Fas Protein Expression Research Articles

Porphyromonas gingivalis infection facilitates immune escape of esophageal cancer by enhancing YTHDF2-mediated Fas degradation

To investigate the effect of Porphyromonas gingivalis (Pg) infection on immune escape of oesophageal cancer cells and the role of YTHDF2 and Fas in this regulatory mechanism. We examined YTHDF2 and Fas protein expressions in esophageal squamous cell carcinoma (ESCC) tissues with and without Pg infection using immunohistochemistry and in Pg-infected KYSE150 cells using Western blotting. The interaction between YTHDF2 and Fas was investigated by co-immunoprecipitation (Co-IP). Pg-infected KYSE150 cells with lentivirus-mediated YTHDF2 knockdown were examined for changes in expression levels of YTHDF2, cathepsin B (CTSB), Fas and FasL proteins, and the effect of E64 (a cathepsin inhibitor) on these proteins were observed. After Pg infection and E64 treatment, KYSE150 cells were co-cultured with human peripheral blood mononuclear cells (PBMCs), and the expressions of T cell-related effector molecules were detected by flow cytometry. ESCC tissues and cells with Pg infection showed significantly increased YTHDF2 expression and lowered Fas expression. The results of Co-IP demonstrated a direct interaction between YTHDF2 and Fas. In Pg-infected KYSE150 cells with YTHDF2 knockdown, the expression of CTSB was significantly reduced while Fas and FasL expressions were significantly increased. E64 treatment of KYSE150 cells significantly decreased the expression of CTSB without affecting YTHDF2 expression and obviously increased Fas and FasL expressions. Flow cytometry showed that in Pg-infected KYSE150 cells co-cultured with PBMCs, the expressions of Granzyme B and Ki67 were significantly decreased while PD-1 expression was significantly enhanced. Pg infection YTHDF2-dependently regulates the expression of Fas to facilitate immune escape of esophageal cancer and thus promoting cancer progression, suggesting the key role of YTHDF2 in regulating immune escape of esophageal cancer.

Analyzing the impact of acupuncture in conjunction with a Quantum lipid-lowering device on hyperlipidemic rats: insights into adenosine monophosphate-activated protein kinase signaling, blood lipids, and gut microbiota.

We explored the impact of acupuncture (ACUP) in conjunction with a quantum lipid-lowering device (Quantum) on the blood lipids and gut microbiota in hyperlipidemic rats, focusing on the adenosine monophosphate- (AMP)-activated protein kinase (AMPK) signaling pathway. Fifty Sprague-Dawley rats were randomly allocated into five groups: Normal, Model, Acup + Quantum, Acup, and Quantum. Hyperlipidemic models were established in all groups except Normal. The Model group did not receive any intervention after modeling. The Acup + Quantum group received both treatments, the Acup group received only acupuncture, and the Quantum group received only the quantum lipid-lowering device. We used ELISA to measure serum lipid and liver enzyme levels, hematoxylin and eosin (HE) staining for liver pathology, Western blot for protein expression, and 16S rRNA sequencing to analyze intestinal microbiota diversity in rats. Elisa results showed that compared with the model group, Acup + Quantum group could reduce the levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein-cholesterol (LDL-C), aspartate transaminase (AST) and aspartate transaminase (ALT) in rats with hyperlipidemia (P<0.01), and increase the level of high-density lipoprotein-cholesterol (HDL-C) (P<0.01). HE staining results showed that compared with the model group, the hepatocytes of rats in the Acup + Quantum group looked round and full, the liver plates were arranged regularly and neatly, and there was no obvious abnormality in the liver sinusoids. Western blot results showed that compared with the model group, the Acup + Quantum group inhibited AMPK activation, increased P-AMPK/AMPK protein expression (P<0.05), and decreased phospho-acetyl-CoA carboxylases (P-ACC/ACC), Sterol regulatory element-binding transcription factor-1C (SREBP-1C), and FAS protein expression (P<0.05; P<0.01; P<0.01), which resulted in lipid-lowering effect. The results of intestinal flora showed that Acup + Quantum group improved the intestinal microbial microenvironment of hyperlipidemic rats by regulating the structure of intestinal microflora, increasing the abundance of Firmicutes flora, and decreasing the abundance of harmful bacteria, such as Bacteroidetes and Proteobacteria. Acupuncture combined with quantum lipid-lowering device can improve the blood lipid and liver function levels and regulate the intestinal microbial microenvironment of hyperlipidemic rats. This therapeutic outcome is likely achieved through the activation of the AMPK pathway and the beneficial modulation of the intestinal microbiota of rats.

