNAs （miRNAs） between the active stage and the remission stage of ulcerative colitis （UC） was analyzed by bioinformatics method， and the regulatory relationship was constructed by screening the differentially expressed genes （DEGs）. The mechanism of Xizhuo Jiedu recipe in the treatment of UC was speculated and verified by animal experiments. MethodThe miRNAs data set of colonic mucosa tissue of UC patients was obtained from the gene expression database （GEO）， and the most differentially expressed miRNAs were screened by GEO2R， Excel， and other tools as research objects. TargetScan， miRTarbase， miRDB， STRING， TRRUST， and Matescape databases were used to screen key DEGs， predict downstream transcription factors （TFs）， gene ontology （GO）， and conduct Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis. The key signaling pathways were selected for animal experiments. In animal experiments， the UC mouse model was prepared by making the mouse freely drink 2.5% dextran sodium sulfate （DSS）. Xiezhu Jiedu recipe and mesalazine were given by gavage for seven days， and the inflammatory infiltration of colonic mucosa was observed by hematoxylin-eosin （HE） staining. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of miR-155-5p in colon tissue. Immunohistochemistry and Western blot were used to detect the protein expression levels of cytokine signal transduction inhibitor （SOCS1）， phosphorylated transcriptional signal transductor and activator 3 （p-STAT3）， phosphorylated Janus kinase 2 （p-JAK2）， and retinoic acid-associated orphan receptor-γt （ROR-γt）. The expression levels of transforming growth factor-β （TGF-β）， interleukin-17 （IL-17）， interleukin-6 （IL-6）， and interleukin-10 （IL-10） in serum were detected by enzyme linked immunosorbent assay （ELISA）. ResultThe GSE48957 dataset was screened from the GEO database， and miR-155-5p was selected as the research object from the samples in the active and remission stages. 131 DEGs were screened. The GO/KEGG enrichment analysis was closely related to biological processes such as positive regulation of miRNA transcription and protein phosphorylation， as well as signaling pathways such as stem cell signaling pathway， IL-17 signaling pathway， and helper T cell 17 （Th17） cell differentiation. The Matescape database was used to screen out 10 key DEGs， among which SOCS1 was one of the key DEGs of miR-155-5p. Further screening of the TFS of key DEGs revealed that STAT3 was one of the main TFs of SOCS1. The results of animal experiments showed that Xiezhu Jiedu Recipe could effectively down-regulate the mRNA expression of miR-155-5p and protein expression of p-STAT3， p-JAK2， and ROR-γt in colon tissue of UC mice and the expression of IL-17 and IL-6 in serum of UC mice， up-regulate the protein expression of SOCS1 and the expression of TGF-β and IL-10， increase the level of anti-inflammatory factors， and reduce inflammatory cell infiltration. ConclusionIt is speculated that Xizhuo Jiedu recipe may interfere with SOCS1 by regulating the expression of miR-155-5p in UC mice， inhibit the phosphorylation of STAT3， inhibit the differentiation of CD4+ T cells into Th17 cells， reduce the levels of pro-inflammatory factors （IL-17 and IL-6）， and increase the levels of anti-inflammatory factors （TGF-β and IL-10）. As a result， the inflammation of colon mucosa in UC mice was alleviated.
