Effects of Silybin inhibiting Fas/FasL signaling pathway on inflammatory injury and macrophage apoptosis in pulmonary tuberculosis rats / 中国免疫学杂志

Objective:To explore the effects of Silybin on pulmonary inflammatory injury and macrophage apoptosis in pulmo-nary tuberculosis(TB)rats,and its regulation on death receptor Fas and its ligand FasL.Methods:TB rat model was prepared by tail vein injection of Mycobacterium tuberculosis(Mtb).Rats were randomly separated into model group,Silybin group,Fas overexpres-sion recombinant protein(pcDNA-Fas)group,pcDNA-Fas negative control(pcDNA-NC)group and Silybin+pcDNA-Fas group,with 15 rats in each group,and another 15 rats were selected as normal control group.Acid-fast staining was used to measure infection of lung tissue;HE staining was performed to observe pathological changes of lung tissue;expressions of TNF-α and IL-6 in lung tissue were detected by ELISA;apoptosis rate of alveolar macrophages was detected by flow cytometry combined with Annexin V-FITC/PI staining;expression levels of Fas,FasL,caspase8,caspase3 and macrophage inflammatory protein-2(MIP-2)were detected by Western blot.Results:Compared with normal control group,expressions of inflammatory factors in lung tissue and apoptotic rate of alveolar macrophages were increased in model group,Mtb infection and caseous necrosis in lung tissue were severe,and Fas/FasL-mediated caspase8/3 apoptotic pathway was activated(P<0.05).Compared with model group,expressions of inflammatory factors in lung tissue and apoptosis rate of alveolar macrophages in Silybin group were reduced,Mtb infection and caseous necrosis in lung tissue were alleviated,and the activity of Fas/FasL-mediated caspase8/3 apoptosis pathway decreased(P<0.05).pcDNA-Fas was able to further activate Fas/FasL-mediated caspase8/3 apoptotic pathway,aggravate lung tissue Mtb infection and caseous necrosis,promote inflammatory damage in lung tissue and macrophage apoptosis,and weaken the anti-Fas/FasL activation,anti-inflammatory and anti-apoptotic effects of Silybin(P<0.05).Conclusion:Silybin may play an anti-Mtb infection,anti-apoptosis of lung tissue macro-phages and anti-inflammatory effects by inhibiting the Fas/FasL signaling pathway.

Hei Xiaoyaosan Regulates Fas/FasL/Caspase-8/Caspase-3 Signaling Pathway to Inhibit Neuronal Apoptosis in AD Rats / 中国实验方剂学杂志

ObjectiveTo explore the effect and mechanism of Hei Xiaoyaosan in regulating the tumor necrosis factor receptor superfamily member 6 （Fas）/Fas ligand （FasL）/cysteine protease-8 （Caspase-8）/cysteine protease-3 （Caspase-3） signaling pathway to intervene in neuronal apoptosis and prevent Alzheimer's disease （AD）. MethodNinety SPF-grade SD male rats of 4 months old were selected and randomly grouped as follows： 10 rats in the blank group， 10 rats in the sham group （bilateral hippocampus injected with 1 μL normal saline）， and 70 rats in the modeling group ［bilater hippocampus injected with 1 μL amyloid-beta protein 1-42 （Aβ1-42） solution for the modeling of AD］. Fifty successfully modeled rats were selected and randomly assigned into model， donepezil hydrochloride （0.45 mg·kg-1）， and high-， medium-， and low-dose （15.30， 7.65， 3.82 g·kg-1） Hei Xiaoyaosan groups. Rats were administrated with corresponding agents by gavage once a day for 42 days. Terminal-deoxynucleoitidyl transferase-mediated nick end labeling （TUNEL） was employed to observe the apoptosis of neurons in the cortex and hippocampus， and immunohistochemistry （IHC） was used to detect the expression of B-cell lymphoma-2 （Bcl-2） and Bcl-2-associated X protein （Bax） in the hippocampus. Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was employed to determine the mRNA levels of Fas， FasL， and Fas-associated protein with death domain （Fadd）. Western blot was used to determine the protein levels of Fas， FasL， Fadd， Caspase-3， cleved Caspase-3， Caspase-8， and cleved Caspase-8. ResultCompared with the blank group and sham group， the model group showed increased apoptosis rate in the cortex and hippocampus （P<0.01）， elevated Bax level （P<0.01）， lowered Bcl-2 level （P<0.01）， up-regulated mRNA levels of Fas， FasL， and Fadd in the hippocampus （P<0.01）， and up-regulated protein levels of Fas， FasL， Fadd， cleaved Caspase-3， and cleaved Caspase-8 （P<0.01）. Compared with the model group， donepezil hydrochloride and Hei Xiaoyaosan at high and medium doses decreased the apoptosis rate in the cortex and hippocampus （P<0.05， P<0.01）， lowered the Bax level （P<0.01）， elevated the Bcl-2 level （P<0.01）， and down-regulated the mRNA levels of Fas， FasL， and Fadd and the protein levels of Fas， FasL， Fadd， cleaved Caspase-3， and cleaved Caspase-8 （P<0.05， P<0.01） in the hippocampus. Low-dose Hei Xiaoyaosan decreased the apoptosis rate in the cortex and hippocampus （P<0.05， P<0.01）， lowered the Bcl-2 level （P<0.01）， and down-regulated the mRNA levels of FasL and Fadd （P<0.05） and the protein levels of Fas， FasL， Fadd， cleaved Caspase-3， and cleaved Caspase-8 （P<0.05） in the hippocampus. ConclusionHei Xiaoyaosan can protect neurons in the cortex and hippocampus of AD rats by inhibiting the apoptosis mediated by the Fas/FasL/Caspase-8/Caspase-3 signaling pathway.