Puberty as a DOHaD programming window: high-fat diet induces long-term hepatic dysfunction in male rats.

The aim of this study was to evaluate whether high-fat (HF) diet intake during puberty can program obesity as well as generate glucose imbalance and hepatic metabolic dysfunctions in adult life. Male Wistar rats were randomly assigned into two groups: rats fed standard chow (NF) and rats fed a HF from postnatal 30-day-old (PND30) until PND60. Then, both groups were fed a standard chow from PND60 until PND120. Euthanasia and samples collections occurred at PND120. HF animals were overweight (+11%) and had increased adiposity, hyperphagia (+12%), hyperglycaemia (+13%), hyperinsulinemia (+69%), and hypertriglyceridemia (+34%). Plasma glucose levels during intravenous glucose tolerance test (ivGTT) and intraperitoneal insulin tolerance test (ipITT) were also higher in the HF group, whereas Kitt was significantly lower (-34%), suggesting reduced insulin sensitivity. In the same sense, HF animals present pancreatic islets hypertrophy and high β-cell mass. HF animals also had a significant increase in blood glucose levels during pyruvate tolerance test, indicating increased gluconeogenesis. Hepatic morphology analyses showed an increase in lipid inclusion in the HF group. Moreover, PEPCK and FAS protein expression were higher in the livers of the HF animals (+79% and + 37%, respectively). In conclusion, HF during puberty causes obese phenotype leading to glucose dyshomeostasis and nonalcoholic fatty liver disease, which can be related to the overexpression of proteins PEPCK and FAS.

Gastrodia elata extract exerts anti-obesity effects on obese mice caused by a high-starch diet by suppressing SREBP-1 and ChREBP

ObjectiveGastrodia elata (GE), a natural treatment, is extensively used in traditional Korean medicine. The study's goal was to look into how GE affected obese mice that had been fed a high-starch diet (HSD). MethodsThe C57BL/6 J mice used in this investigation were given HSD to make them obese. Gastrodia elata extract (GEE) was administered orally to the mice throughout the course of the next five weeks in doses of 100, 200, and 500 mg/kg. When the experiment was over, we measured the animals body weight, liver and fat weight, blood biochemical parameters, and gene expression that is related to obesity. ResultsThe overall body weight of animals was dramatically lowered via GEE oral treatment. In comparison to the HSD group, the serum and liver TG levels were considerably lower in the GEE-treated group. Additionally, regional fatty tissues and liver weight markedly dropped in the GEE-treated batch in comparison to the HSD batch. We observed considerably lower levels of PPAR-γ, C/EBP-α, and ChREBP mRNA expression level in the GEE-treated group in addition to lower levels of SREBP-1, ACC, and FAS protein expression. ConclusionThe studies show that GEE has anti-obesity advantages in the mouse model of obesity caused by HSD. Despite the fact that we utilized mice as a model for the experiment rather than people, who have different biochemical pathways, the finding has significant implications for how GEE generally functions.

