Genetic evidence for a causal link between gut microbiota and arterial embolism and thrombosis: a two-sample Mendelian randomization study.

Background: Previous research has hinted at a crucial link between gut microbiota and arterial embolism and thrombosis, yet the causal relationship remains enigmatic. To gain a deeper understanding, we aimed to comprehensively explore the causal relationship and elucidate the impact of the gut microbiota on the risk through a two-sample Mendelian randomization (MR) study. Methods: Genetic instrumental variables for gut microbiota were identified from a genome-wide association study (GWAS) of 18,340 participants. Summary statistics for IBS were drawn from a GWAS including 1,076 cases and 381,997 controls. We used the inverse-variance weighted (IVW) method as the primary analysis. To test the robustness of our results, we further performed the weighted median method, MR-Egger regression, and MR pleiotropy residual sum and outlier test. Results: We identified three bacterial traits that were associated with the risk of arterial embolism and thrombosis: odds ratio (OR): 1.58, 95% confidence interval (CI): 1.08-2.31, p = 0.017 for genus Catenibacterium; OR: 0.64, 95% CI: 0.42-0.96, p = 0.031 for genus Dialister; and OR: 2.08, 95% CI: 1.25-3.47, p = 0.005 for genus Odoribacter. The results of sensitivity analyses for these bacterial traits were consistent (P<0.05). Conclusion: Our systematic analyses provided evidence to support a potential causal relationship between several gut microbiota taxa and the risk of arterial embolism and thrombosis. More studies are required to show how the gut microbiota affects the development of arterial embolism and thrombosis.

Effect of γδ T cells on the Proliferation, Apoptosis and Autophagy of Multiple Myeloma Cells / 中国实验血液学杂志

AbstractObjective: To investigate the effect of γδ T cells on the proliferation, apoptosis and autophagy of multiple myeloma cells.@*METHODS@#Peripheral blood mononuclear cells (PBMNC) were isolated from healthy volunteers, and stimulated with zoledronic acid (Zol) in combination with rhIL-2. Flow cytometry analysis was used to detected the purity of γδ T cells. γδ T cells were collected and co-cultured with RPMI-8226 or U-266 cells at different effector target ratios. The proliferation of RPMI-8226 or U-266 cell lines were detected by CCK-8. Cell cycle and cell apoptosis were detected by flow cytometry and Western blot．The expressions of autophagy-related proteins were detected by Western blot.@*RESULTS@#γδ T cells can be expanded in vitro. γδ T cells could inhibit the proliferation of RPMI-8226 or U-266 cells, induced cell cycle arrest and promoted apoptosis in an effector target-dependent manner. In addition, γδ T cells could induce autophagy of myeloma cells, inhibited the expression of autophagy-related PI3K, P-AKT and P-mTOR, while increased the expression of AMPK and Beclin-1.@*CONCLUSION@#γδ T cells can inhibit the proliferation of RPMI-8226 and U-266 myeloma cells, induce cell cycle arrest, promote apoptosis, and enhance autophagy in vitro. The mechanism may be related to inhibition of PI3K/AKT/mTOR signaling pathway and/or activation of AMPK/Beclin-1 signaling pathway.

Effect of MiR-29b-3p Targeting STAT3 on Proliferation and Apoptosis of Acute Myeloid Leukemia Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of miR-29b-3p on apoptosis and proliferation of acute myeloid leukemia (AML) cells by targeting signal transducer and activator of transcription 3 (STAT3).@*METHODS@#TargetScan and miRanda online databases were used to predict the binding sites of miR-29b-3p and STAT3 3'UTR. The targeting relationship between them was estimated by Dual-Luciferase reporter assay experiment. After miR-29b-3p over-expression, qPCR and Western blot were used to detect the expression of STAT3 mRNA and proteins, flow cytometry to determine the apoptosis of AML cells, and MTS to detect the changes of cell proliferation in each group.@*RESULTS@#Dual-Luciferase reporter assay confirmed that STAT3 was the target gene of miR-29b-3p. After miR-29b-3p overexpression, the expression of STAT3 mRNA and protein decreased. Compared with the control groups, the proliferation of AML cells in the overexpression group decreased and the apoptosis increased (P<0.05).@*CONCLUSION@#MiR-29b-3p can inhibit the proliferation and induce apoptosis of AML cells by down-regulating STAT3.