Correlation between UGRP1 and apoptosis pathway mediated by Fas in hashimoto thyroiditis / 安徽医科大学学报

Objective To investigate the correlation between uterine globulin associated protein 1(UGRP1)and Fas mediated apoptosis pathway in hashimoto thyroiditis(HT).Methods The expression of UGRP1 in thyroid cells of normal people and HT patients was detected by immunohistochemistry(IHC).FRTL-5 cells were transfect-ed by plasmids in vitro,and control group,UGRP1 group,Fas group were established respectively.Real-time fluo-rescent quantitative reverse transcription PCR(RT-qPCR)was used to detect the expression of Fas and UGRP1 mRNA in each group.Results UGRP1 expression was positive in thyroid cells of HT patients and negative in that of normal people.There were no significant differences between control group and UGRP1 group in Fas gene ex-pression(1.085 0±0.124 9 vs 1.021 0±0.113 9).Compared with the control group,the expression of UGRP1 gene increased significantly in Fas group(P＜0.000 1,5.807 0±0.323 2 vs 0.752 7±0.076 0).Conclusion The high expression of UGRP1 in HT may be related to apoptosis pathway mediated by Fas.

Andrographolide reduces cisplatin resistance of endometrial cancer Ishikawa/DPP cells by inhibiting the Fas/FasL signaling axis / 中国肿瘤生物治疗杂志

@#目的：探讨穿心莲内酯（Andro）调节脂肪酸合成酶（Fas）/脂肪酸合成酶配体（FasL）信号轴对子宫内膜癌Ishikawa细胞顺铂（DDP）耐药性的影响。方法：采用0、5、10、20 μg/mL DDP分别处理Ishikawa细胞和顺铂耐药的Ishikawa/DPP细胞，0、5、10、25、50 μmol/L Andro处理Ishikawa/DDP细胞，MTT法检测细胞增殖情况并为后续实验选择合适的给药剂量。将Ishikawa/DDP细胞随机分为对照组、DDP组（DDP干预）、Andro组（DDP、Andro干预）、pcDNA3.1-NC组（转染pcDNA3.1+DDP、Andro干预）、pcDNA3.1-Fas/FasL组（转染pcDNA3.1-Fas/FasL+DDP、Andro干预），24 h后，采用qPCR法检测Fas、FasL mRNA的表达，平板克隆形成实验、Transwell实验和流式细胞术分别检测细胞克隆能力、细胞迁移与侵袭和细胞凋亡，WB法检测增殖细胞核抗原（PCNA）、BAX、Bcl-2、MMP-2、PD-L1、多药耐药蛋白-1（MDR-1）及Fas、FasL蛋白表达。结果：DDP以剂量依赖的方式抑制Ishikawa和Ishikawa/DPP细胞增殖，并且与Ishikawa细胞比较，Ishikawa/DPP细胞对DDP的敏感性更低（均P<0.05）；Andro以剂量依赖性的方式抑制Ishikawa/DPP细胞的增殖（均P<0.05）。Ishikawa/DPP细胞中Fas、FasL的表达水平均高于Ishikawa细胞（均P<0.05）。选取20 μg/mL DDP和25 μmol/L Andro为干预剂量，干预时间24 h。与对照组比较，DDP组Ishikawa/DPP细胞中PD-L1、MDR-1、Fas、FasL mRNA及蛋白表达水平显著升高（P<0.05），而克隆形成率、迁移与侵袭细胞数、凋亡率差异均无统计学意义（均P>0.05）；与DDP组比较，Andro组Ishikawa/DPP细胞中Fas、FasL mRNA表达水平、细胞克隆形成率、迁移与侵袭细胞数、PCNA、Bcl-2、MMP-2、PD-L1、MDR-1、Fas、FasL蛋白表达水平显著降低，BAX蛋白表达水平及凋亡率显著升高（P<0.05或P<0.01），pcDNA3.1-NC组与Andro组类似；与pcDNA3.1-NC组比较，pcDNA3.1-Fas/FasL组Ishikawa/DPP细胞上述指标变化均被逆转（P<0.05）。结论：Andro可能通过抑制Fas/FasL信号轴来抑制Ishikawa/DPP细胞增殖、迁移与侵袭，促进凋亡，从而降低细胞对DDP的耐药性。

To Explore the Effect of Xiangpi Shengji Ointment on the Apoptosis of Wound Cells in Model Rats after Anal Fistula Operation based on Fas/Fas L Pathway / 世界科学技术-中医药现代化

Objective To explore the effect of Xiangpi Shengji Ointment on wound healing and apoptosis-related Fas/Fas L pathway in model rats after anal fistula operation.Methods Thirty-six SD rats were used to construct the anal fistula model by using steel wire hanging line and indwelling for 30 days.After successful modeling,27 rats with anal fistula were randomly selected for"fistulectomy"to construct a postoperative wound model.After operation,the wound model rats were randomly divided into three groups,9 rats in each group,which were Xiangpi Shengji Ointment Group,Vaseline Group and Model Group,and the remaining 9 rats with anal fistula were sham operation group.The rats in the Xiangpi Shengji Ointment group were externally applied with Xiangpi Shengji Ointment gauze,while those in the Vaseline group were externally applied with Vaseline gauze.The rats in the model group were only disinfected and rinsed.No special treatment was given to the rats in the sham operation group.The wound healing was observed on the 3rd,5th,7th and 10th day after medication intervention,and the wound healing rate was calculated.After 10 days of continuous intervention,wound tissues were taken from each group,and the histopathological changes,the number of apoptosis,the expressions of Fas,Fas L,caspase-8 and cyto-c in wound tissues were observed by HE staining,TUNEL staining and immunohistochemistry respectively,and the mRNA expressions of Fas,Fas L and cyto-c in wound tissues were detected by RT-PCR.Results Compared with the model group,Xiangpi Shengji ointment group and Vaseline group significantly promoted wound healing at 7 and 10 days after intervention(P<0.01),and the wound healing rate of Xiangpi Shengji ointment group was significantly higher than that of Vaseline group(P<0.01).After 10 days of drug intervention,compared with sham operation group,the apoptosis rate of Xiangpi Shengji ointment group,Vaseline group and model group increased significantly(P<0.01),and the relative expressions of Fas,Fas L and cyto-c mRNA and the expression levels of Fas,Fas L,caspase-8 and cyto-c protein in wound tissue increased significantly(P<0.05,P<0.01).Compared with the model group,the apoptosis rate,the relative expression of Fas,Fas L and cyto-c mRNA and the expression level of Fas,Fas L,caspase-8 and cyto-c protein in Xiangpi Shengji ointment and Vaseline groups decreased significantly(P<0.05,P<0.01).Compared with Vaseline group,the apoptosis rate of Xiangpi Shengji ointment group decreased significantly(P<0.01),and the relative expression of Fas,Fas L and cyto-c mRNA and the expression level of Fas,Fas L,caspase-8 and cyto-c protein decreased significantly(P<0.05,P<0.01).Conclusion Xiangpi Shengji Ointment can inhibit the activation of Fas/Fas L pathway,reduce the apoptosis of wound tissue cells and promote wound healing after anal fistula operation.

