Protein Expression Levels Of Bcl-2 Research Articles

Acupotomy relieves pain and improves motor function by regulating autophagy and apoptosis of cartilage in rabbits with knee osteoarthritis apoptosis

To observe the effect of acupotomy on the expression of Beclin-1, Bcl-2 and Caspase-3 in the cartilage tissue in rabbits with knee osteoarthritis (KOA), so as to explore its mechanism underling improvement of KOA. Twenty-four healthy male New Zealand rabbits were randomly and equally divided into blank control, model and acupotomy groups, with 8 rabbits in each group. By using the modified Videman's methods, the KOA model was established by left hind limb immobilization with a plaster cast for 6 weeks. The severity of KOA (knee pain, swelling and motor function) was assessed using Lequesne score, and the rabbits with a score below 4 were excluded. The acupotomy was applied to "Hedingci" (the attachment of the quadriceps tendon to the patella at the upper edge), "Binneixia" (the medial patellar supporting band attachment of medial inferior patellar margin), "Binwaixia" (the lateral patellar supporting band attachment of the lower lateral patellar margin), "Chengfeijian" (the lateral collateral ligament of the knee passes over the lateral joint space), "Weiyangci" (the medial margin of biceps femoris at the lateral end of popliteus), "Yinlingci" (the medial tibial attachment of anserinus tendon) on the left hind limb once a week for 4 weeks. One week after the last intervention, the left knee joint dysfunction severity(pain, maximum walking distance, and some activities of daily living) was evaluated by using modified Lequesne score. Histopathological changes of the cartilage were observed under light microscope after H.E. staining. The apoptosis of chondrocytes was observed after terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick-end labeling (TUNEL) staining. The autophagolysosomes of chondrocytes were observed using transmission electron microscopy. The expression levels of Beclin-1, Bcl-2 and Caspase-3 (related factors of autophagy and apoptosis) were detected using Real-time PCR and Western blot separately. In comparison with the blank control group, the Lequesne score, apoptosis rate, expression levels of Caspase-3 mRNA and protein were significantly increased (P<0.001), and the number of autophagolysosomes, expression levels of Beclin-1 and Bcl-2 mRNAs and proteins considerably decreased (P<0.001) in the model group. Relevant to the model group, the acupotomy group had an obvious decrease in Lequesne score, rate of apoptosis, and expression levels of Caspase-3 mRNA and protein (P<0.001) and an apparent increase in the number of autophagolysosomes and expression levels of Beclin-1 and Bcl-2 mRNAs and proteins (P<0.001). Findings of H.E. staining showed severe damaged cartilage surface, with a large number of exfoliation defects, few chondrocytes on the surface and disordered arrangement of transitional cells in the model group, which was relatively milder in the acupotomy group. Acupotomy can mitigate knee-joint pain and improve functional activity in KOA rabbits, which may be associated with its functions in promoting autophagy and suppressing apoptosis by up-regulating expressions of Beclin-1 and Bcl-2 mRNAs and proteins and down-regulation of Caspase-3 mRNA and protein.

Royal jelly arranges apoptotic and oxidative stress pathways and reduces damage to liver tissues of rats by down-regulation of Bcl-2, GSK3 and NF-κB and up-regulation of caspase and Nrf-2 protein signalling pathways

Introduction Royal jelly (RJ) from the honey bee, Apis mellifera, is a traditional product that is widely used as a food supplement to support the medical treatment of various diseases. Material and methods Our study continued for 8 weeks. 42 Wistar albino (8 weeks old) male rats were used in the study. The study included 6 groups; Group 1: Control group (fed with standard diet), Group 2: RJ (100 mg/kg, bw), Group 3: F-50 (50 mg/kg, bw), group 4: F-100 (100 mg/kg, bw) group 5: F-50 (50 mg/kg, bw) + RJ (100 mg/kg, bw) Group 6: F-100 (100 mg/kg, bw) + RJ (100 mg/kg, bw). Malondialdehyde (MDA), catalase (CAT) and glutathione (GSH) activities in liver tissue were determined by spectrophotometer. Liver tissue samples were examined histopathologically and various protein levels were determined by Western blotting technique. Results RJ caused a significant decrease in MDA level, Bcl-2, GSK3 and NF-κB protein expression levels, whereas induced a significant increase in GSH level, CAT activities and Bax, BDNF, caspase-6, caspase-3, Nrf-2 protein expression levels. Conclusion Our findings suggest RJ to be used as a hepatoprotective agent in the clinic to modulate the toxic effects of fluoride and other chemicals in the future.