Effect of SARI Overexpression on Apoptosis of CBFL Cells and Its Mechanism / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of SARI overexpression on the proliferation and apoptosis of core binding factor leukemia (CBFL) cells and explore the potential molecular mechanisms.@*METHODS@#C-KIT N822K mutation status in Kasumi-1 cell line was detected by exon 17 sequencing. Then the SARI lentiviral vector (pGC-FU-SARI) was constructed, meanwhile Kasumi-1 cells were transfected with the SARI lentiviral vector. Quantitative PCR and Western blot were employed to identify efficacy of SARI overexpression after the transfection of cells. Cells were divided into three groups, including the cells infected with pGC-FU-SARI (OE group), the cells infected with pGC-FU-GFP (NC group) and the untreated cells (blank control group). Cell proliferative activity was tested by MTT assay, cell apoptosis was measured by flow cytometry (FCM) and the expression of apoptosis-related proteins: BCL-2,BAX,Cyto C,Caspase 9,Caspase 3,cleaved-Caspase 3,PARP and cleaved-PARP as well as PI3K/Akt pathway proteins: PI3K（p85）,p-PI3K（p85）,Akt and p-Akt were detected by Western blot.@*RESULTS@#The Kasumi-1 cells were detected to bear c-KIT N822K (T>A) mutation. The Kasumi-1 cells with SARI was overexpression were construeted successfully. Compared with NC group, the cell proliferation was decreased and cell apoptosis was increased; BCL-2 expression was reduced, BAX expression was enharued; cyto C expression appeared; the expression of Caspase 9 and Caspase 3 was down-regulated, the expression of cleaved Caspase 3 was up-regulated; the PARP expression was decreased, cleaved PARP expression was increased; the phosphorylation level of PI3K/Akt pathway proteins: p-PI3K/PI3K, p-Akt/Akt was down-regulated in OE group (P<0.05).@*CONCLUSION@#SARI gene may suppress the proliferation of CBFL cells, and induce their apoptosis through the mitochondrial pathway, which may be related with the inhibition of PI3K/Akt pathway.

Comparison of the serum metabolic signatures based on 1H NMR between patients and a rat model of deep vein thrombosis.

Deep vein thrombosis (DVT) and pulmonary embolism (PE) have high morbidity, reduce quality of life, and can cause death. Biomarkers or genetic risk factors have not been identified in patients with DVT. In present study, serum of 61 patients suffering from DVT and a rat DVT model (n = 10) were assayed by a proton nuclear magnetic resonance (1H NMR) metabolomics technique combing with multivariate statistical analysis to identify the metabolites. The MetPA platform was used to identify differences in the metabolic pathways between the rat model and patients. The metabolomics results discovered that 11 different metabolites in rats and 20 different metabolites in DVT patients. Seven metabolites both altered in the rats and patients. Moreover, we observed changes in the metabolic pathways, including carbohydrate metabolism, lipid metabolism, and amino acid metabolism that were induced immediately by the thrombosis. Pathway of aminoacyl-tRNA biosynthesis perturbed only in the patients which was associated with the genetic risk factor of DVT. The study demonstrated that serum 1H NMR metabolomics can be used to diagnose DVT in the clinic. The altered pathways related to thrombosis and genetics will provide a foundation and new strategies for understanding the pathological mechanism and pharmacological targets of DVT.

Effects of Xingbi Wenmin Gel on Serum Levels of IL-4, TGF-β1 and Nasal Mucosa Eosinophils in Allergic Rhinitis Rats / 中国中医药信息杂志

Objective To observe the effects of Xingbi Wenmin Gel on serum levels of IL-4, TGF-β1 and nasal mucosa eosinophils (EOS) in allergic rhinitis (AR) rats; To discuss its mechanism of action. Methods Intraperitoneal injection of egg albumin and aluminum hydroxide based sensitized to ovalbumin challenge 2％ local AR model was established successfully. The experimental rats were randomly divided into normal group, model group, positive control group, and Xingbi Gel low-, medium-, and high-dosage groups. Each medication group was given relevant medicine for intervention. The rat symptom scores in each group were compared, and serum IL-4, TGF-β1, and nasal mucosa EOS counts were detected. Results Compared with the normal group, the serum levels of IL-4 and TGF-β1 in model group increased significantly (P<0.01), and the nasal mucosa EOS counts increased significantly (P<0.05). Compared with model group, the serum levels of IL-4 and TGF-β1 in Xingbi Gel medium-dosage group and positive control group decreased significantly (P<0.01), and the nasal mucosa EOS counts decreased significantly (P<0.05). Conclusion Xingbi Wenmin Gel may be effective through down-regulation of IL-4, TGF-β1 levels of inflammatory factors, and reduction of EOS infiltration in nasal mucosa.