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

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.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.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."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.目的：基于死亡受体通路Fas/Fas相关死亡域蛋白（FADD）/半胱氨酸天冬氨酸蛋白酶-8（Caspase-8）探讨“秩边”透“水道”针刺对早发性卵巢功能不全（POI）大鼠卵巢功能的影响及可能机制。方法：将48只雌性SD大鼠随机分为空白组、模型组、药物组和针刺组，每组12只。除空白组外，其余各组大鼠均采用腹腔注射环磷酰胺制备POI模型。针刺组大鼠于造模后采用“秩边”透“水道”针刺干预4周，每日1次，每次30 min；药物组予戊酸雌二醇片（0.09 mg•kg-1•d-1）灌胃4周。比较各组大鼠一般情况和动情周期；采用ELISA法检测大鼠血清卵泡刺激素（FSH）、黄体生成素（LH）和雌二醇（E2）水平，HE染色法观察大鼠卵巢组织形态，免疫组化法和Western blot法检测大鼠卵巢组织Fas、FADD、Caspase-8蛋白表达。结果：造模后，除空白组外，其他各组大鼠毛发干焦、脱毛，精神萎靡，进食减少，小便增多，大便稀软不成形；干预后，针刺组、药物组大鼠大便逐渐成形。干预后，与空白组比较，模型组大鼠动情周期紊乱率升高（P<0.01）；与模型组比较，针刺组、药物组大鼠动情周期紊乱率降低（P<0.01）。与空白组比较，模型组大鼠体质量和血清E2水平降低（P<0.01），血清FSH、LH水平和卵巢组织Fas、FADD、Caspase-8蛋白表达升高（P<0.01）；与模型组比较，针刺组、药物组大鼠体质量和血清E2水平升高（P<0.01），血清FSH、LH水平和卵巢组织Fas、FADD、Caspase-8蛋白表达降低（P<0.01）。结论：“秩边”透“水道”针刺可有效改善POI大鼠卵巢功能，其机制可能为调节POI大鼠血清性激素水平，降低卵巢组织Fas、FADD、Caspase-8表达水平，减缓卵巢细胞凋亡。.

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

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. 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. 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. "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.

Effect of Fas and Bcl-2 DNA Damages Response Expression in Stem Cells on Apoptosis of Nucleus Pulposus of Intervertebral Disc.

The nucleus pulposus is an elastic jelly composed of crisscross fibrous reticular structures, namely, chondrocytes and proteoglycan mucoid matrix. Embryo and adult SC can resist the accumulation of genetic damage and repair them through various DNA repair mechanisms, thus preventing them from spreading to daughter cells. Fresh medullary tissue was fixed with 10% formaldehyde solution, embedded in paraffin, and sectioned at 4 m. The nucleus pulposus was stained with HE, and its degeneration was observed under light microscope. The average apoptotic index (AI) of 20 denatured nuclei was 50.230, the percentage of Fas-positive cells was 74.255%, and the percentage of Bcl-2-positive cells was 55.370%. The average apoptotic index (AI) was 28.317. The percentage of Fas-positive cells, Fas protein-positive cells, and Bcl-2 protein-positive cells in six normal nuclei was 41.717%, 41.717%, and 27.167%, respectively. The average AI value, Fas protein expression, and Bcl-2 protein expression in the two groups were significantly different (P < 0.05).

Effect of electroacupuncture of "Zusanli"(ST36) and "Guanyuan" (CV4) on apoptosis and expression of apoptosis-related proteins of synoviocytes in adjuvant-induced arthritis rats