Effects of polyphyllin Ⅵ on the proliferation and apoptosis of glioma cells and potential mechanism / 中国药房

OBJECTIVE To investigate the effects of polyphyllin Ⅵ（PPⅥ） on the proliferation and apoptosis of glioma cells and potential mechanism. METHODS Using human glioma LN229 cells as objects， MTT assay was used to detect the survival rate after treated with different concentrations of PPⅥ [0 （control group）， 1， 2， 4， 8， 16， 32， 64 μmol/L] for different time （24， 48， 72 h）. The clone formation experiments were adopted to detect the number of cell clones and clone formation rate after being treated with different concentrations of PPⅥ [0 （control group）， 2， 4， 8 μmol/L] for 14 days. The flow cytometry and Western blot assay were used to detect the apoptotic rate of cells， the expressions of apoptosis-related protein [B cell lymphoma-2 （Bcl-2）， Bcl-2 associated X protein （Bax）， cleaved caspase-3]， and the expressions of related proteins of Fas/Fas ligand （FasL） death receptor pathway and protein kinase B （Akt）/glycogen synthesis kinase-3β （GSK-3β） pathway after being treated with different concentrations of PPⅥ [0（control group）， 4， 8 μmol/L] for 24 h. RESULTS Compared with the control group， the survival rate of cells， the number of clones and clone formation rate， the protein expression of Bcl-2， and the phosphorylation levels of Akt and GSK-3β protein were decreased significantly in different concentration groups of PPⅥ （P＜0.05 or P＜0.01）. The apoptotic rate， the protein expressions of Bax， cleaved caspase-3， Fas， FasL and cleaved caspase-8 were increased significantly （P＜0.05 or P＜ 0.01）. CONCLUSIONS PPⅥ can inhibit the proliferation and induce the apoptosis of human glioma LN229 cells， which may be related to the activation of the Fas/FasL death receptor pathway and the inhibition of the Akt/GSK-3β pathway.

Effect of "Zhibian" (BL 54)-to-"Shuidao" (ST 28) needle insertion on the expression of Fas/FADD/Caspase-8 of death receptor pathway in rats with primary ovarian insufficiency / 中国针灸

OBJECTIVE@#To explore the effect of "Zhibian" (BL 54)-to-"Shuidao" (ST 28) needle insertion on the ovarian function in the rats with primary ovarian insufficiency (POI) and the potential effect mechanism based on the Fas/FADD/Caspase-8 of death receptor pathway.@*METHODS@#Forty-eight female SD rats were randomly divided into a blank group, a model group, a medication group and an acupuncture group, with 12 rats in each group. Except in the blank group, the rats in the other groups were intraperitoneally injected with cyclophosphamide to establish the POI model. In the acupuncture group, after successful modeling, the intervention was given with "Zhibian" (BL 54)-to- "Shuidao" (ST 28) needle insertion, once daily, 30 min in each intervention; and the duration of intervention was 4 weeks. In the medication group, estradiol valerate tablets were administered intragastrically, 0.09 mg•kg-1•d-1, for 4 weeks. The general situation and the estrous cycle of the rats were compared among groups. Using ELISA, the levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2) in the serum were detected. HE staining was adopted to observe the morphological changes of ovarian tissue of rats. The protein expression of Fas, FADD and Caspase-8 in ovarian tissue was detected with immunohistochemistry and Western blot.@*RESULTS@#After modeling, except the rats of the blank group, the rats of the other groups had dry fur, lost hair, low spirits, reduced food intake, increased urination and loose stool. After intervention, the stool became regular gradually in the acupuncture group and the medication group. The percentage of estrous cycle disturbance was increased in the rats of the model group when compared with the blank group (P<0.01); in comparison with the model group, the percentages of estrous cycle disturbance were reduced in the acupuncture group and the medication group after intervention (P<0.01). When compared with the blank group, the body mass and E2 content in the serum were lower (P<0.01), the levels of FSH and LH in the serum and the protein expression levels of Fas, FADD and Caspase-8 were increased (P<0.01) in the model group. Compared with the model group, the body mass and E2 contents in the serum were higher (P<0.01), the levels of FSH and LH in the serum and the protein expression levels of Fas, FADD and Caspase-8 were reduced (P<0.01) in the acupuncture group and the medication group.@*CONCLUSION@#"Zhibian" (BL 54)-to-"Shuidao" (ST 28) needle insertion can effectively improve the ovarian function of POI rats, and its effect mechanism may be related to regulating the serum sex hormone levels, reducing the expression of Fas, FADD and Caspase-8 in ovarian tissue and retarding apoptosis of ovarian cells.

Targeting Kindlin-2 in adipocytes increases bone mass through inhibiting FAS/PPARγ/FABP4 signaling in mice

Osteoporosis (OP) is a systemic skeletal disease that primarily affects the elderly population, which greatly increases the risk of fractures. Here we report that Kindlin-2 expression in adipose tissue increases during aging and high-fat diet fed and is accompanied by decreased bone mass. Kindlin-2 specific deletion (K2KO) controlled by Adipoq-Cre mice or adipose tissue-targeting AAV (AAV-Rec2-CasRx-sgK2) significantly increases bone mass. Mechanistically, Kindlin-2 promotes peroxisome proliferator-activated receptor gamma (PPARγ) activation and downstream fatty acid binding protein 4 (FABP4) expression through stabilizing fatty acid synthase (FAS), and increased FABP4 inhibits insulin expression and decreases bone mass. Kindlin-2 inhibition results in accelerated FAS degradation, decreased PPARγ activation and FABP4 expression, and therefore increased insulin expression and bone mass. Interestingly, we find that FABP4 is increased while insulin is decreased in serum of OP patients. Increased FABP4 expression through PPARγ activation by rosiglitazone reverses the high bone mass phenotype of K2KO mice. Inhibition of FAS by C75 phenocopies the high bone mass phenotype of K2KO mice. Collectively, our study establishes a novel Kindlin-2/FAS/PPARγ/FABP4/insulin axis in adipose tissue modulating bone mass and strongly indicates that FAS and Kindlin-2 are new potential targets and C75 or AAV-Rec2-CasRx-sgK2 treatment are potential strategies for OP treatment.