Xinyang Tablet attenuates chronic hypoxia-induced right ventricular remodeling via inhibiting cardiomyocytes apoptosis

BackgroundHypoxia-induced pulmonary hypertension (HPH) is one of the fatal pathologies developed under hypobaric hypoxia and eventually leads to right ventricular (RV) remodeling and RV failure. Clinically, the mortality rate of RV failure caused by HPH is high and lacks effective drugs. Xinyang Tablet (XYT), a traditional Chinese medicine exhibits significant efficacy in the treatment of congestive heart failure and cardiac dysfunction. However, the effects of XYT on chronic hypoxia-induced RV failure are not clear.MethodsThe content of XYT was analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC–MS). Sprague–Dawley (SD) rats were housed in a hypobaric chamber (equal to the parameter in altitude 5500 m) for 21 days to obtain the RV remodeling model. Electrocardiogram (ECG) and hemodynamic parameters were measured by iWorx Acquisition & Analysis System. Pathological morphological changes in the RV and pulmonary vessels were observed by H&E staining and Masson’s trichrome staining. Myocardial apoptosis was tested by TUNEL assay. Protein expression levels of TNF-α, IL-6, Bax, Bcl-2, and caspase-3 in the RV and H9c2 cells were detected by western blot. Meanwhile, H9c2 cells were induced by CoCl2 to establish a hypoxia injury model to verify the protective effect and mechanisms of XYT. A CCK-8 assay was performed to determine the viability of H9c2 cells. CoCl2-induced apoptosis was detected by Annexin-FITC/PI flow cytometry and Hoechst 33,258 staining.ResultsXYT remarkably improved RV hemodynamic disorder and ECG parameters. XYT attenuated hypoxia-induced pathological injury in RV and pulmonary vessels. We also observed that XYT treatment decreased the expression levels of TNF-α, IL-6, Bax/Bcl-2 ratio, and the numbers of myocardial apoptosis in RV. In H9c2 myocardial hypoxia model, XYT protected H9c2 cells against Cobalt chloride (CoCl2)-induced apoptosis. We also found that XYT could antagonize CoCl2-induced apoptosis through upregulating Bcl-2, inhibiting Bax and caspase-3 expression.ConclusionsWe concluded that XYT improved hypoxia-induced RV remodeling and protected against cardiac injury by inhibiting apoptosis pathway in vivo and vitro models, which may be a promising therapeutic strategy for clinical management of hypoxia-induced cardiac injury.

Novel benzo chromene derivatives: design, synthesis, molecular docking, cell cycle arrest, and apoptosis induction in human acute myeloid leukemia HL-60 cells

A series of benzo[h]chromenes, benzo[f]chromenes, and benzo[h]chromeno[2,3-d]pyrimidines were prepared. All the newly synthesised compounds were selected by National Cancer Institute for single-dose testing against 60 cell lines. Benzo[h]chromenes 5a and 6a showed promising anti-cancer activity and selected for the five-dose testing. Compounds 5a and 6a suppressed cell growth in HL-60 by the induction of cell cycle arrest, which was confirmed using flow cytometry and Annexin V-FITC/PI assays showed at the G1/S phase by regulating the expression of CDK-2/CyclinD1, triggering cell apoptosis by activating both the extrinsic (Fas/Caspase 8) and intrinsic (Bcl-2/Caspase 3) apoptosis pathways, which were determined by the western blot. Benzo[h]chromenes 5a and 6a decreased the protein expression levels of Bcl-2, CDK-2, and CyclinD1 and increased the expression of caspase 3, caspase 8, and Fas. In silico molecular analysis of compounds 5a and 6a in CDK-2 and Bcl-2 was performed.

Effects of matrine combined with LY294002 on proliferation, apoptosis and cell cycle of human myeloid leukemia K562 cells

