Inflammatory bowel disease and risk of autoimmune hepatitis: A univariable and multivariable Mendelian randomization study.

OBJECTIVE: This study aimed to use Mendelian randomization (MR) to investigate the potential causal association between inflammatory bowel disease (IBD) and autoimmune hepatitis (AIH). METHODS: Two-sample MR was performed to estimate the causal effect of IBD on AIH. The primary analysis employed the inverse variance weighted (IVW) method in univariable MR analysis, supplemented by additional methods including MR-Egger, weighted median, simple mode, and weighted mode. The p values were adjusted by FDR p-value adjustment. In the replication analysis, the primary IVW analysis was repeated and then pooled by meta-analysis. Sensitivity analyses were performed using Cochran's Q test, MR-Egger intercept test, MR-PRESSO, leave-one-out, and funnel plot analysis to evaluate the robustness of the MR findings. Additionally, multivariable MR (MVMR) was employed to estimate the direct causal effect of IBD on the risk of AIH. RESULTS: In univariable MR analysis, a significant positive causal association was observed between IBD (both Crohn's disease (CD) or ulcerative colitis (UC)) and the risk of AIH (for CD and AIH, the IVW odds ratio (OR) = 1.10, 95% confidence interval (CI) = 1.00-1.16, P = 0.045, FDR P = 0.045; for UC and AIH, the IVW OR = 1.07, 95% CI = 1.00-1.13, P = 0.038, FDR P = 0.076). Furthermore, no significant positive correlation between IBD and the risk of AIH (OR = 1.13, 95% CI = 0.94-1.35, P = 0.194). Sensitivity analysis revealed no pleiotropic bias. MVMR analysis further confirmed the direct causal effect of CD or UC on the risk of AIH after adjusting for the common risk factors (cigarettes per day and osteoporosis). In the replication analysis, the positive causal association between UC and the risk of AIH remain significant (the IVW odds ratio (OR) = 1.32, 95% CI = 1.18-1.48, P = 2.90E-06). While no significant positive association was observed between CD or IBD and the risk of AIH in the replication analysis, a suggestive positive association between the identified risk factors (UC, CD, and IBD) and the risk of AIH was detected in the meta-analysis (OR = 1.09, 95% CI = 1.05-1.13, P<0.0001). CONCLUSION: This MR study revealed a positive impact of the identified risk factors (CD, UC and IBD) on the risk of AIH within the European population.

Application and evaluation of nine-grid thinking model in biochemistry teaching.

Biochemistry is an important professional course to undergraduates majoring in rehabilitation therapy in medical colleges and universities. To deepen students' understanding of the taught content, enhance their application ability and cultivate their high-level thinking ability, we investigated the effect of integration of the nine-grid thinking model into the teaching process. With the inline and divergence of relevant knowledge as the guiding ideology, students' understanding of knowledge points was deepened through thinking visualization. According to the questionnaire survey, 75% of the students believed that the application of the nine-grid thinking model was an effective teaching method for improving the efficiency of teaching and enhancing the teaching effect. In addition, a team of four students from the investigated class were granted by the 2021 Shanxi University Student Innovation and Entrepreneurship Training Program and awarded RMB 6000 as a research fund (20210563). According to them, the application of the nine-grid thinking model in teaching is of great significance for cultivating students' higher-order thinking ability. The findings of this study might provide a new, effective approach to college course teaching.

The imbalance of liver resident macrophages polarization promotes chronic autoimmune hepatitis development in mice.