Fas cell surface death receptor/Fas ligand genetic variants in gastric cancer patients: A case-control study

Background & objectives: Various studies have suggested a correlation between Fas cell surface death receptor/Fas ligand (FAS/FASL) variants and multiple types of cancers. The present study aimed to investigate the association between FAS-670A/G and FASL-844C/T and the synergistic effects of both variants on the risk of gastric cancer (GC) in the Kurdish population of west of Iran. Methods: This study was conducted by polymerase chain reaction-restriction fragment length polymorphism technique using MvaI and BsrDI restriction enzymes in 98 GC patients and 103 healthy control individuals. Results: According to the obtained results, a significant association (P=0.008) of FASL polymorphism among GC patients and the control group was detected. Furthermore, no significant differences were found in the FAS polymorphism frequencies between GC patients and the control group. Codominant and dominant models in FASL polymorphism showed significant protective effects against GC [odds ratio (OR)=0.307, 95% confidence interval (CI) (0.134-0.705), P=0.005; OR=0.205, 95% CI (0.058-0.718), P=0.013 and OR=0.295, 95% CI (0.129-0.673), P=0.004 for models of codominant CC vs. CT, codominant CC vs. TT and dominant, respectively]. Furthermore, the presence of both FAS-670G and FASL-844T alleles represented a significant protective effect against GC occurrence [OR=0.420, 95% CI (0.181-0.975), P=0.043]. Interpretation & conclusions: So far, we believe this is the first study, the results of which suggest that FASL gene variation and its synergistic effects with FAS gene could be associated with the risk of GC in the Kurdish population in the west of Iran

Estimación de la tasa de filtración glomerular: concordancia de la ecuación del European Kidney Function Consortium de 2020 con CKD-EPI y FAS en adultos jóvenes

Contexto: la TFG es un indicador de la función renal y se estima por ecuaciones TFGe, la mayoría son aplicables en un rango etario, aunque se producen discrepancias en los valores al cambiar de fórmula por cruzar un límite de edad. Así, la ecuación CKD-EPI sobreestima la TFG en adultos jóvenes, mientras que la ecuación FAS la sobreestima para creatininemias bajas. Para minimizar sus limitaciones, el European Kidney Function Consortium propuso la ecuación EKFC combinando características de diseño de FAS y CKD-EPI. Objetivo: evaluar el comportamiento de las ecuaciones EKFC vs. CKD-EPI y FAS en jóvenes, las diferencias en TFGe y la concordancia en asignación a categorías de TFG. Metodología: estudio analítico aprobado por el Comité Asesor de Ética y Seguridad de la Investigación de la Facultad de Bioquímica y Ciencias Biológicas de la UNL, con una muestra de 157 estudiantes voluntarios, de entre 18 y 37 años. Para la medición de la creatininemia se utilizó el método Jaffé cinético trazable a Isotopic Dilution Mass Spectroscopy, con el programa estadístico MedCalc. Resultados: EKFC: TFGe menores que CKD-EPI y FAS, total y por sexo. Media de las diferencias (mL/min/1,73 m2): (CKD-EPI - EKFC) totales = 10,42; 18-20 años = 11,91; 21-30 años = 11,10; 31-37 años = 8,96 / (FAS-EKFC) totales = 2,79; FAS ≤ 110 mL/min/1,73 m2 y mayor: 1,1 y 9,0 respectivamente. Asignación a categorías G: kappa menores EKFC vs. CKD-EPI que vs. FAS. Recategorización: 13,4 % en G1 por CKD-EPI categorizados G2 por EKFC; 0,6 % respecto a FAS en igual sentido. Asignación a categorías ≥ 75mL/min/1,73 m2 o menor: buena concordancia. Conclusiones: en la muestra, EKFC cumple los objetivos de su diseño. La sobreestimación de TFGe por CKD-EPI en adultos jóvenes disminuyó, más fuertemente hacia los 18 años, y corrigió la de FAS para creatininemias bajas. Es importante desarrollar estimadores de TFG basados en creatininemia que cubran todo el rango de edades y estados de función renal.

Introduction: GFR is a kidney function indicator. The estimation of the GFR (eGFR) is carried out by equations. Most of them are applicable with in an age range. Discrepancies between the values are found when crossing a limit of age. CKD-EPI overestimates GFR in young adults; FAS overestimates it for low creatininemias. To minimize these limitations, the European Kidney Function Consortium proposed the EKFC equation that combines design features of FAS and CKD-EPI. Objective: The performance of EKFC vs. CKD-EPI and FAS in young people was evaluated: differences in eGFR and agreement in the allocation to GFR categories were found. Methods: Analytical study approved by the Ethics Committee. Sample: 157 volunteer students, 18-37 years old. Creatininemia: kinetic Jaffé method traceable to Isotopic Dilution Mass Spectroscopy. Program: MedCalc. Results: EKFC: eGFR lower than CKD-EPI and FAS, total and by sex. Means of the differences (mL/min/1.73m2): total (CKD-EPI - EKFC) = 10.42; 18-20 years = 11.91; 21-30 years = 11.10; 31-37 years = 8.96 // (FAS-EKFC) total = 2.79; FAS≤ 110 mL/min/1.73m2 and higher: 1.1 and 9.0 respectively. Allocation to G categories: lower kappa EKFC vs. CKD-EPI than vs. FAS. Recategorization: 13.4% in G1 by CKD-EPI categorized G2 by EKFC; 0.6% compared to FAS, in the same sense. Allocation to categories ≥75mL/min/1.73 m2 or less: good agreement. Conclusions: In the sample, EKFC meets the objectives of its design. The overestimation of eGFR by CKD-EPI in young adults decreased, even more around 18 years of age, and corrected that of FAS for low creatininemias. It is important to develop GFR estimators based on creatininemia that cover the entire range of ages and renal function status.