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Objective:To discuss the regulatory effect of physiological tensile stress on the differentiation of chondrocytes,and to clarify the associated signaling pathway mechanism.Methods:The ATDC5 chondrocytes were cultured in vitro and subjected to physiological tensile stress by four-point bending cell mechanical loading device.Initially,the cells were divided into control group and tensile stress group(2 000 μstrain/2 h group),and further divided into different stress magnitudes(1 000,2 000,and 3 000 μstrain)for 2 h,and 2 000 μstrain for different duration time(1,2,and 4 h)groups;the cells without tensile stress were used as control group.Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of type Ⅱ collagen(Col-Ⅱ),type Ⅹ collagen(Col-Ⅹ),aggregated proteoglycom(Aggrecan),sex-determining region Y-box protein 9(SOX9),vascular endothelial growth factor(VEGF),proliferating cell nuclear antigen(PCNA),Nel-like molecule tyep 1(Nell-1),Runt-related transcription factor 2(Runx2),Indian hedgehog(Ihh),patched homolog 1(Ptch-1),GLI family zinc finger protein 1(Gli-1),and hedgehog interacting protein 1(Hhip-1)mRNA in the cells in various groups;Western blotting method was used to detect the expression levels of Nell-1,Runx2,and Ihh proteins in the cells in various groups.The ATDC5 cells were divided into control group,cyclopamine group,tensile stress group,and cyclopamine + tensile stress group.RT-qPCR method was used to detect the expression levels of Nell-1,Ihh,Ptch-1,Gli-1,and Hhip-1 mRNA in the cells in various groups;Western blotting method was used to detect the expression levels of Nell-1 and Ihh proteins in the cells in various groups.Results:Compared with control group,the expression levels of Col-Ⅱ,Col-Ⅹ,Aggrecan,SOX9,VEGF,and PCNA mRNA in the cells in 2 000 μstrain/2 h group were significantly increased(P<0.01);after treated with 2 000 μstrain tensile stress for different duration time(1,2,and 4 h)or different tensile stresses(1 000,2 000,and 3 000 μstrain)for 2 h,compared with control group,the expression levels of Runx2 mRNA in the cells in other groups were increased with the prolongation of time or the increasing of tensile stress(P<0.01),and the expression levels of Nell-1,Ihh,Ptch-1,Gli-1,and Hhip-1 mRNA were gradually increased(P<0.01),the expression levels reached the peaking at 2 000 μstrain/2 h,and then decreased but remained significantly higher than that in control group(P<0.01).The Western blotting results showed that the expression levels of Nell-1,Runx2,and Ihh proteins in the cells were consistent with the change trend of mRNA expression levels.After pre-treated with cyclopamine,compared with control group,the expression levels of Ihh,Ptch-1,Gli-1,and Hhip-1 mRNA in the cells in cyclopamine group were significantly decreased(P<0.01),and the expression levels of Ihh,Ptch-1,Gli-1,and Hhip-1 mRNA in the cells in tensile stress and cyclopamine+tensile stress groups were significantly increased(P<0.01);compared with cyclopamine group,the expression levels of Nell-1,Ihh,Ptch-1,Gli-1,and Hhip-1 mRNA in the cells in cyclopamine+tensile stress group were significantly increased(P<0.01);compared with tensile stress group,the expression levels of Ihh,Ptch-1,Gli-1,and Hhip-1 mRNA in the cells in cyclopamine + tensile stress group were significantly decreased(P<0.01).Compared with control group,the expression level of Ihh protein in the cells in cyclopamine group was significantly decreased(P<0.01),but there was no significant difference in expression level of Nell-1 protein in the cells between control group and cyclopamine group(P>0.05),while the expression levels of Nell-1 and Ihh proteins in the cells in tensile stress group and cyclopamine + tensile stress group were significantly increased(P<0.01);compared with cyclopamine group,the expression levels of Nell-1 and Ihh proteins in the cells in tensile stress group and cyclopamine + tensile stress group were significantly increased(P<0.01);compared with tensile stress group,in the expression levels of Nell-1 and Ihh proteins in the cells in cyclopamine + tensile stress group had no significant differences(P>0.05).Conclusion:After stimulated with physiological tensile stress,Nell-1 can activate the Ihh signaling pathway upstream,and regulate the differentiation of the ATDC5 chondrocytes.