Background: Autoimmune hepatitis (AIH) is a chronic immune-mediated inflammatory liver disease. At present, it is largely unknown how the innate immune cells influence AIH development. Objective: To inquiry about mechanism of liver resident macrophages in AIH development, thus offering a new direction for AIH targeted treatment. Methods: The liver resident macrophages were eliminated by clodronate liposomes in AIH liver tissues, followed by HE and Picrosirius assay to detect liver fibrosis and lymphocyte infiltration. The liver resident macrophages polarization was detected by Immunohistochemistry and qPCR. The collagenase digestion was used to isolate Kupffer cells from AIH mice liver tissues and pro-/anti-inflammatory cytokines were determined by qPCR. Results: M2 macrophages were the dominant phenotype at early immune response stage and hepatic inflammation was progressively aggravated after depletion of liver resident macrophages. M2 macrophages could effectively delay the development of AIH and could be polarized to M1 macrophages at the disease progresses. TLR2 ligands could promote M2 macrophages producing anti-inflammatory cytokines, whereas TLR4 ligands could promote M1 macrophages producing proinflammatory cytokines. The change of TLR2 and TLR4 ligands could lead to continuous high expression of TLR4 and decreased expression of TLR2 in macrophages to further affect liver resident macrophages polarization state. Conclusion: TLR2 and TLR4 ligands mediated liver resident macrophages polarization to favor chronic autoimmune hepatitis development.

RAE1 is a prognostic biomarker and is correlated with clinicopathological characteristics of patients with hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a primary malignant tumor that accounts for approximately 90% of all cases of primary liver cancer worldwide. Microtubule alterations may contribute to the broad spectrum of resistance to chemotherapy, tumor development, and cell survival. This study aimed to assess the value of ribonucleic acid export 1 (RAE1), as a regulator of microtubules, in the diagnosis and prognosis of HCC, and to analyze its correlation with genetic mutations and pathways in HCC. RESULTS: The mRNA and protein levels of RAE1 were significantly elevated in HCC tissues compared with those in normal tissues. The high expression level of RAE1 was correlated with T stage, pathologic stage, tumor status, histologic grade, and alpha-fetoprotein level. HCC patients with a higher expression level of RAE1 had a poorer prognosis, and the expression level of RAE1 showed the ability to accurately distinguish tumor tissues from normal tissues (area under the curve (AUC) = 0.951). The AUC values of 1-, 3-, and 5-year survival rates were all above 0.6. The multivariate Cox regression analysis showed that RAE1 expression level was an independent prognostic factor for a shorter overall survival of HCC patients. The rate of RAE1 genetic alterations was 1.1% in HCC samples. Gene ontology and kyoto encyclopedia of genes and genomes pathway enrichment analyses indicated the co-expressed genes of RAE1 were mainly related to chromosome segregation, DNA replication, and cell cycle checkpoint. Protein-protein interaction analysis showed that RAE1 was closely correlated with NUP205, NUP155, NUP214, NUP54, and NXF1, all playing important roles in cell division and mitotic checkpoint. CONCLUSION: RAE1 can be a potential diagnostic and prognostic biomarker associated with microtubules and a therapeutic target for HCC.

[Ponatinib inhibits the proliferation of SNU-449 human hepatocellular cancer cells and blocks MAPK and PDK1/AKT/mTOR signaling pathways].

Objective To investigate the effects of ponatinib (a multi-target kinase inhibitor) on the proliferation of SNU-449 human hepatocellular cancer cells and the underlying mechanism. Methods SNU-449 hepatocellular cancer cells were treated with 16 tyrosine kinase inhibitors for 72 hours. Then MTT assay was used to detect the effects of ponatinib on the survival and proliferation of the cancer cells. Ponatinib was the most sensitive drug to SNU-449 cells and the IC50 value was obtained. SNU-449 cells were cultured and treated with (0.06, 0.3, 0.6) µmol/L ponatinib, and the control group was treated with DMSO. Colony formation assay and inverted microscope were applied to observe the effects of ponatinib on the colony formation ability and morphology of SNU-449 cells. Flow cytometry was used to detect the effects of ponatinib on the apoptosis and cell cycle of SNU-449 cells. Western blotting was performed to examine the expression of Src, phosphorylated Src (p-Src), mitogen-activated protein kinase kinase (MEK), phosphorylated MEK (p-MEK), extracellular signal-regulated kinase (ERK), phosphorylated ERK (p-ERK), phosphoinositide 3-kinase (PI3K), phosphorylated PI3K (p-PI3K), phosphoinositide-dependent protein kinase 1 (PDK1), phosphorylated PDK1 (p-PDK1), AKT, p-AKT, mammalian target of rapamycin (mTOR) and phosphorylated mTOR (p-mTOR). Results MTT assay showed that ponatinib displayed the best inhibitory effects on SNU-449 cells in 16 tyrosine kinase inhibitors. Ponatinib promoted cell apoptosis in a concentration-dependent manner and induced cell cycle arrest at the G1 phase in SNU-449 cells. A number of kinase signaling pathways were inhibited by ponatinib, including the Src signaling pathway, MAPK pathway and PDK1/AKT/mTOR pathway. Conclusion Ponatinib can inhibit the proliferation, promote the apoptosis and cell cycle arrest of hepatocellular cancer cells and block MAPK and PDK1/AKT/mTOR signaling pathways, which might be a potential agent for liver cancer treatment.