To observe the effect of electroacupuncture (EA) of "Zusanli"(ST36) and "Guanyuan"(CV4) on the apoptosis rate of synoviocytes and protein expression of Fas, FasL and Caspase-3 in synovial tissue of adjuvant-induced arthritis (AIA) rats, so as to explore its mechanisms underlying improvement of rheumatoid arthritis （RA）. A total of 24 rats were randomly divided into normal, model, medication and EA groups, with 6 rats in each group. The AIA model was established by injection of complete Freund's adjuvant (CFA, 0.1 mL) into the left hindlimb paw. The rats in the medication group received intraperitoneal injection of 0.35 mg/kg of methotrexate, twice a week for 4 weeks. The rats in the EA group received EA stimulation of ST36 and CV4 (20 Hz/50 Hz, 1 mA) for 20 min, 6 times a week for 4 weeks. The left hind paw volume was measured using a paw volume meter, and histopathological changes of synovial tissue were observed by light microscope after H.E. staining. The serum contents of tumor necrosis factor-α（TNF-α） and interleukin-1 （IL-1） were measured by ELISA. The apoptosis of synoviocytes was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL), and the expression of apoptosis-associated proteins Fas, FasL and Caspase-3 in synovium were detected by Western blot. Compared with the normal group, the left hind paw volume from day 3 to 24 after administration of CFA, serum IL-1 and TNF-α contents were significantly increased (P<0.01), while the expressions of Fas, FasL and Caspase-3 proteins and apoptotic rate of synoviocytes were significantly decreased in the model group (P<0.01). In comparison with the model group, the paw volume from day 17 to 24 after modeling, the serum IL-1 and TNF-α contents were significantly reduced (P<0.01), while the apoptotic rate of synoviocytes, expressions of Fas protein in both medication and EA groups, Caspase-3 protein in the acupuncture group and FasL protein in the medication group were increased (P<0.01, P<0.05). Compared with the medication group, the expression of FasL protein was decreased in EA group (P<0.05). H.E. stain showed obvious hyperplasia of the synovial lining layer, and disordered arrangement of synovial cells, with edema and enlargement in some cells in the model group, which was relatively milder in both medication and EA groups. EA of ST36 and CV4 can promote the apoptosis of synoviocytes and the expressions of Fas and FasL proteins in AIA rats, which may contribute to its role in relieving synovitis through activating Fas/FasL signaling.

A Study on How Methionine Restriction Decreases the Body's Hepatic and Lipid Deposition in Rice Field Eel (Monopterus albus).

Methionine restriction reduces animal lipid deposition. However, the molecular mechanism underlying how the body reacts to the condition and regulates lipid metabolism remains unknown. In this study, a feeding trial was performed on rice field eel Monopterus albus with six isonitrogenous and isoenergetic feeds that included different levels of methionine (0, 2, 4, 6, 8, and 10 g/kg). Compared with M0 (0 g/kg), the crude lipid and crude protein of M. albus increased markedly in M8 (8 g/kg) (p < 0.05), serum (total cholesterol, triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and non-esterified free fatty acids), and hepatic contents (hepatic lipase, apolipoprotein-A, fatty acid synthetase, total cholesterol, triglyceride, and lipoprteinlipase). However, in the serum, very-low-density lipoprotein and hepatic contents (hormone-sensitive triglyceride lipase, Acetyl CoA carboxylase, carnitine palmitoyltransterase, and mirosomal triglygeride transfer protein) decreased markedly in M8 (p < 0.05). The contents of hepatic C18:2n-6, C22:6n-3, and n-3PUFA in the M8 group were significantly higher than those in M0 (p < 0.05), and the contents of lipid droplets in M8 were higher than those in M0. Compared with M0, the hepatic gcn2, eif2α, hsl, mttp, ldlrap, pparα, cpt1, and cpt2 were remarkably downregulated in M8, while srebf2, lpl, moat2, dgat2, hdlbp, srebf1, fas, fads2, me1, pfae, and icdh were markedly upregulated in M8. Moreover, hepatic SREBP1 and FAS protein expression were upregulated significantly in M8 (p < 0.01). In short, methionine restriction decreased the lipid deposition of M. albus, especially for hepatic lipid deposition, and mainly downregulated hepatic fatty acid metabolism. Besides, gcn2 could be activated under methionine restriction.