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Objective To investigate the predictive value of the expression levels of YY1 transcription fac-tor(YY1)and microRNA(miR)-181a-5p in peripheral blood mononuclear cell for adverse pregnancy out-comes in gestational diabetes mellitus(GDM).Methods A total of 200 patients with GDM were enrolled as the GDM group.100 healthy pregnant women who underwent prenatal examinations during the same period were selected as the control group.The expressions levels of YY1 and miR-181a-5p in peripheral blood mono-nuclear cell were detected by fluorescent quantitative PCR.Receiver operating characteristic(ROC)curve was drawn to analyze the predictive value of YY1 and miR-181a-5p for adverse pregnancy outcomes in GDM pa-tients.Results Compared with the control group,the expression levels of YY1 and miR-181a-5p in peripheral blood mononuclear cell of GDM group were obviously decreased(P＜0.05),and the incidence rates of post-partum hemorrhage,macrosomia and neonatal hypoglycemia in GDM group were obviously higher(P＜0.05).Multivariate Logistic regression analysis showed that age and poor blood glucose control were inde-pendent risk factors for adverse pregnancy outcomes in GDM patients(P＜0.05),and the expression levels of peripheral blood mononuclear cell YY1 and miR-181a-5p were independent protective factors for adverse preg-nancy outcomes in GDM patients(P＜0.05).ROC curve results showed that the area under the curve(AUC)of the expression levels of YY1 and miR-181a-5p in peripheral blood alone and in combination in predicting ad-verse pregnancy outcomes in GDM patients was 0.717,0.751 and 0.832,respectively,and the AUC of their combination was obviously higher than that of the two alone(P＜0.05).Conclusion The decreased expres-sion levels of YY1 and miR-181a-5p in peripheral blood mononuclear cell of GDM patients could increase the risk of adverse pregnancy outcomes,YY1 and miR-181a-5p are closely related to adverse pregnancy outcomes in GDM patients,and both could be used as predictors of adverse pregnancy outcomes in GDM patients.
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Objective To investigate the impact of ampelopsin(AMP)on oxygen glucose deprivation/reperfusion(OGD/R)induced neuronal damage and its mechanism,and to lay a foundation for the study of neonatal hypoxic-ischemic brain damage.Methods Neurons of newborn SD rats were isolated and cultured in vitro,and they were divided into 5 groups:control group(AMP 0 μmol/L),OGD/R group,low dose AMP group(OGD/R+AMP 20 μmol/L),high dose AMP group(OGD/R+AMP 30 μmol/L)and JAK2/STAT3 activator group(OGD/R+AMP 30 μmol/L+Coumermycin A1 10 μmol/L).CCK-8 method was used to de-tect the cell viability of different treatment groups,the lactate dehydrogenase(LDH)kit was used to detect the cell activity of LDH in the medium,flow cytometry was used to detect the apoptosis rate,enzyme-linked immunosorbent assay was used to detect the levels of interleukin-6(IL-6),interleukin-10(IL-10)and tumor necrosis factor α(TNF-α),the kit was used to detect the levels of reactive oxygen species(ROS),malondial-dehyde(MDA)and superoxide dismutase(SOD),and Western blotting was used to detect the expression of apoptosis related proteins B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),enzymatic cleavage of cysteine containing aspartate protein hydrolase-3(C-caspase-3),tyrosine kinase 2(J AK2),phosphorylated JAK2(p-JAK2),signal transduction and transcription activating factor 3(STAT3)and phosphorylated STAT3(p-STAT3).Results Compared with the concentration of AMP of 0 μmol/L,the cell viability in con-centration of AMP of 5-30 μmol/L was not obvious different(P＞0.05),when the concentration of AMP was 40 μmol/L,the cell viability decreased obviously(P＜0.05).Compared with the control group,the cell viability,the levels of SOD fluorescence intensity,IL-10 and Bcl-2 in OGD/R group decreased obviously,the LDH activity,cell apoptosis rate,the levels of ROS,MDA,IL-6,TNF-α,Bax,C-caspase-3,p-JAK2/JAK2,and p-STAT3/STAT3 increased obviously(P＜0.05).Compared with OGD/R group,the cell viability,the levels of SOD fluorescence intensity,IL-10 and Bcl-2 in low and high dose AMP groups increased,the LDH activity,cell apoptosis rate,the levels of ROS,MDA,IL-6,TNF-α,Bax,C-caspase-3,p-JAK2/JAK2,and p-STAT3/STAT3 decreased(P＜0.05),and JAK2/STAT3 activator was able to reverse the protective effect of AMP on OGD/R induced neuronal.Conclusion AMP attenuates OGD/R induced neuronal by reducing oxidative stress and inflammatory response,and its mechanism may be related to inhibition of JAK2/STAT3 signal pathway phosphorylation.