MiR-125b inhibits growth of gastric cancer cells by targeting Fas/FasL signaling pathway / 中国医师杂志

Objective:To investigate the regulation effect of miR-125b in the gastric cancer cell growth mediated by apoptosis related protein (Fas)/apoptosis related protein ligand (FasL) signal.Methods:Gastric cancer SGC-7901 cells were cultured in vitro. MiR-125b inhibitor sequence, NC sequence and transfection reagent were transfected into SGC-7901 cells and divided into three groups: miR-125b inhibited group, NC group and control group. The expression of miR-125b in transfected cells was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR), and cell proliferation was detected by cell counting kit-8 (CCK-8) method. The colony formation was detected by plate cell clone formation assay. Cell apoptosis and cycle were detected by flow cytometry. The protein expression of Fas and FasL was detected by Western blot. The targeted regulation of Fas by miR-125b was detected by luciferase activity assay. Results:The expression level of miR-125 and the number of cell colony in miR-125b inhibited group was significantly lower than those in control group and NC group, and the inhibition rate of cell proliferation and apoptosis rate were significantly higher than that in control group and NC group (all P<0.05). The DNA content in G 1 phase in miR-125b inhibited group was significantly higher than that in control group and NC group, and the DNA content in S phase in miR-125b inhibited group was significantly lower than that in control group and NC group (all P<0.05). The expression of Fas and FasL protein in miR-125b inhibited group was significantly higher than that in control group and NC group (all P<0.05). The target site of miR-125b was found in 3′-UTR of Fas mRNA, and compared with the NC+ Fas 3′UTR-Wt group, the activity of luciferase in the miR-125b inhibited group+ Fas 3′-UTR-Wt group decreased significantly ( P<0.05). Conclusions:Inhibition of miR-125b expression can activate Fas/FasL signal and inhibit SGC-7901 cell proliferation, induce G 1 phase arrest of cell cycle and promote apoptosis.

Autoimmune lymphoproliferative syndrome: analysis of 1 pedigree and review of literature / 白血病·淋巴瘤

Objective:To improve the understanding of autoimmune lymphoproliferative syndrome (ALPS).Methods:The clinical data of the proband and his family members in Children's Hospital Affiliated to Zhengzhou University in August 2018 were retrospectively analyzed, and the peripheral blood DNA of the proband, his parents and siblings was extracted. High-throughput next-generation sequencing was used to make gene analysis and validation. Phenotype and genotype of them were also analyzed. Relevant literature was reviewed.Results:The proband was a 1-year and 1-month old boy with hemolytic anemia, thrombocytopenia and splenomegaly as the main manifestations. The double negative T cells and the Vitamin B 12 of the proband were significantly increased and the autoantibodies were positive. The boy's father had a history of splenomegaly. His elder brother and sister had similar clinical manifestations. The results of next-generation sequencing showed that the FAS gene frameshift mutation (c.648delT) was detected in this boy and his father, elder brother and sister, which was a new mutation. After immunosuppressive treatment, the symptoms of the boy improved and the blood cells increased. Conclusions:The frameshift mutation of FAS gene may be the cause of the disease in this ALPS pedigree. Clinically, it is necessary to consider ALPS for children with unexplained hemocytopenia and hepatosplenomegaly. Double-negative T cells, autoantibodies, Vitamin B 12 should be tested, and high-throughput gene sequencing should be performed if necessary.

Investigation of FAS and IL-6 expression in placentas with HELLP syndrome / Investigación de la expresión de FAS e IL-6 en placentas con síndrome de HELLP

SUMMARY: The aim of our study was to investigate the effect of inflammation in the placenta on the pro-apoptotic development after severe preeclampsia. Placenta tissue samples of 15 HELLP syndrome and 15 healthy 35-38th week-pregnant women were involved in the study. Tissue samples were taken only from the maternal side of the placenta and fixed in 10 % formaldehyde, then blocked in paraffin wax and 4-6 mm-thick sections were cut and stained with Harris Hematoxylene-Eosin. Antigen retrieval was performed for sections, incubated with FAS antibody and anti-IL-6 antibody. After the application of streptavidin peroxidase followed by AEC chromogen solution, sections were counterstained with Harris hematoxylin. Significant thickening of the fibrinoid layer, degeneration and apoptotic change in decidua cells, marked increase in the hyalinized area, degenerative changes in the syncytial regions of the chorionic villus and an increase in syncytial nodes and bridges and IL- expression were observed as positive. FAS expression was positive in the pycnotic nuclei of decidual cells in the maternal region and in the syncytial regions. It was observed that the proapoptotic process increased as a result of severe preeclampsia. It was concluded that the control of cytokine activity and reduction of pro-apoptotic signal during the inflammation process will slow down the development of HELLP syndrome.

RESUMEN: El objetivo de nuestro estudio fue investigar el efecto de la inflamación en la placenta sobre el desarrollo proapoptótico después de la preeclampsia severa. Se recogieron muestras de tejido de placenta de 15 mujeres con síndrome de HELLP y 15 mujeres sanas con un embarazo de 35 a 38 semanas. Se tomaron muestras de tejido solo del lado materno de la placenta y se fijaron en formaldehído al 10 %, luego se bloquearon en parafina y se cortaron secciones de 4-6 mm de espesor y se tiñeron con hematoxilena-eosina de Harris. La recuperación del antígeno se realizó para secciones, incubadas con anticuerpo FAS y anticuerpo anti-IL-6. Después de la aplicación de estreptavidina peroxidasa seguida de solución de cromógeno AEC, las secciones se contrastaron con hematoxilina de Harris. Se observó como positivo un engrosamiento significativo de la capa fibrinoide, degeneración y cambio apoptótico en las células de la decidua, aumento marcado en el área hialinizada, cambios degenerativos en las regiones sincitiales de la vellosidad coriónica y un aumento en los nódulos y puentes sincitiales y la expresión de IL-6. La expresión de FAS fue positiva en los núcleos picnóticos de las células deciduales en la región materna y en las regiones sincitiales. Se observó que el proceso proapoptótico se incrementó como consecuencia de la preeclampsia severa. Se concluyó que el control de la actividad de las citocinas y la reducción de la señal proapoptótica durante el proceso de inflamación ralentizarán el desarrollo del síndrome de HELLP.