C6orf120 gene knockout in rats mitigates concanavalin A‑induced autoimmune hepatitis via regulating NKT cells

ObjectiveTo elucidate the role of the functional unknown gene C6orf120 in the pathogenesis of AIH and its mechanism of action, using C6orf120 knockout rats. MethodsAn autoimmune hepatitis model was established with 35 mg/kg intravenous injection of concanavalin A (Con A) in C6orf120-knockout (C6orf120-/-) and wild-type (WT) rats. Rats were sacrificed after administering Con A for 0, 12, and 24 h. The peripheral blood, liver, spleen, and mesenteric lymph nodes were collected for follow-up studies. ResultsC6orf120 knockout significantly decreased the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and improved the histological damage in Con A-induced autoimmune liver injury.Loss of C6orf120 function significantly increased the frequency of CD3+ CD161+ NKT cells in the peripheral blood, liver, and spleen; downregulated the expression of CD314 (NKG2D) in the liver, spleen, and mesenteric lymph nodes; reduced the expression of inflammatory cytokines and chemokines; and suppressed the mRNA and protein expression of Fas and FasL in the liver. Additionally, C6orf120 knockout significantly downregulated the expression of p-JAK1, p-JAK2, p-STAT1, and p-STAT3 in liver tissue. ConclusionThe protective effect of C6orf120 knockout against Con A-induced hepatitis may be due to the inhibition of NKT cell activation, restriction of cytokine and chemokine activities, inhibition of JAK-STAT and Fas/FasL signaling pathway activation, and reduction in liver inflammation and hepatocyte apoptosis.

Effect of Copper-Containing Stainless Steel on Apoptosis of Coronary Artery Smooth Muscle Cells.

Background:We aimed to investigate the effect of copper stainless steel on apoptosis of vascular smooth muscle cells in coronary artery.Methods:The study was carried out in 2019 at Hubei University of Medicine, Xiangyang, China. The rat coronary artery smooth muscle cell was used for cell resuscitation and culture. MTT method was used to visualize cell growth curve and to detect the cell survival and growth. The incubated cells were randomly divided into copper-containing stainless-steel group, ordinary stainless-steel group, and control group. The cells were made into single cell suspension, which were intervened by experimental group and incubated in incubator with CO2 for 48 hours. TUNEL method was used to detect the apoptosis. The number of apoptotic cells in five high power fields (×200) was counted. The expression of Fas protein in three groups of cells was detected by Western blot.Results:The growth curves of rat coronary artery smooth muscle cells showed that the OD value of the cells reached the plateau 7 days after inoculation, indicating that the cells grew well. TUNEL staining showed the apoptosis in all three groups. The apoptotic index in copper-containing group was significantly higher than that in common stainless-steel group (P <0.01). The results of the Fas protein expression level through Western blot showed that the level in the copper-containing group was significantly higher than that in the common stainless-steel group (P<0.01).Conclusion:Copper-containing stainless steel can promote apoptosis of coronary artery smooth muscle cells. The material could prevent stent restenosis.

Effect of Chronic Western Diets on Non-Alcoholic Fatty Liver of Male Mice Modifying the PPAR-γ Pathway via miR-27b-5p Regulation.

Western diets contribute to metabolic diseases. However, the effects of various diets and epigenetic mechanisms are mostly unknown. Here, six week-old C57BL/6J male and female mice were fed with a low-fat diet (LFD), high-fat diet (HFD), and high-fat high-fructose diet (HFD-HF) for 20 weeks. We determined that HFD-HF or HFD mice experienced significant metabolic dysregulation compared to the LFD. HFD-HF and HFD-fed male mice showed significantly increased body weight, liver size, and fasting glucose levels with downregulated PPARγ, SCD1, and FAS protein expression. In contrast, female mice were less affected by HFD and HFD-HF. As miR-27b contains a seed sequence in PPARγ, it was discovered that these changes are accompanied by male-specific upregulation of miR-27b-5p, which is even more pronounced in the HFD-HF group (p < 0.01 vs. LFD) compared to the HFD group (p < 0.05 vs. LFD). Other miR-27 subtypes were increased but not significantly. HFD-HF showed insignificant changes in fibrosis markers when compared to LFD. Interestingly, fat ballooning in hepatocytes was increased in HFD-fed mice compared to HFD-HF fed mice, however, the HFD-HF liver showed an increase in the number of small cells. Here, we concluded that chronic Western diet-composition administered for 20 weeks may surpass the non-alcoholic fatty liver (NAFL) stage but may be at an intermediate stage between fatty liver and fibrosis via miR-27b-5p-induced PPARγ downregulation.