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Objective To establish a rapid detection method for zika virus based on direct amplification re-al-time fluorescent quantitative reverse transcription polymerase chain reaction(RT-PCR)technique.Methods A direct amplification RT-PCR technique for the rapid detection of zika virus in 5 samples(whole blood,serum,saliva,throat swab and urine)was established by using a special function DNA polymerase and a preferred PCR enhancer.Results The detection limits of the 5 samples were 103 PFU/mL in serum,102 PFU/mL in urine,throat swab,and saliva,and 104 PFU/mL in whole blood.The coefficient of goodness-fit of stand-ard curves was above 0.98,and the amplification efficiency was 90％-110％.Zika virus nucleic acid was suc-cessfully amplified,but non-zika virus nucleic acid was not amplified.Based on the repeatable detection of sam-ples from urine,whole blood,and saliva,the variation coefficient of 6 repeated Ct values at 106 PFU/mL and 102 PFU/mL concentrations were all＜5％.The zika virus detection method established by the direct amplifi-cation RT-PCR technique was consistent with the detection results of conventional RT-PCR technique.Only two serum samples were detected in eight zika virus samples,and the remaining 62 non-zika virus samples and 12 negative samples were not amplified.Conclusion A rapid detection method for zika virus based on direct ampli-fication RT-PCR technique is successfully established.The method is simple,rapid,sensitive and specific.
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Objective To investigate the relationship between the expression levels of thyroid transcription factor-1(TTF-1)and Galectin-3 in differentiated thyroid carcinoma(DTC)tissues and clinical manifestations and prognosis of patients.Methods A total of 76 DTC patients admitted to the hospital from January 1,2017 to May 30,2020 were selected as the study objects.Cancer tissue specimens obtained during surgery were in-cluded in the DTC group(n=76),and corresponding paracancer tissue specimens were included in the para-cancer group(n=76).The expressions of TTF-1 and Galectin-3 in DTC group and paracancer group were de-tected by immunohistochemistry,and the relationship between the expression levels of TTF-1 and Galectin-3 and the clinicopathological characteristics of DTC patients was analyzed.Multivariate Cox regression analysis was used to investigate the prognostic factors of DTC patients.Results The positive expression rates of TTF-1 and Galectin-3 in DTC group were higher than those in paracancer group,and the difference was statistically significant(P＜0.05).The TTF-1 positive expression rate and Galectin-3 positive expression rate in DTC pa-tients with TNM stage Ⅲ to Ⅳ,low differentiation,tissue type of papillary thyroid carcinoma and lymph node metastasis were higher than those in DTC patients with TNM stage Ⅰ to Ⅱ,medium/high differentiation,tis-sue type of thyroid follicular carcinoma and no lymph node metastasis.The difference was statistically signifi-cant(P＜0.05).The 3-year overall survival rate of TTF-1 negative and Galectin-3 negative DTC patients was higher than that of TTF-1 positive and Galectin-3 positive DTC patients,and the difference was statistically significant(P＜0.05).Multivariate Cox regression analysis showed that lymph node metastasis,positive TTF-1 and positive Galectin-3 were prognostic factors in DTC patients(P＜0.05).Conclusion TTF-1 and Galectin-3 are related to TNM stage,differentiation degree,tissue type,lymph node metastasis and 3-year sur-vival rate of DTC patients,and have important reference value for the diagnosis and prognosis evaluation of DTC patients.