Effect of PI3K/Akt/mTOR signal pathway-mediated N822K mutation on apoptosis induced by c-KIT inhibitor in AML cells / 中国免疫学杂志

Objective:To investigate the effect of c-KIT N822K mutation on the apoptosis of AML cells induced by c-KIT inhibitor and to explore the underlying molecular mechanisms.Methods: Kasumi-1 cells that carry the c-KIT N822K mutation were used as experimental group,and HL-60 and NB4 cells with non-c-KIT N822K mutation were used as control group.These AML cells were treated with 0,0.04,0.16 and 0.64 μmol/L c-KIT inhibitor sunitinib for 24 h,respectively.Apoptosis-related proteins and PI3K/Akt/mTOR pathway proteins were detected by Western blot,compared the changes of cell-related signal pathway proteins in each group.Results: With the increase of sunitinib concentration,the expression of apoptosis-related proteins Bax,CytoC,Caspase-9, Actived-Caspase-3 and PARP in HL-60 and NB4 cells were increased (P<0.05),and the expression of anti-apoptotic protein Bcl-2 was down-regulated (P<0.01).However,the trend of this change was obviously weakened in Kasumi-1 cells with N822K mutation.In Kasumi-1 cells,the phosphorylation levels of PI3K/Akt/mTOR pathway proteins such as PI3K,Akt,4EBP1 and mTOR were down-regulated in a dose-dependent manner(P<0.05),but not in HL-60 cells and NB4 cells.Conclusion:The constitutive activation of c-KIT induced by N822K mutation may affect the apoptosis induction of c-KIT inhibitor sunitinib to Kasumi-1 cells,which may be related to the inhibition of PI3K/Akt/mTOR pathway.

Effect of SU11248 on leukemia cell line K562 and its molecular mechanisms / 中国实验血液学杂志

This study was aimed to investigate the effect of SU11248 on proliferation and apoptosis of leukemia cell line K562 in vitro and its mechanism. The inhibitory effect of 3.2 µg/ml SU11248 on K562 proliferation was tested by MTT assay. The ability of SU11248 to induce apoptosis of K562 cells was examined by TUNEL and DNA ladder. The expression of C-MYC, hTERT and BCR-ABL mRNA in K562 cells was detected by RT-PCR. The protein expression of Akt and p-Akt in K562 cells was detected by Western blot. The results showed that the proliferation of K562 cells was obviously inhibited by 3.2 µg/ml SU11248 in a time-dependent manner. SU11248 could induce K562 cells apoptosis in dose-and time-dependent manner. The mRNA expression of C-MYC, hTERT and BCR-ABL was reduced significantly by SU11248 in a time-dependent manner (P < 0.05). Western blot detection showed that the expression of p-Akt protein in K562 cells decreased in dose-and time-dependent manner after SU11248 treatment, but the expression of Akt was not significantly changed. It is concluded that SU11248 can inhibit the growth of K562 cells efficiently through inducing apoptosis, its mechanism may be closely relate with the expression down-regulation of C-MYC, hTERT, BCR-ABL and the inhibition of Akt phosphorylation.

Acquisition of anoikis resistance through CD147 upregulation: A new mechanism underlying metastasis of hepatocellular carcinoma cells.

Acquisition of anoikis resistance is a prerequisite for the metastasis of hepatocellular carcinoma (HCC) cells. Activation of growth factor signaling pathways and rearrangement of the cytoskeleton have been reported as vital steps in this process. However, key molecules involved in anoikis resistance remain to be determined. The aim of this study was to investigate the effect of CD147 on HCC cells resistant to anoikis. The human SMMC-7221 human HCC cell line was used. Immunofluorescence was used to investigate the expression levels of CD147. Anoikis-induced cell death was assessed using trypan blue exclusion. In the present study, the results showed that SMMC-7721 HCC cells exhibited significant morphological changes when suspended in culture medium supplemented with 1% methocel and a subpopulation of cells resistant to anoikis was acquired with higher viability and invasion ability. CD147 was identified to be significantly increased in cells resistant to anoikis, when compared to the parental cells. CD147 knockdown by siRNA notably induced cell anoikis, partially through the inactivation of PI3K/Akt pathway. All of these evidence provide a novel CD147-related mechanism underlying the metastasis of HCC cells.