Danggui Niantong decoction induces apoptosis by activating Fas/caspase-8 pathway in rheumatoid arthritis fibroblast-like synoviocytes

To explore the effect of Danggui Niantong decoction (DGNTD) on cell apoptosis and TNF receptor super family 6 (Fas)/caspase-8 pathway in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). 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. 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 (P &lt; 0.05). Hoechst 33342 staining and flow cytometry showed that the sera containing different doses of DGNTD significantly promoted apoptosis of FLS (P &lt; 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 (P &lt; 0.05). The application of KR-33493 obviously reversed the effects of DGNTD on the FLS (P &lt; 0.05). DGNTD can induce apoptosis of the FLS by activating Fas/caspase-8 signaling pathway.

OGR1 negatively regulates β-casein and triglyceride synthesis and cell proliferation via the PI3K/AKT/mTOR signaling pathway in goat mammary epithelial cells

Goat milk in some cases is less allergenic than cow milk, therefore, more people drink goat milk in the world, so it is necessary for us to improve the yield and quality of goat milk. Previous studies have shown that some genes are closely related to lactation. Ovarian cancer G protein-coupled 1 (OGR1) is a G protein-coupled receptor discovered recently. OGR1 is widely found in various tissues of organisms and is involved in cell skeleton reorganization, carcinogenesis, cell proliferation, and apoptosis by regulating multiple signaling pathways in cells. However, the modulating effect of OGR1 in lactation is still unknown. Therefore, the objective of this study is to investigate the function of OGR1 in goat mammary epithelial cells (GMECs). Flow cytometry, CCK8, EDU, enzyme-linked immunosorbent assay, and triglyceride test kit assays were performed and we found that OGR1 regulated Bcl-2/Bax ratio, Fas protein expression as well as the phosphorylation of AKT and mammalian target of rapamycin (mTOR). si-OGR1 could enhance the proliferation of GMECs by promoting G1/S phase progression and the synthesis of β-casein and triglyceride. By contrast, OGR1 repressed GMECs proliferation and down-regulated the synthesis of β-casein and triglyceride by blocking the PI3K/AKT/mTOR signaling pathway in GMECs.

Anti-adipogenic and anti-oxidant effects of a standardised extract of Moro blood oranges (Citrus sinensis (L.) Osbeck) during adipocyte differentiation of 3T3-L1 preadipocytes

Particular features in the pulp of blood oranges are marked levels of anthocyanin, a class of polyphenolic compounds well known to exert numerous health-promoting actions on human wellbeing including anti-obesity effects. In this study, we investigated in vitro, the antioxidant and anti-adipogenic activities of Morosil®, a standardised extract of Moro blood oranges. During adipocyte differentiation, 3T3-L1 cells were treated with concentrations of extract containing 2.5, 5, 10, 25 μM of anthocyanins. After seven days of treatment and differentiation, we measured reactive oxygen species production, non-proteic thiol groups content, adipokine secretion and triglyceride accumulation together with mRNA expression of adipogenic transcription factors such as PPARγ, C/EBPα, SREBP-1c. Furthermore, both ACCα, FAS protein expression and citrate synthase activity were measured. Results show that Morosil® exerts antioxidant and anti-adipogenic activities during adipocyte differentiation of 3T3-L1 pre-adipocytes.