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Objective To investigate the ameliorative effect of sulforaphane on inflammatory response and airway remodeling in rats with chronic obstructive pulmonary disease(COPD).Methods Seventy-five SD rats were randomly divided into the normal group,the model group,and the low-,medium-,and high-dose groups of sulforaphane,with 15 rats in each group.Except for the normal group,the COPD model was prepared in the remaining group using aroma smoke inhalation combined with intratracheal droplet lipopolysaccharide(LPS)method.After the successful modelling,the rats were administered the drug by gavage for 28 days.At the end of the administration,the general conditions of the rats in each group were observed,and the lung function[forced vital capacity(FVC),peak expiratory flow-rate(PEF),forceful expiratory volume in 1 second(FEV1)]was examined,and the pathological changes of the lung tissues were observed by hematoxylin-eosin(HE)staining method,and the indexes of airway remodeling(thickness of the bronchial wall,thickness of the smooth muscle)were measured;the enzyme-linked immunosorbent assay(ELISA)was used to examine the lung function of the rats.The levels of inflammatory factors[tumor necrosis factor α(TNF-α),interleukin 1β(IL-1β)]were detected in lung tissue by enzyme-linked immunosorbent assay(ELISA),and changes in the protein expressions of Toll-like receptor 4(TLR4),myeloid differentiation factor 88(MyD88),and nuclear transcription factor κB(NF-κB)were detected in lung tissue by Western Blot.Results(1)The rats in the model group had dry and lack of glossy fur,obvious coughing and nose scratching,shortness of breath,slow movement,and preferred to arch their backs and lie curled up;the rats in the low-,medium-and high-dose groups of sulforaphane showed significant improvement in shortness of breath,coughing,and other abnormal manifestations.(2)HE staining showed that the airway wall and smooth muscle of rats in the model group were thickened,the airway epithelium was damaged,and alveolar destruction,fusion,and massive infiltration of inflammatory cells were seen;the histopathological changes in the lungs of rats in the low-,medium-and high-dose groups of sulforaphane improved to varying degrees,with the airway wall becoming thinner,the degree of alveolar destruction being reduced,and the infiltration of inflammatory cells being reduced.(3)Compared with the normal group,FVC,PEF and FEV1 were significantly reduced in the model group(P＜0.05),and the levels of TNF-α and IL-1β,bronchial wall thickness,smooth muscle thickness,and the expression levels of TLR4,MyD88 and NF-κB were significantly increased in the model group(P＜0.05);and in comparison with the model group,the levels of FVC,PEF,and FEV1 were significantly increased in the rats in the sulforaphane low-,medium-,and high-dose groups(P＜0.05),and the levels of TNF-α,IL-1β,bronchial wall thickness,smooth muscle thickness,and the expression levels of TLR4,MyD88,and NF-κB were significantly decreased(P＜0.05)compared with the model group.Conclusion Sulforaphane helps to inhibit the inflammatory response,attenuate airway remodeling,and improve the pathological injury and lung function of lung tissue in rats with COPD,and its mechanism may be related to the inhibition of TLR4,MyD88,and NF-κB protein expressions.