SMYD5 acts as a potential biomarker for hepatocellular carcinoma.

Determining the prognosis of patients remains a challenge due to the phenotypic and molecular diversities of hepatocellular carcinomas (HCC). We aimed to evaluate the role of SMYD5 in HCC. Wilcoxon signed-rank test and logistic regression analyzed the relationship between clinical pathologic features and SMYD5. We found that increased expression of SMYD5 in HCC was closely associated with high histologic grade, stage, T stage and nodal stage. Kaplan-Meier method, Cox regression, univariate analysis and multivariate analysis detected overall survival of TCGA-HCC patients. It turned out that high expression of SMYD5 predicted a worse prognosis in HCC. Gene Set Enrichment Analysis (GSEA) was applied via TCGA data set, which indicated that complement and coagulation cascades, fatty acid metabolism, primary bile acid biosynthesis, drug metabolism cytochrome P450, PPAR signaling pathway and retinol metabolism were differentially enriched in SMYD5 high expression phenotype. Interestingly, we proved that SMYD5 upregulation in HCC cells was induced by promoter hypo-methylation. Moreover, functional experiments demonstrated that SMYD5 silencing abrogated cell proliferation, migration and invasion and enhanced paclitaxel sensitivity in HCC. All findings implied that SMYD5 might be an underlying biomarker for prognosis and treatment of HCC.

EZH2-mediated H3K27me3 promotes autoimmune hepatitis progression by regulating macrophage polarization.

Autoimmune hepatitis (AIH) is a chronic progressive liver disease related to abnormal immune stimulation, leading to liver cirrhosis, liver cancer and liver failure. There is an urgent need to find novel biomarkers and potential drug targets for effective treatment of the disease. Although previous studies have shown that EZH2, as a histone methyltransferase, plays critical roles in tumor and autoimmune diseases, its role in autoimmune hepatitis remains largely unknown. In this study, we reported that the EZH2 and H3K27me3 expression level was significantly upregulated in liver tissues during the progression of AIH. High expression of EZH2 enhanced autoimmune hepatitis, immune response and liver fibrosis through H3K27me3. EZH2 inhibition induced the phenotype of hepatic macrophages to switch from M1 to M2 in the development of AIH. These findings indicated that EZH2-mediated H3K27me3 promoted autoimmune hepatitis by regulating the polarization of hepatic macrophages. EZH2 may be a promising therapeutic target for the prevention or treatment of autoimmune hepatitis.

[Multi-target kinase inhibitor dasatinib inhibits cell proliferation, dissociation and migration via up-regulating E-cadherin expression in SK-Hep-1 human hepatocellular cancer cells].