Effects of Xinfeng capsule on the Fas/FasL-mediated apoptotic pathway in patients with rheumatoid arthritis

OBJECTIVETo observe the effects of Xinfeng capsule on the apoptosis of peripheral blood CD4 + T lymphocytes and changes in the Fas/FasL-mediated apoptotic pathway in patients with rheumatoid arthritis (RA). METHODSA total of 28 RA patients were included in the study; they were randomly divided into the Xinfeng capsule (XFC) group (3 capsules, 3 per day) and the leflunomide (LEF) group (1 pellet, once per night). The treatment course in each groups was 12 weeks. The normal control (NC) group consisted of 10 healthy people. The apoptotic rate was examined using flow cytometry. Fas, FasL, caspase 8, caspase 3, bcl-2, and bax mRNA were examined using qRT-PCR. Apoptotic proteins Fas, FasL, caspase 8, and caspase 3 were examined using western blotting. RESULTSAfter treatment, patients in the two groups all showed some trend of improvement. Disease activity indexes, joint morning stiffness time, joint swelling/tenderness number, health assessment questionnaire (HAQ) score, RA quality of life (RAQOL) questionnaire, and self-rating anxiety scale (SAS), as well as all apoptotic related indicators were reduced in both groups after treatment with no significant difference between groups. But the improvement in terms of the self-rating depression scale (SDS) in the XFC group was better than in the LEF group. RA patients showed lower apoptotic rates in CD4+ T cells, lower bax, Fas, caspase 8, and caspase 3 mRNA, and less protein expression of Fas, caspase 8, and caspase 3 than in the NC group. These indicators increased after treatment. However, the level of Bcl-2 mRNA was higher in the XFC group than in the NC group before treatment, and it subsequently decreased. The XFC group expressed lower Bcl-2 mRNA than the LEF group. Negative correlations were found between ESR and the apoptotic rate in CD4+ T cells, Fas, and caspase 3; CRP and Fas; and, swollen joint count and Bax, while positive correlations were found between ESR and Bcl-2. CONCLUSIONXFC can regulate the Fas/FasL system and promote CD4+ T cell apoptosis and thus reduce the abnormal immune response, which can improve symptoms in RA patients.

The loss of P2X7 receptor expression leads to increase intestinal glucose transit and hepatic steatosis

In intestinal epithelial cells (IEC), it was reported that the activation of the P2X7 receptor leads to the internalization of the glucose transporter GLUT2, which is accompanied by a reduction of IEC capacity to transport glucose. In this study, we used P2rx7−/− mice to decipher P2X7 functions in intestinal glucose transport and to evaluate the impacts on metabolism. Immunohistochemistry analyses revealed the presence of GLUT2 at the apical domain of P2rx7−/− jejunum enterocytes. Positron emission tomography and biodistribution studies demonstrated that glucose was more efficiently delivered to the circulation of knockout animals. These findings correlated with increase blood glucose, insulin, triglycerides and cholesterol levels. In fact, P2rx7−/− mice had increased serum triglyceride and cholesterol levels and displayed glucose intolerance and resistance to insulin. Finally, P2rx7−/− mice developed a hepatic steatosis characterized by a reduction of Acaca, Acacb, Fasn and Acox1 mRNA expression, as well as for ACC and FAS protein expression. Our study suggests that P2X7 could play a central role in metabolic diseases.

The effect of interleukin-17 on podocyte-associated factors and apoptosis and molecular mechanism