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Objective To investigate the repair mechanism of baicalin on gastric mucosa of chronic atrophic gastritis mice based on the network pharmacology and animal experiments.Methods(1)Applied network pharmacology to predict and analyze the potential key targets of baicalin in the treatment of chronic atrophic gastritis.(2)Animal experiment:40 C57BL/6N mice were randomly divided into normal group,model group,Vitacoenzyme group and baicalin group,10 mice in each group.Except for the normal group,the other three groups of mice were treated with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)by gavage combined with hunger and satiety disorder method to construct a chronic atrophic gastritis model.At the end of drug administration,the histopathological changes of gastric mucosa were observed by hematoxylin-eosin(HE)staining,the changes of gastrin(GAS)and prostaglandin E2(PGE2)levels in serum were detected by enzyme-linked immunosorbent assay(ELISA),and the mRNA and protein expression levels of Janus tyrosine kinase 1(JAK1),signal transducer and activator of transcription 3(STAT3)in the gastric mucosa were detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)and protein immunoblotting(Western Blot)methods,respectively.Results The results of network pharmacology showed that baicalin could spontaneously bind to the core targets JAK1 and STAT3.The results of animal experiments showed that compared with the normal group,the gastric mucosa of mice in the model group suffered from atrophy,disordered gland arrangement,the presence of a large number of lymphocytes,a significant increase in apoptotic index of the gastric mucosa(P＜0.05),a significant decrease in the levels of GAS and PGE2 in serum(P＜0.05),and a significant increase in the levels of mRNA and protein expressions of JAK1 and STAT3 in the gastric mucosa(P＜0.05);compared with the model group,the pathological changes of gastric mucosa in the Vitacoenzyme group and baicalin group were alleviated,the glands were arranged relatively neatly,the structure was more intact,the apoptosis index of gastric mucosal cells was significantly decreased(P＜0.05),the levels of GAS and PGE2 in serum were significantly increased(P＜0.05),and the mRNA and protein expression levels of JAK1 and STAT3 in gastric mucosa were significantly decreased(P＜0.05).There was no significant difference in the above-mentioned indexes between the baicalin group and the Vitacoenzyme group(P＞0.05).Conclusion Baicalin can effectively repair gastric mucosal lesions in mice with chronic atrophic gastritis,and its mechanism may be related to the down-regulation of mRNA and protein expressions of JAK1 and STAT3.
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Objective To investigate the therapeutic effects and mechanisms of Maxing Shigan Decoction on cough variant asthma(CVA)rats.Methods Sixty rats were randomly divided into normal group,model group,low and high dose groups of Maxing Shigan Decoction,and high-dose of Maxing Shigan Decoction + signal transducer and activator of transcription 3(STAT3)activator Colivelin(Col)group,12 rats in each group.Except for the normal group,the CVA model was constructed by intraperitoneal injection of ovalbumin combined with moxa fumigation in all other groups of rats.After the corresponding treatment,the rats were observed for signs and cough counts,airway resistance(RE)was detected by pulmonary function meter,eosinophils(EOS)were counted by Diff-Quik staining,histopathological features of the lungs and bronchial tubes were observed by hematoxylin-eosin(HE)staining method,and the lung tissues were detected by enzyme-linked immunosorbent assay(ELISA)for monocyte chemotactic protein 1(MCP-1),and tumor necrosis factor α(TNF-α),and the protein expression levels of interleukin 6(IL-6),STAT3,and transient receptor potential vanilloid-1 channel(TRPV1)were detected by Western Blot.Results Compared with the normal group,rats in the model group showed obvious asthma symptoms,severe inflammatory cell infiltration was seen in the lung tissue,bronchial epithelial cell necrosis,ciliated adhesion,mucus,and RE,EOS number,MCP-1 and TNF-α contents,and protein expression levels of IL-6,STAT3,TRPV1 were elevated(P＜0.05);compared with the model group,rats in the low-and high-dose groups of Maxing Shigan Decoction showed significant improvement in asthma symptoms,reduction in lung and bronchial injury,and dose-dependent reduction in RE,EOS number,MCP-1 and TNF-α contents,and protein expression levels of IL-6,STAT3,and TRPV1(P＜0.05);compared with the high-dose group of Maxing Shigan Decoction,the rats in the high-dose Maxing Shigan Decoction+Col group showed increased asthma,increased lung and bronchial injury,and increased RE,EOS number,MCP-1 and TNF-α contents,and protein expression levels of IL-6,STAT3,and TRPV1(P＜0.05).Conclusion Maxing Shigan Decoction can effectively improve cough variant asthma in rats,and its mechanism is related to the inhibition of IL-6/STAT3 signaling pathway and the high expression of TRPV1.