Objective To investigate the effects of dasatinib (a multi-target kinase inhibitor) on the proliferation, adhesion and migration ability of SK-Hep-1 human hepatocellular cancer cells and the underlying mechanism. Methods SK-Hep-1, Bel-7402, SNU-423, SNU-387 and Huh-7 hepatocellular cancer cells were treated with dasatinib. Then MTT assay was used to detect the effect of dasatinib on the survival and proliferation of cancer cells, and the sensitive cells were obtained. SK-Hep-1 cells were cultured and treated with (0.5, 1, 2) µmol/L dasatinib, and the control group was treated with DMSO. Slow aggregation assay and dissociation assay were used to detect the effects of dasatinib on the adhesion ability of SK-Hep-1 cells. Wound healing assay was applied to observe the effect of dasatinib on the migration ability of the cancer cells. Western blotting was performed to detect the effect of dasatinib on the expression of E-cadherin. Results MTT assay showed that the proliferation of the liver cancer cells was obviously inhibited by dasatinib and SK-Hep-1 was the most sensitive cells to dasatinib. Dasatinib promoted the aggregation of SK-Hep-1 cells, inhibited cell dissociation and migration, and up-regulated E-cadherin expression. Conclusion Dasatinib can promote the aggregation and adhesion of SK-Hep-1 cells, and inhibit cell proliferation, dissociation and migration via up-regulating E-cadherin expression.

Systematic Investigation of the Effect of Powerful Tianma Eucommia Capsule on Ischemic Stroke Using Network Pharmacology.

BACKGROUND: Ischemic stroke (IS) is a serious disease with a high rate of death and disability, and a growing number of people are becoming victims. Existing drugs not only have limited therapeutic effects but also have obvious side effects. Most importantly, drug resistance due to long-term or improper use of drugs is detrimental to patients. Therefore, it is urgent to find some alternative or supplementary medicines to alleviate the current embarrassment. Powerful Tianma Eucommia Capsule (PTEC) is mainly used to treat IS in China for thousands of years; however, the molecular mechanism is not clear. METHODS: Pharmacology ingredients and target genes were filtered and downloaded from websites. A pharmacology ingredient-target gene network was constructed to predict the molecular interactions between ingredients and target genes. Enrichment analysis was performed to explore the possible signal pathways. LeDock was used to simulate the interaction form between proteins and main active ingredients and to deduce key amino acid positions. RESULTS: Two hundred eighty-nine target genes and seventy-four pharmacological ingredients were obtained from public databases. Several key ingredients (quercetin, kaempferol, and stigmasterol) and primary core target genes (PTGS1, NCOA2, and PRSS1) were detected through ingredient-target gene network analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis demonstrated that ingredients affect networks mainly in nuclear receptor activity and G protein-coupled amine receptor activity; besides, fluid shear stress and atherosclerosis, human cytomegalovirus infection, and hepatitis B signaling pathways might be the principal therapy ways. A series of presumed key amino acid sites (189ASP, 190SER, 192GLN, 57HIS, and 99TYE) were calculated in PRSS1. Six of the target genes were differentially expressed between male and female patients. CONCLUSIONS: Seven new putative target genes (ACHE, ADRA1A, AR, CHRM3, F7, GABRA1, and PRSS1) were observed in this work. Based on the result of GO and KEGG analysis, this work will be helpful to further demonstrate the molecular mechanism of PTEC treatment of IS.

Dasatinib inhibits proliferation of liver cancer cells, but activation of Akt/mTOR compromises dasatinib as a cancer drug.

Dasatinib is a multi-target protein tyrosine kinase inhibitor. Due to its potent inhibition of Src, Abl, the platelet-derived growth factor receptor (PDGFR) family kinases, and other oncogenic kinases, it has been investigated as a targeted therapy for a broad spectrum of cancer types. However, its efficacy has not been significantly extended beyond leukemia. The mechanism of resistance to dasatinib in a wide array of cancers is not clear. In the present study, we investigated the effect of dasatinib on hepatocellular carcinoma cell growth and explored the underlying mechanisms. Our results showed that dasatinib potently inhibited the proliferation of SNU-449 cells, but not that of other cell lines, such as SK-Hep-1, even though it inhibited the phosphorylation of Src on both negative and positive regulation sites in all these cells. Dasatinib activated the phosphoinositide-dependent protein kinase1 (PDK1)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway in SK-Hep-1 cells, but not in SNU-449 cells. Blocking the Akt/mTOR signaling pathway strongly promoted the efficacy of dasatinib in SK-Hep-1 cells. In SNU-449 cells, dasatinib promoted apoptosis and the cleavage of caspase-3 and caspase-7, induced cell cycle arrest in the G1 phase, and inhibited the expression of Cyclin-dependent kinase (CDK4)/6/CyclinD1 complex. These findings demonstrate that dasatinib exerts its anti-proliferative effect on hepatocellular cell proliferation by blocking the Src family kinases; however, it causes Akt activation, which compromises dasatinib as an anti-cancer drug.