Effect of fucoxanthin on insulin resistance in obese mice induced by high fat diet / 中国中药杂志

The aim of this paper was to study the effect and mechanism of fucoxanthin on insulin resistance of obese mice induced by high-fat diet. Fifty C57 BL/6 J male mice were randomly divided into control group and high-fat diet group. The insulin resistance model was induced with high-fat diet for 12 weeks, and model mice were randomly divided into model group, fucoxanthin-0.2% group, fucoxanthin-0.4% group and metformin group. After dietary treatment for 6 weeks, the body weight and epididymal fat weight in each group were measured. Fasting blood glucose(FBG), fasting insulin(FINS), total cholesterol(TC), triglyceride(TG), low-density lipoprotein(LDL-C) and high-density lipoprotein(HDL-C) were measured, and insulin resistance index(HOMA-IR) was calcula-ted. The pathological morphology in liver was observed by hematoxylin eosin staining, and the expressions of some key proteins in insulin receptor substrate 1(IRS-1)/posphoinositide 3-kinase(PI3 K)/serine-threonine kinase(Akt) and peroxisome proliferators-activated receptor-γ(PPARγ)/sterol regulatory element binding protein-1(SREBP-1)/fatty acid synthetase(FAS) pathways in liver were detected by Western blot. According to the findings, compared with the model group, levels of body weight, epididymal fat weight, FBG, FINS, TC, TG, LDL-C and HOMA-IR, as well as protein expressions of PPARγ, SREBP-1 and FAS in liver were significantly reduced(P<0.05 or P<0.01), while level of HDL-C and protein expressions of p-IRS-1, IRS-1, PI3 K and p-Akt in liver were signi-ficantly increased after treatment with fucoxanthin(P<0.05 or P<0.01). And the pathological changes of liver tissue in fucoxanthin-treated mice were also improved obviously. The results showed that fucoxanthin could improve obesity, hyperglycemia and hyperlipidemia, and alleviate insulin resistance in obese mice, and its mechanism is possibly related to the regulation of IRS-1/PI3 K/Akt and PPARγ/SREBP-1/FAS pathways.

Effect of Danggui Niantongtang on Key Factors of Extrinsic and Intrinsic Apoptotic Pathway in Synovial Tissue of Adjuvant Arthritis Rats / 中国实验方剂学杂志

Objective:To observe the effect of Danggui Niantongtang on the protein and mRNA expression of key regulatory factors of the extrinsic and intrinsic apoptotic pathway in synovial tissue of adjuvant arthritis （AA） rats， and to further explore the mechanism of Danggui Niantongtang in the prevention and treatment of rheumatoid arthritis. Method:The general condition of AA rats， including its body weight， were observed. The changes of toe volume were detected by toe volume meter. Histopathological changes of synovium of knee joint were observed by hematoxylin-eosin （HE） staining. The mRNA and protein expression levels of tumor necrosis factor receptor super family 6 （Fas）， Fas-associating protein with a novel death domain（FADD）， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax） and cysteinyl aspartate specific proteinase Caspase-3 （Caspase-3） were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot. Result:Compared with the normal group， the toe volume of the model group increased significantly （<italic>P</italic><0.01）， with significantly proliferated synovial cells， significantly reduced mRNA and protein expression levels of Fas， FADD， Bax and Caspase-3 in synovial tissues（<italic>P</italic><0.05，<italic> P</italic><0.01）， and significantly increased Bcl-2 level （<italic>P</italic><0.01）. Compared with the model group， the swelling degree of toes in Danggui Niantongtang group and Tripterygium group was significantly alleviated （<italic>P</italic><0.01）， with significantly improved synovial hyperplasia， significantly increased mRNA and protein expression levels of Fas， FADD， Bax and Caspase-3 （<italic>P</italic><0.05， <italic>P</italic><0.01）， and significantly decreased expression levels of bcl-2 mRNA and protein （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:Danggui Niantongtang can effectively reduce joint swelling and abnormal proliferation of synovial tissue in AA rats. Its mechanism may be related to regulating the expression of Fas， FADD， Bax， Bcl-2 and Caspase-3， and promoting the apoptosis of synovial cells.

Effect of Shaoyaotang on Expressions of CD14, FADD and Caspase-8 in Colonic Tissues of Rats with Large Intestinal Damp-heat Syndrome of Ulcerative Colitis / 中国实验方剂学杂志

Objective:To observe the effect of Shaoyaotang on the contents of cell adhesion molecule-1 （ICAM-1） and transforming growth factor-<italic>β</italic>₁ （TGF-<italic>β</italic>₁） in serum of large intestine damp-heat syndrome of ulcerative colitis （UC） in rats， and the gene and protein expressions of leukocyte differentiation antigen14 （CD14）， Fas-related death domain protein （FADD） and cysteinyl aspartate specific protease-8 （Caspase-8） in the focal colon tissue. Method:A total of 80 SPF Wistar rats were randomly divided into the blank group （<italic>n</italic>=10） and modeling group （<italic>n</italic>=70）. The large intestine damp-heat syndrome of UC rats was replicated by the combination of disease and syndrome， which was high-fat， high-sugar and spicy diets combined with 2， 4-dinitrobenzene sulfonic acid （DNBS） and ethanol. After successful modeling， the modeled groups were divided into model group， sulfasalazine （SASP）control group， and low， medium and high-dose Shaoyaotang groups by the method of random number table， with14 rats in each group. Low， medium and high doses of Sulfasalazine 0.2 g·kg^-1·d^-1 and Shaoyaotang （6， 12， 24 g·kg^-1·d^-1）were given by gavage. The blank group and the model group were given equal volume of normal saline for 21 days. The contents of serum ICAM-1 and TGF-<italic>β</italic>₁ were detected by enzyme-linked immunosorbent assay （ELISA）， the expressions of CD14， FADD and Caspase-8 mRNA in colon tissues were detected by Real-time quantitative polymerase chain reaction （Real-time PCR）， and the expressions of CD14， FADD and Caspase-8 protein in colon tissues were detected by Western blot. Result:Compared with the blank group， the serum ICAM-1 level in the model group were significantly increased， whereas the content of TGF-<italic>β</italic>₁ were significantly decreased （<italic>P</italic><0.05）. The relative expression levels of CD14， FADD， Caspase-8 mRNA and protein were significantly increased （<italic>P</italic><0.05）. Compared with the model group， the content of ICAM-1 in the serum of the rats in the medium， high-dose Shaoyaotang groups and the SASP group were significantly decreased， while the content of TGF-<italic>β</italic>₁ in the serum of the rats in the low， medium， high-dose Shaoyaotang groups and the SASP group were significantly increased （<italic>P</italic><0.05）. The expression levels of CD14， FADD， Caspase-8 mRNA and protein in each intervention group were significantly decreased （<italic>P</italic><0.05）， especially in the high-dose Shaoyaotang group and the SASP group. Conclusion:Shaoyaotang has a certain intervention effect on UC rats with large intestine damp-heat syndrome， and its mechanism may be related to the inhibition of CD14， FADD and Caspase-8 genes and proteins expression.