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Objective To observe the regulatory mechanism of drug-containing serum of Jinghou Zengzhi Prescription based on qi and blood replenishing method on the expression of growth and differentiation factor 9(GDF9)and apoptosis of ovarian granulosa cells in rats with controlled ovarian hyperstimulation(COH).Methods Serum of COH rats(blank serum)and serum of COH rats gavaged by the Jinghou Zengzhi Prescription were prepared.A COH rat model was established and ovarian granulosa cells were collected.The experiment was divided into 5 groups:blank serum group,drug-containing serum group,drug-containing serum+SB203580[p38 mitogen-activated protein kinase(p38MAPK)inhibitor]group,drug-containing serum + PDTC[nuclear transcription factor κB(NF-κB)inhibitor]group,drug-containing serum + SB203580 + PDTC group.The mRNA expression levels of p38MAPK,casein kinase 2(CK2),nuclear transcription factor κB inhibitor α(IκBα),NF-κB and GDF9 were detected by real-time quantitative polymerase chain reaction(qRT-PCR),and GDF9 protein expression level was detected by Western Blot,and ovarian granulosa cell apoptosis was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL).Results The drug-containing serum of Jinghou Zengzhi Prescription decreased the mRNA expressions of p38MAPK and NF-κB,elevated the mRNA expressions of CK2 and IκBα,increased the mRNA and protein expression levels of GDF9,and decreased the apoptosis rate of ovarian granulosa cells in COH rats.The addition of p38MAPK inhibitor SB203580 alone and the addition of NF-κB inhibitor PDTC alone both promoted the mRNA and protein expressions of GDF9 and reduced the apoptosis rate of granulosa cells.Conclusion The drug-containing serum of Jinghou Zengzhi Prescription based on qi and blood replenishing method can promote the expression of GDF9 and inhibit the apoptosis of ovarian granulosa cells in rats with COH,and its mechanism may be related to the regulation of the expression of genes of the dual signaling pathways of p38MAPK and NF-κB.
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Objective To explore the effect of endoplasmic reticulum stress activating transcription factor 6(ATF6)on the expression of reproduction related gene heat shock protein family A member 1 like(HSPA1L)and preliminari-ly clarify its regulatory molecular mechanism.Methods The ATF6 over-expression plasmid was transfected into HEK-293T cells and the over-expression efficiency was verified by RT-qPCR and Western blot.The transcriptome sequen-cing information of testis tissue of male ATF6 knockout mice was used to screen five reproduction related genes down-stream of ATF6.The dual luciferase reporter assay selected the downstream genes with high promoter activity and de-tected the effect of over-expression of ATF6 on the promoter activity of downstream genes.The possible binding sites of ATF6 and downstream gene promoters were predicted by gene-regulation.RT-qPCR and Western blot were used to detect the effect of over-expression of ATF6 on downstream gene expression in HEK-293T cells.Whether ATF6 binds to downstream gene promoters was determined by electrophoretic mobility shift assay(EMSA).Results The expres-sion of ATF6 mRNA(P＜0.001)and protein(P＜0.001 and P＜0.05)in HEK-293T cells was significantly increased after transfection.HSPA1L(P＜0.001 and P＜0.05),a reproductive related gene downstream of ATF6 was screened by transcriptome sequencing and dual luciferase reporter assay.ATF6 promoted the truncated promoter activity of HSPA1L(P＜0.001).After over-expression of ATF6,the expression of HSPA1L was significantly increased(P＜0.001).The differences were statistically significant.ATF6 protein could bind to the aagtcgtcac DNA sequence of HSPA1L promoter.Conclusions ATF6,a key molecule of endoplasmic reticulum stress,regulates the expression of reproduction related gene HSPA1L by binding to the promoter of HSPA1L.This result will lay a foundation for further research on the prevention or treatment of endoplasmic reticulum stress(ERS)related male infertility.