IL-35 promotes EMT through STAT3 activation and induces MET by promoting M2 macrophage polarization in HCC.

The tumor microenvironment and interplay with cancer cells could promote tumor growth and metastasis. Here we report that polarization state of macrophages could affect epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). IL-35 level secreted by M1 macrophage was significantly higher than M2 macrophage and it facilitated EMT process through activation of STAT3 in hepatocellular carcinoma cells. Interestingly, IL-35 could not directly promote MET, but it could indirectly induce MET of HCC cells through M2 macrophage polarization. These results indicated the level of IL-35 in tumor microenvironment may fluctuate at different stages of oncogenesis to regulate epithelial plasticity of HCC and provide potential therapeutic targets for tumor metastasis.

Identification of a 14-Gene Prognostic Signature for Diffuse Large B Cell Lymphoma (DLBCL).

Although immunotherapy is a potential strategy to resist cancers, due to the inadequate acknowledge, this treatment is not always effective for diffuse large B cell lymphoma (DLBCL) patients. Based on the current situation, it is critical to systematically investigate the immune pattern. According to the result of univariate and multivariate cox proportional hazards, LASSO regression and Kaplan-Meier survival analysis on immune-related genes (IRGs), a prognostic signature, containing 14 IRGs (AQP9, LMBR1L, FGF20, TANK, CRP, ORM1, JAK1, BACH2, MTCP1, IFITM1, TNFSF10, FGF12, RFX5, and LAP3), was built. This model was validated by external data, and performed well. DLBCL patients were divided into low- and high-risk groups, according to risk scores from risk formula. The results of CIBERSORT showed that different immune status and infiltration pattern were observed in these two groups. Gene set enrichment analysis (GSEA) indicated 12 signaling pathways were significantly enriched in the high-risk group, such as natural killer cell-mediated cytotoxicity, toll-like receptor signaling pathway, and so on. In summary, 14 clinically significant IRGs were screened to build a risk score formula. This formula was an accurate tool to provide a certain basis for the treatment of DLBCL patients.

Chemical induced inflammation of the liver breaks tolerance and results in autoimmune hepatitis in Balb/c mice.

Autoimmune hepatitis (AIH) is a chronic liver disease mediated by immunity, and could lead to liver fibrosis and hepatocellular carcinoma. However, the mechanisms for breaking hepatic tolerance and driving AIH still remain elusive. We herein reported that the non-specific liver inflammation triggered by carbon tetrachloride (CCl4) recruited high numbers of CD4+T, CD8+T and B cells, and elevated the expression of proinflammaitory cytokines in Balb/c mice, further breaking liver tolerance and inducing autoimmune response, AIH inflammation and liver fibrosis in the presence of CYP2D6 antigen mimicry. In contrast, adenovirus infection could not break liver tolerance and induce AIH in Balb/c mice even in the presence of CYP2D6 antigen mimicry. These results suggested that genetic predisposition could determine liver tolerance in Balb/c mice. The chemical induced inflammation in the liver breaks tolerance and might be considered important for the initiation and development of AIH in Balb/c mice.

CRISPR/Cas9-mediated knockout of the PDEF gene inhibits migration and invasion of human gastric cancer AGS cells.