Objective To investigate the effect of interleukin(IL)-17 on podocyte-associated factors and apoptosis in podocytes and explore the molecular mechanism. Methods The podocytes were used as the object of study.The apoptosis of podocytes induced by IL-17 in a dose(1 nm/ml, 10 nm/ml, 50 nm/ml, 100 nm/ml) and time(12 h, 24 h, 48 h, 72 h) way and the protein expression of Fas and FasL of podocytes induced by IL-17 were detected by flow cytometrey.The podocytes were divided into the blank control group and 100 ng/ml IL-17 induction group.The expressions of mRNA of Nephrin, WT1, Synaptopodin, Podocylaxin, Fas and FasL were measured by Real-time RT PCR.The proteins of WT1, Caspases8, Caspases3 in podocyte were detected by immunocytochemistry method. Results IL-17 promoted the apopotosis of podocyte in a dose and time way(P 0.05). Conclusion IL-17 may cause renal injure by inducing the apoptosis of podocyte and decreasing the expression of Podocylaxin in podocyte. Key words: Interleukin-17; Podocyte; Apoptosis; Fas; Podocylaxin,

Effect of Icariin on apoptosis and expression of Fas, Fas ligand, B cell lymphoma, and Bcl-2-associated X protein in CD4 + T lymphocytes from patients with ankylosing spondylitis

ObjectiveTo investigate the effects of icariin on apoptosis and the expression of Fas, Fas ligand (FasL), B cell lymphoma (Bcl-2), and Bcl-2-associated X protein (Bax) in CD4+ T lymphocytes from patients with ankylosing spondylitis. MethodsPrimary cultures of peripheral blood CD4+ T lymphocytes were established and treated with icariin at high, medium, and low doses (0.5, 0.25, and 0.125 mg/mL). Sulfasalazine treated and helthy cells were used as controls. Apoptosis of treated cells was determined by flow cytometry. Reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assays were used to determine the effects of icariin on the expression of Fas, FasL, Bcl-2, and Bax. The activity of caspase 8 and caspase 3 was determined by a colorimetric assay. ResultsThe mRNA and protein expression of Fas, and activity of caspase 8 and caspase 3 in CD4 + T lymphocytes were increased by icariin (P < 0.05). Conversely, the mRNA and protein expression of Bcl-2 was decreased (P < 0.05). The expression of FasL and Bax were not significantly different between groups. The proapoptotic effects of icariin were dose-dependent. ConclusionIcariin induces the apoptosis of CD4 + T cells from patients with AS comparing to normal control. Therefore, the induction of apoptosis may be the likely mechanism of action of icariin's antirheumatics activities.

Specific MicroRNAs comparisons in hypoxia and morphine preconditioning against hypoxia-reoxgenation injury with and without heart failure

AimsIschemia reperfusion (I/R) injury is an inevitable event arising during the cardiovascular diseases development and the process of potent surgical treatments. microRNAs (miRNAs) are critical regulators of multiple cell processes including I/R injury. The present study aims to quantify miRNA alterations and regulated genes upon hypoxia-reoxygenation (H/R) injury in a rat heart failure model comparing with normal cardiomyocytes. Main methodsChronic heart failure was established by injecting doxorubicin (2mg/kg/week) for 6weeks, then H/R was performed on primary cultured cardiomyocytes isolated from normal and failed heart. Cellular injury was evaluated by detecting LDH release levels, cell variability and apoptotic rate. Dysregulated miRNAs in control, hypoxia preconditioning (HPC) and morphine preconditioning (MPC) groups under two conditions were quantified by microarray analysis. Fas protein expression was analyzed using Western Blotting analysis. Key findingsChronic heart failure was confirmed with lower ejection fraction (EF), and significant cellular injury. HPC could reverse the injury induced by H/R in normal heart rather than failed heart, otherwise, MPC significantly attenuated cellular injury dose dependently in both conditions. There was 12 miRNAs significantly altered after doxorubicin injection, 7 downregulated and 5 upregulated. miR-133b-5p, miR-6216, miR-664-1-5p and let7e-5p were differentially expressed after HPC and MPC treatments. The direct interaction between miR-133b-5p and target gene Fas were established. The Fas protein expression was manipulated by MPC not HPC affording protective effect against H/R injury. SignificanceWe investigated that miR-133b-5p might play a particularly important role in the cardioprotective effect of MPC by regulating the target gene Fas.