Yin-Yang-1 decreases Fas-induced apoptosis in acute lymphoblastic leukemia under hypoxic conditions: its implications in immune evasion / YY1 induce la disminución de la apoptosis a través de Fas en la leucemia linfoblástica aguda en condiciones hipóxicas: implicaciones en la evasión de la respuesta inmunitaria

Abstract Background: Acute lymphoblastic leukemia (ALL) is an aggressive malignant disease with high prevalence in pediatric patients. It has been shown that the downregulation of Fas expression is correlated with an inadequate response in ALL, although these mechanisms are still not well understood. Several reports demonstrated that hypoxia is involved in dysfunctional apoptosis. Yin-Yang-1 (YY1) transcription factor is involved in resistance to apoptosis, tumor progression, and it is increased in different types of cancer, including leukemia. The regulatory mechanism underlying YY1 expression in leukemia is still not understood, but it is known that YY1 negatively regulates Fas expression. The study aimed to evaluate the effect of YY1 on Fas expression under hypoxic conditions in ALL. Methods: Leukemia cell line RS4; 11 was cultured under normoxic and hypoxic conditions. YY1, Fas receptor, and hypoxia-inducible factor (HIF-1α) expression were analyzed. After treatment with a Fas agonist (DX2), apoptosis was analyzed through the detection of active caspase 3. Data were analyzed using Pearson’s correlation. Results: Leukemia cells co-expressed both HIF-1α and YY1 under hypoxia, which correlated with a downregulation of Fas expression. During hypoxia, the levels of apoptosis diminished after DX2 treatment. The analysis revealed that patients with high levels of HIF-1α also express high levels of YY1 and low levels of Fas. Conclusions: These results suggest that YY1 negatively regulates the expression of the Fas receptor, which could be involved in the escape of leukemic cells from the immune response contributing to the ALL pathogenesis.

Resumen Introducción: La leucemia linfoblástica aguda (LLA) es una enfermedad con alta prevalencia en la población pediátrica. El mecanismo por el cual el receptor de Fas participa en la regulación inmunitaria en los tumores es desconocido, pero se sabe que está subexpresado en LLA. El factor de transcripción Ying-Yang-1 (YY1) está involucrado en la resistencia a la apoptosis y la progresión tumoral; se encuentra aumentado en diferentes tumores, incluida la LLA. Aunque los mecanismos que regulan la expresión de YY1 en LLA son desconocidos, se sabe que YY1 regula la expresión del receptor de Fas. El objetivo de este trabajo fue evaluar el efecto de YY1 en la expresión de Fas en condiciones de hipoxia en la LLA. Métodos: Se cultivaron células RS4;11 en condiciones de hipoxia y se analizó la expresión de YY1, receptor de Fas y HIF-1α. La apoptosis fue inducida usando un agonista de Fas (DX2) y se analizó con la detección de caspasa 3 activa. Los datos se analizaron mediante correlación de Pearson. Resultados: Las células RS4;11 coexpresaron HIF-1αy YY1 en hipoxia, lo cual correlaciona con una baja expresión de Fas. La apoptosis se encontró disminuida durante condiciones de hipoxia, después del tratamiento con DX2. El análisis bioinformático mostró que los pacientes con altos niveles de HIF-1αpresentan YY1 elevado y bajos niveles del receptor de Fas. Conclusiones: Estos resultados sugieren que YY1 regula negativamente la expresión del receptor de Fas, lo cual podría estar involucrado en el escape de las células leucémicas a la respuesta inmunitaria, contribuyendo a la patogénesis de la LLA.

Anti-tumor effect and its related mechanisms of cinobufotalin combined with cisplatin on H22 liver cancer mice / 中国中药杂志