Gastric cancer is one of the most common malignant tumors worldwide and has the second highest incidence and mortality rate among malignant tumors in China. Prostate-derived Ets factor (PDEF) is a member of the Ets family of transcription factors. Although PDEF plays an important role in tumorigenesis, its biological function in gastric cancer is still unclear. Here, we evaluated PDEF expression in 30 cases of human gastric carcinoma and the corresponding peritumoral tissues, using immunohistochemistry and immunofluorescence. Significantly higher levels of PDEF were detected in tumors compared to peritumoral tissues. We then investigated PDEF expression in the gastric cancer cell lines SGC and AGS and the normal gastric epithelial cell line GES; The CRISPR/Cas9 genome-editing system was used to knockout PDEF in AGS cells as a model for gastric cancer. Cell proliferation, apoptosis, migration, and invasion of PDEF-knockout AGS cells were evaluated using CCK-8, flow cytometry, scratch wound, and transwell assays, respectively. The results illustrated that PDEF-knockout inhibited AGS cell proliferation, migration, and invasion. Taken together, the results imply that PDEF plays important roles in the proliferation, migration, and invasion of AGS cells and may serve as a new treatment target in gastric cancer.

[Establishment of a mouse model of autoimmune hepatitis induced by human CYP2D6].

Objective To investigate the effect of human cytochrome P450 family 2 subfamily D member 6 (CYP2D6) on the establishment of animal model of autoimmune hepatitis (AIH) in C57BL/6 and BALB/c mice. Methods Adenovirus (Ad) and plasmid pCYP2D6 were injected into the tail vein of mice. Serum anti-CYP2D6 antibody and hepatic protein (HP) autoantibody were detected by ELISA. HE staining was used to detect the degree of liver AIH inflammation, and the degree of liver fibrosis was detected by sirius red staining. The level of alanine aminotransferase (ALT) was determined by microplate method. Results Ad and self-mimicking antigen (human CYP2D6) induced the secretion of HP autoantibody, AIH, hepatic injury and liver fibrosis in C57BL/6 mice, whereas only anti-CYP2D6 antibody was generated in BALB/c mice; no HP autoantibody, AIH or liver fibrosis was induced. Conclusion In the presence of AD, the continuous expression of self-mimicking antigen (human CYP2D6) could induce AIH in C57BL/6 mice, but not in BALB/c mice. Thus, the genetic background in the different strains of mice could influence the occurrence and development of AIH.

[Over-expressions of p-eIF4E and p-4EBP1 are associated with poor prognosis in nasopharyngeal carcinoma].

Objective To observe the expressions of phosphorylated eukaryotic translation initiation factor 4E (p-eIF4E) and phospho-eIF4E-binding protein 1 (p-4EBP1) and investigate their associations with clinicopathological features and prognosis in nasopharyngeal carcinoma (NPC). Methods We detected the expressions of p-eIF4E and p-4EBP1 proteins by immunohistochemistry in NPC tissues (n=314 cases), and analyzed the associations between the expressions of p-eIF4E and p-4EBP1 proteins and clinicopathological features and prognosis in NPC by SPSS16.0. Results Positive rates of p-eIF4E and p-4EBP-1 proteins in NPC were 74.2% (233/314) and 72.9% (229/314), respectively. There were significantly higher expressions of p-eIF4E and p-4EBP-1 proteins in the cases with lymph node metastasis than in those without lymph node metastasis. However, no significant correlations were observed between the expressions of p-eIF4E and p-4EBP-1 proteins and the age, gender, histological type, and clinical stage. There was significantly positive relationship between p-eIF4E and p-4EBP1 protein expressions in NPC. The overall survival rates were higher in the NPC patients with negative expressions of p-eIF4E and p-4EBP1 proteins than in those with positive expressions. The positive expressions of p-eIF4E and p-4EBP1 proteins were identified as the independent poor prognostic factors in NPC. Conclusion The over-expression of p-eIF4E and p-4EBP-1 proteins may be closely related to the occurrence, development and adverse prognosis of NPC. Hence, p-eIF4E and p-4EBP1 proteins are expected to be molecular markers for clinical diagnosis and prognosis of NPC.