[Application of timolol in preventing variceal hemorrhage in patients with portal hypertension].

OBJECTIVE: To determine the effectiveness of timolol in preventing first variceal hemorrhage in portal hypertensive patients with esophageal varices. METHODS: A total of 42 cirrhotic patients with esophageal varices were enrolled in this study and received timolol or band ligation therapy randomly,with 21 patients in each group. The diameters of portal vein (PV), superior mesenteric vein (SMV), and splenic vein (SPV) as well as the portal venous flow and the splenic venous flow were measured. The first esophageal variceal bleeding of the two groups was recorded. RESULTS: The diameters of PV, SMV, and SPV as well as the flow of PV and SPV showed no significant difference before and after treatment in band ligation group (P>0.05). In timolol group,however,the diameter of portal vein significantly decreased after treatment [(14.11±2.96) mm vs. (12.15±1.61)mm, P<0.05], and the average blood flow of portal vein also significantly decreased after treatment [(1277.33±495.19) ml/min vs. (719.17±245.16)ml/min, P<0.05]. Both timolol and band ligation effectively prevented esophageal variceal bleeding, and the risk of first esophageal variceal bleeding in these two groups were not significantly different (15% vs. 10%, P<0.05). CONCLUSIONS: Timolol is safe and effective in preventing the first variceal bleeding in portal hypertensive patients with esophageal varices.

Killing effect of ZnPcH(1)-PDT on lymphoma cells / 中国实验血液学杂志

The objective of this study was to investigate the effect of ZnPcH(1)-PDT on the lymphoma cells and its mechanism. Human Burkitt's lymphoma cell line CA46 and mouse lymphoma cell line P388 were selected as objects for study. The killing effect of ZnPcH(1)-PDT on cells were assessed by MTT method and colony formation assay; the cell death patterns were analyzed by AO/EB fluorescence stain, TdT-mediated dUTP nick end labeling (TUNEL), DNA ladder assay; and the different proportions of each death pattern were determined by Annexin-V(-FITC)/PI double stains. The results showed that ZnPcH(1)-PDT displayed anti-proliferation effect on both CA46 cells and P388 cells in dose-dependent manner. CA46 cells were less sensitive to PDT than P388 cells (p < 0.05). Furthermore, PDT could induce cell apoptosis in time-dependent manner. The rate of cell apoptosis increased in the PDT-treated cells. The results of Annexin-V(-FITC)/PI stain indicated that early apoptosis was the main death pattern in the PDT-treated CA46 cells, while early apoptosis and necrosis were the main death model in the PDT-treated P388 cells. It is concluded that ZnPcH(1)-PDT can effectively inhibit lymphoma cell proliferation and induce cell apoptosis.

Expression of P27(kip1) and cyclin G in patients with acute leukemia and its correlation / 中国实验血液学杂志

This study was purposed to explore the expression of P27(kip1)and cyclin G in patients with acute leukemia (AL) and its correlation. The reverse polymerase chain reaction (RT-PCR) was used to analyse the expression of P27(kip1) and cyclin G mRNA in 89 AL patients and 10 normal persons; Western blot was used to analyze the expression of P27(kip1) and cyclin G protein in 39 AL patients and 10 normal persons. The results showed that the cyclin G mRNA and protein expressions in new diagnosed/relapsed cases of AL were significantly higher than those in patients with remission and normal controls (p < 0.05 and p < 0.01), but there was no difference between remission cases and normal controls (p > 0.05). The expression of P27(kip1) mRNA in newly diagnosed/relapsed patients with AL was not significantly different from patients with remission and normal controls (p > 0.05), while the P27(kip1) protein expression in remission cases of AL and normal controls was significantly higher than that in new diagnosed/relapsed cases (p < 0.05), but there was no difference between remission cases and normal controls (p > 0.05). The expression of P27(kip1) negatively and lowly correlated with the expression of cyclin G in patients with AL. It is concluded that the low expression of P27(kip1) and the high expression of cyclin G in patients with AL may have some correlation with genesis and development of AL and may be an indication for poor prognosis of AL.