In order to observe the anti-tumor effect of cinobufotalin on H22 liver cancer mice and to explore its regulatory mechanism, 50 Kunming mice were subcutaneously inoculated with H22 intraperitoneal passage cells under the armpit to establish H22 hepatocellular carcinoma model. They were then randomly divided into model group, cinobufotalin low dose group, cinobufotalin high dose group, cisplatin group and cisplatin+cinobufotalin group, which received 0.01% ethanol solution, 1 mg·kg~(-1) cinobufotalin, 5 mg·kg~(-1) cinobufotalin, 5 mg·kg~(-1) cisplatin, 5 mg·kg~(-1)cisplatin + 5 mg·kg~(-1) cinobufotalin respectively for 10 days. The general condition of mice during the intervention was observed, and the inhibition rate, tumor mass, thymus index, histopathological changes of the tumors, apoptotic rate of the tumors, the expressions of phosphatidylinositol 3-kinase(PI3 K), protein kinase B(Akt), apoptosis related gene(Fas), Fas ligand(FasL) mRNA and protein phosphorylated Akt(pAkt) protein in the tumors of each group were compared. The results showed that during the modeling period, the mice showed a decline in food intake, dark fur, poor mental status, and gradually worsened over time. The mental status of mice in each intervention group was improved gradually, especially in the cisplatin+cinobufotalin group. As compared with the model group, the tumor mass of each intervention group was lower(P<0.05). As compared with the cinobufotalin low dose group, the tumor mass was lower and inhibition rate was higher in the cinobufotalin high dose group, cisplatin group and cisplatin+cinobufotalin group(P<0.05). As compared with the cinobufotalin high dose group and the cisplatin group, the tumor mass was lower and the inhibition rate was higher in cisplatin+cinobufotalin group(P<0.05). As compared with the model group, the thymus index was higher in cinobufotalin high dose group and cisplatin + cinobufotalin group, while was lower in cisplatin group(P<0.05). As compared with the cinobufotalin low dose group, the thymus index was higher in the cinobufotalin high dose group and lower in the cisplatin group(P<0.05). As compared with the cinobufotalin high dose group, the thymus index was lower in cisplatin group(P<0.05). As compared with cisplatin group, the thymus index was higher in cisplatin+cinobufotalin group(P<0.05). Pathological staining showed that a large number of heterogeneous cells and mitotic phenomena were observed in the model group. Cell fragments and neutrophils were observed in the tumor tissues of the intervention groups, showing diffuse necrosis, and the diffuse necrosis was more obvious in the cisplatin+cinobufotalin group. As compared with the model group, the apoptotic rate of the tumors and the relative expressions of Fas mRNA and protein were higher in the intervention groups, while the relative expressions of PI3 K, FasL mRNA and protein and the relative expression of pAkt protein were lower in the intervention groups(P<0.05). As compared with the cinobufotalin low dose group, the apoptotic rate of the tumors and relative expression of Fas and protein were higher in the cinobufotalin high dose group, cisplatin group and cisplatin+cinobufotalin group, while the relative expressions of PI3 K, FasL mRNA and protein and pAkt protein were lower(P<0.05). As compared with the cinobufotalin high dose group and the cisplatin group, apoptotic rate of the tumors and the relative expression of Fas mRNA and protein were higher in the cisplatin+cinobufotalin group, while the relative expressions of PI3 K, FasL mRNA and protein and pAkt protein were lower in the cisplatin+cinobufotalin group(P<0.05). In summary, cinobufotalin has significant anti-tumor effect on H22 liver cancer mice, and can enhance the immune function of mice and synergistically enhance the effect of chemotherapy. Its mechanism may be associated with regulating PI3 K/Akt/Fas/FasL signaling pathway related genes and protein expression.

A new perspective of triptolide-associated hepatotoxicity: the relevance of NF- B and NF- B-mediated cellular FLICE-inhibitory protein

Previously, we proposed a new perspective of triptolide (TP)-associated hepatotoxicity: liver hypersensitivity upon lipopolysaccharide (LPS) stimulation. However, the mechanisms for TP/LPS-induced hepatotoxicity remained elusive. The present study aimed to clarify the role of LPS in TP/LPS-induced hepatotoxicity and the mechanism by which TP induces liver hypersensitivity upon LPS stimulation. TNF- inhibitor, etanercept, was injected intraperitoneally into mice to investigate whether induction of TNF- by LPS participated in the liver injury induced by TP/LPS co-treatment. Mice and hepatocytes pretreated with TP were stimulated with recombinant TNF- to assess the function of TNF- in TP/LPS co-treatment. Additionally, time-dependent NF-B activation and NF-B-mediated pro-survival signals were measured and . Finally, overexpression of cellular FLICE-inhibitory protein (FLIP), the most potent NF-B-mediated pro-survival protein, was measured and to assess its function in TP/LPS-induced hepatotoxicity. Etanercept counteracted the toxic reactions induced by TP/LPS. TP-treatment sensitized mice and hepatocytes to TNF-, revealing the role of TNF- in TP/LPS-induced hepatotoxicity. Mechanistic studies revealed that TP inhibited NF-B dependent pro-survival signals, especially FLIP, induced by LPS/TNF-. Moreover, overexpression of FLIP alleviated TP/LPS-induced hepatotoxicity and TP/TNF--induced apoptosis . Mice and hepatocytes treated with TP were sensitive to TNF-, which was released from LPS-stimulated immune cells. These and other results show that the TP-induced inhibition of NF-B-dependent transcriptional activity and FLIP production are responsible for liver hypersensitivity.

decoction induces apoptosis by activating Fas/caspase-8 pathway in rheumatoid arthritis fibroblast-like synoviocytes / 南方医科大学学报

OBJECTIVE@#To explore the effect of decoction (DGNTD) on cell apoptosis and TNF receptor super family 6 (Fas)/caspase-8 pathway in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS).@*METHODS@#FLS isolated from the synovial tissue of RA patients were cultured and identified using immunofluorescence staining. The cells were treated with 10% blank serum (blank control group), 10% sera containing low, moderate or high doses of DGNTD, or 20 μmol/mL KR-33493 (a Fas inhibitor) combined with 10% serum containing high-dose DGNTD. MTT assay was used to detect the proliferation of the cells after the treatments. Apoptosis of the cells was detected at 48 h in each group using Hoechst 33342 staining and flow cytometry with annexin V-FITC/PI staining. The mRNA and protein expressions of Fas, FADD, caspase-8 and caspase-3 in the cells at 48 h were detected using qPCR and Western blotting.@*RESULTS@#Immunofluorescence staining identified the cultured cells as FLS. Treatment with DGNTD-containing sera significantly inhibited the proliferation of FLS, and the inhibitory effects were enhanced as the dose and intervention time increased ( < 0.05). Hoechst 33342 staining and flow cytometry showed that the sera containing different doses of DGNTD significantly promoted apoptosis of FLS ( < 0.05). The expression levels of Fas, FADD, caspase-8, and caspase-3 at both mRNA and protein levels were significantly increased in the cells after treatment with different doses of DGNTD-containing sera ( < 0.05). The application of KR-33493 obviously reversed the effects of DGNTD on the FLS ( < 0.05).@*CONCLUSIONS@#DGNTD can induce apoptosis of the FLS by activating Fas/caspase-8 signaling pathway.