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Objective To investigate the effect of cisplatin treatment on the transcriptional level of human liver cancer cells by conducting transcriptome sequencing analysis after treating human liver cancer cell lines with differ-ent concentrations of cisplatin(CDDP).Methods Liver cancer cell lines HepG2 and Huh7 were incubated with cisplatin at different final concentrations of 0,20,50,100 and 200 μmol/L.After 12 hours,cell viability,immuno-fluorescence and RNA-sequencing(RNA-seq)were performed.Differential gene expression analysis(DEG),KEGG pathway analysis,and protein-protein interaction network analysis were conducted.Results Cisplatin de-creased cell viability and increased DNA damage in HepG2,Huh7 cells.Among the genes regulated after cisplatin treatment at different concentrations,59 genes were commonly up-regulated in both HepG2 and Huh7 cells,while 81 genes were commonly down-regulated.The commonly upregulated genes were mainly enriched in cancer initiation and progression pathways.The 81 commonly down-regulated genes were mainly enriched in Rap1 signaling pathway,Ras signaling pathway,signaling pathways regulating pluripotency of stem cells,axon guidance,and cell adhesion-related pathways.Survival analysis of key nodes in the protein-protein interaction network of commonly up-regulated and downregulated genes revealed a significant correlation between high expression of Jun proto-oncogene,AP-1 transcription factor subunit(JUN)and prolonged patient survival and a significant correlation between low ex-pression of growth arrest and DNA damage inducible alpha(GADD45A)and prolonged patient survival.Conclu-sions The study revealed common transcriptional changes in liver cancer cells under cisplatin treatment.Differential expression of JUN and GADD45A is a potential core mechanism to explain drug resistance.This conclusion provides some important prognostic indicators for clinical treatment.
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Objective To investigate the association of 13 single nucleotide polymorphism(SNP)sites in 6 phalange-bone development related genes[fibroblast growth factor receptor 2(FGFR2),indian hedgehog signaling molecule(IHH),Msh homeobox 1(MSX1),Runx family transcription factor 2(RUNX2),SRY-box transcription factor 9(SOX9),Wnt family member 5A(WNT5A)]with human index-ring finger length ratio(2D∶4D).Methods Digital cameras were used to take frontal photographs of the hands of 731 college students(358 males and 373 females)in Ningxia,and image analysis software was used to mark anatomical points and measure finger lengths of index(2th)and ring(4th);genotyping of 13 SNP sites(rs1047057,rs755793,rs41258305,rs3731881,rs3100776,rs12532,rs3821949,rs45585135,rs3749863,rs1042667,rs12601701,rs1829556,rs3732750)for 6 genes by multiplex PCR;One-Way ANOVA or independent sample t-test indirectly assessed the association between 2D∶4D and 13 SNP sites.Results Both left and right hand 2D∶4D were significantly higher in females than males in Ningxia college students(all P<0.01);no statistically significant differences in genotype and allele frequencies of the 13 SNP sites among different sexes(all P>0.05);among different sexes,male left hand 2D∶4D was significantly associated with the genotype of SOX9 gene rs12601701 site(P<0.05)and right hand 2D∶4D was significantly associated with the genotype of WNT5A gene rs1829556 site(P<0.05);the female right hand 2D∶4D was significantly associated with the MSX1 gene rs12532(P<0.01)and rs3821949(P<0.05)sites genotypes.Conclusion SOX9(rs12601701),WNT5A(rs1829556)and MSX1(rs12532 and rs3821949)gene polymorphisms may be associated with the formation of 2D∶4D in Ningxia population.