[COX-2 and mPGES-1 expression in carotid atherosclerotic plaques].

OBJECTIVE: To investigate the changes in the expressions of inducible cyclooxygenase type 2 (COX-2) and membrane associated prostaglandin E-1(mPGES-1) in human carotid atherosclerotic plaques and to explore possible mechanisms of inflammatory process involved in plaque stability. METHODS: The mRNA and protein levels of COX-2 and mPGES-1 were compared between minimally and grossly atherosclerotic arterial tissues. COX-2 and mPGES-1 gene expression were established by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) in 10 mesenchymal artery controls and 24 atherosclerotic specimens. Presence of COX-2 and mPGES-1 protein was assessed by Western blotting. RESULTS: Immunohistochemical staining showed that the COX-2 and mPGES-1 immunoreactive substances were present in the cytoplasm of smooth muscle cell. Compared with the control group, immunostaining positive cells increased in carotid atherosclerotic plaque group. COX-2 and mPGES-1 gene expression was significantly elevated in atherosclerotic plaques (P< 0.05, respectively). The increased mRNA and protein levels of COX-2 and mPGES-1 were correlated in atherosclerotic tissue (P< 0.05). The mRNA and protein levels of COX-2 and mPGES-1 related to degree of pathological damage in atherosclerotic tissue (P< 0.05). COX-2 and mPGES-1 were not found in the control group (mesenteric vascular walls). CONCLUSION: COX-2 and mPGES-1 expression in plaques is significantly higher than that in the control group. These findings suggests that COX-2 and mPGES-1 might play a role in pathogenesis of atheroscleros and modulation of inflammatory process involved in plaque stability, and COX-2 may have proinflammatory enzyme properties.

[Histochemical and ultrastructure study of bile cast in liver transplantation patients].

OBJECTIVE: To investigate the histological components and solubility of the bile-cast, and to study the pathological course of bile cast formation. METHODS: HE staining, bilirubin staining (Gmelin reaction), Masson's staining, alcian blue staining and fibrin staining (weigert's) were performed on the formalin-fixed paraffin-embedded section of the bile cast. Ultrastructure was examined under the scanning electron microscope. Solubility test was also conducted using chymotrypsin, heparin, trypsin solution, HCl and NaOH solution to dissolve the bile-cast. RESULTS: The major components of the bile-cast were bilirubin crystals and collagen fibers. Between the mass of collagen fibers there was certain blood vessel structure. Necrosis bile duct structure was not found in the cast. Under the scanning electron microscope, four kinds of crystal morphologies were viewed. There were some mucoid mass and necrosis defluvium epithelial cells in the bile cast. Solubility test showed that the bile cast could be partial dissolved in NaOH solution (pH = 12.5). No dissolution was found in HCl solution (pH = 5.0), chymotrypsin solution, heparin and trypsin solution. CONCLUSIONS: Collagen fibers work as framework in the bile cast with bilirubin crystal filling between the framework. The emergence of fibroblast and blood vessels indicated the formation of bile cast might be the course of exudation and organization due to bile duct epithelium damage. Bile cast could be partially dissolved in alkaline solution, but could not be dissolved in acid solution, or in chymotrypsin, heparin and trypsin solutions.

[Secretory expression of chimeric Fab antibody HAb18 against human hepatocellular carcinoma in Pichia pastoris].

AIM: To express secretively chimeric Fab antibody HAb18 (cFab) against human hepatocellular carcinoma in Pichia pastoris. METHODS: Genes encoding CL chain and Fd fragment of cFab antibody HAb18 were subcloned into vectors pPIC9K and pPICZalphaA, respectively. After confirmed by DNA sequence analysis, the recombinant plasmids pPIC9K/CL and pPICZalphaA/Fd were transformed into the genome of Pichia pastoris GS115. Mut(+) multiple insert transformants were screened by G418 and Zeocin and then induced with 5 mL/L methanol to express cFab. RESULTS: 4 days after methanol induction, 26 mg/L of the cFab fragment was detected in the culture supernatant. Western blot proved that the expressed protein could specifically bind with HAb18GEF antigen. CONCLUSION: The successful expression of cFab/HAb18 in Pichia pastoris lays the foundation for large-scale production and further application of the antibody.