To investigate the effects of matrine combined with LY294002 on proliferation, apoptosis and cell cycle of human myeloid leukemia K562 cells and explore the underlying mechanism. The effects of different concentrations of matrine alone and in combination with LY294002 on the proliferation of K562 cells were examined with CCK-8 assay. The changes in morphology of K562 cells were observed following treatment for 48 h with 0.4 g/L matrine and 10 μmol/L Y294002, either alone or in combination, and cell apoptosis was detected using flow cytometry with annexin V-FITC/PI double labeling; the changes in cell cycle was detected with PI labeling. Western blotting was performed to examine the effect of matrine combined with LY294002 on expressions of p-mTOR, p-PI3K, Akt, p-Akt, cyclinD1, Bcl-2 and caspase-9 in the cells. Treatment with different concentrations of matrine, both alone and in combination with LY294002, inhibited the proliferation of K562 cells in a time- and concentration-dependent manner. Compared with matrine treatment alone, the combined treatment caused more obvious morphological changes of the cells, significantly increased cell apoptosis (P < 0.01), and induced cell cycle arrest in G0/G1 (P < 0.01). Western blotting showed that the protein expression levels of p-mTOR, cyclinD1, p-PI3K, p-Akt and Bcl-2 in K562 cells increased while the expression level of caspase-9 decreased significantly after the combined treatment (P < 0.01). Matrine combined with LY294002 produces a synergistic inhibitory effect on K562 cells possibly by down-regulating the p-Akt expression in PI3K/Akt signaling pathway, reducing the expressions of p-mTOR, cyclinD1 and Bcl-2, and increasing the expression of caspase-9.

BCL-XL Blockage with A1155463 Significantly Increases Efficacy of Venetoclax in Mantle Cell Lymphoma in Vitro and In Vivo

Mantle cell lymphoma (MCL) is a rare chronically relapsing subtype of aggressive B-cell non-Hodgkin lymphoma characterized by the canonical chromosomal translocation t (11;14) and other molecular cytogenetic aberrations including overexpression of BCL2 protein. Venetoclax (VEN), a BCL2 inhibitor, has demonstrated activity in MCL both as a monotherapy and in combination with other targeted agents. Treatment with single agent venetoclax is, however, hampered by frequent development of drug resistance caused in large part by adaptive upregulation of other anti-apoptotic BCL2 family members. Immunodeficient NOD-SCID-gamma mice were xenografted subcutaneously with MCL cell lines and PDX cells established from patients with relapsed and refractory MCL. The therapy was given for two weeks. VEN was administered by oral gavage (100 mg/kg/d), A1155463 was administered intraperitoneally (10 mg/kg/d). Western blotting was implemented to evaluate expression levels of BCL2 proteins, immunoprecipitation was used to analyze the levels of BIM bound to BCL2 and BCL-XL before and after exposure to VEN. CRISPR-Cas9 was employed to derive clones with BCL-XL knock-out. Blood cell counts in mice on A1155463 +/- VEN therapy were analyzed using the Mindray BC-5300 Auto Hematology Analyzer. In this study, we analyzed molecular mechanisms of in vivo acquired resistance to VEN using a panel of several murine cell line-based xenografts (CDX) and patient-derived xenografts (PDX) of MCL. First, the mice xenotransplanted with VEN-sensitive MCL cells were subject to monotherapy with VEN until development of resistant tumors (VEN-R). Western blot analysis of VEN-R tumors revealed upregulation of BCL-XL in majority of VEN-R tumors compared to controls. Other changes included upregulation of MCL1 and downregulation of BIM proteins in several models. Immunoprecipitation experiments confirmed that BCL-XL indeed serves as a buffer for BIM released from BCL2 after exposure to VEN thereby blocking VEN-triggered apoptosis. Importantly, we demonstrated that the upregulation of BCL-XL caused not only VEN resistance, but also led to BCL-XL-specific pro-apoptotic priming of VEN-R lymphoma cells. In vitro, the combination of VEN and A1155463, a specific BCL-XL inhibitor, induced cytotoxic synergy on a panel of MCL cell lines and primary cells. CRISPR-Cas9-mediated BCL-XL knock-out resulted in marked sensitization to VEN-induced apoptosis in MCL cell lines. In vivo, pharmacological blockage of BCL-XL strongly increased sensitivity to VEN. Despite that A1155463 exerted limited anti-lymphoma activity as monotherapy on tested CDX and PDX models, its combination with VEN was synthetically lethal and exerted significantly enhanced anti-lymphoma activity. The efficacy of the VEN and A1155463 combination was highly effective even in mice bearing VEN-R tumors (tumors with acquired VEN resistance, Figure 1). It has been published that mechanisms responsible for BCL-XL upregulation include activation of NFkappaB signaling via CD40 and hypoxia. Upregulation of BCL-XL induced by these factors in turn caused VEN resistance in vivo. Indeed, we confirmed that upregulation of BCL-XL was observed already after engraftment of VEN-sensitive MCL cells in immunodeficient mice. MCL cells isolated ex vivo from the established CDX tumors were significantly more sensitive to BCL-XL inhibition compared to the corresponding in vitro growing cell lines. Our results thus confirmed increased BCL-XL dependence of MCL cells in vivo compared to in vitro. We suggest that these microenvironmental factors were also critical for the selection of VEN-resistant clones during the therapy with VEN. Historically, experimental therapy of patients with chronic lymphocytic leukemia with navitoclax, a combined inhibitor of BCL2 and BCL-XL, was rater disappointing. The plausible reasons included both low efficacy of navitoclax due to insufficient BCL2 inhibition, and a dose-limiting thrombocytopenia. We demonstrated that the thrombocytopenia associated with continued therapy with A1155463 could be managed by 4 days on / 3 days off treatment strategy, which cannot be applied in case of fixed dual BCL2/BCL-XL inhibitors like navitoclax, or AZD4320. In summary, the combined inhibition of BCL2 and BCL-XL with VEN and A1155463 is a highly effective experimental treatment strategy for R/R MCL with a potential translation to the clinical grounds. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

In Search of Drivers of CD34+/CD38-/CD26+ Leukemia Stem Cells Persistence in CML Patients

Introduction: In Chronic Myeloid Leukemia (CML) patients, we confirmed that the expression of CD26 discriminates CML Leukemic Stem Cells (LSCs) from normal Hematopoietic Stem Cells (HSCs) or from LSCs of other myeloid neoplasms. CD34+CD38-CD26+ cells are easily measurable in Peripheral Blood (PB) at diagnosis, and circulating residual CD26+LSCs persist in most patients during treatment with Tyrosine Kinase Inhibitors (TKIs) and after successful TKI discontinuation. The reason for LCSs persistence may be correlated to mechanisms helping their survival, such as resistance to apoptosis (via BCL-2 or CD47 expression) or immune checkpoint escaping (via PD-L1). Moreover, few is still known about the "activity state" or "quiescence" of CML LSCs. Aim of the study: to unravel phenotypic characteristics that could help CML LSCs persistence, evaluating by flow-cytometry the expression level of BCL-2 protein, PD-L1, CD47 and Ki67 on CD26+ LSCs population. Methods: Flow-cytometry assays were performed on PB from newly diagnosed CML patients by using lyophilized reagents (CD34-FITC, CD26-PE, CD38-APC, CD45-V500) and liquid reagents (CD47-V450, PD-L1-Cy7, BCL-2-FITC, Ki67-PerCP-Cy 5.5). Analysis have been done using a BD FACSLyric cytometer. Results: 47 newly diagnosed CML patients entered this study. With regards to PD-L1 expression on CD26+LSCs, 22/47 (47%) scored negative while 25/47 (53%) resulted positive, albeit with a quite variable percentage of expression (median 28.5 % of CD26+ cells, range 12-82.8 %). BCL-2 protein was expressed in 47/47 (100%) CD26+ LSCs, and the level of expression evaluated comparing the Mean Fluorescent Index (MFI) was found much lower on CD26+ LSCs than lymphocytes (median 5.98, range 1.90-32.14; median 17.20, range 7-76, respectively, p value <0.05). Regarding CD47 expression, 47/47 (100%) patients scored positive, with an MFI that is double on lymphocytes compared to CD26+ LSCs (median 26.58, range 5.70-87.09; median 13.22, range 5.64-33.22, respectively). Lastly, all 47/47 (100%) CD26+ LSCs were found Ki-67 positive. Conclusions: These preliminary results show that in newly diagnosed CML patients, Ki67 is highly expressed on circulating CD26+ LSCs, suggesting an unexpected "active proliferative state" of CD26+ LSCs, rather than a "quiescent" status. The latter may explain the rapid reduction of the bulk of CD26+LSCs we observe already at 3 months of TKI treatment. Both BCL-2 protein and CD47 are expressed at a higher level on lymphocytes than on CD26+ CML LSCs, indicating that the "don't eat me signal" that blocks apoptosis may not be relevant for CML LSCs persistence. On the other hand, CD26+/PD-L1+ LSCs (found in about 53% of CML patients) could theoretically escape the immune checkpoint controls, an aspect that could be taken in consideration for a future Treatment Free Survival (TFR) approach in CML patients as it may lead to a loss of molecular response compared to patients with CD26+ LSCs scoring negative for PD-L1. Additional studies need to be performed on a larger cohort of patients with a longer follow-up to support these hypotheses; however, these preliminary data may start to pave the way to understand the relationship between CD26+ LSC and immunity of CML patients.

Moringa oleifera L. leaf extract induces cell cycle arrest and mitochondrial apoptosis in Dalton's Lymphoma: An in vitro and in vivo study

Ethnopharmacological relevanceThe present work is based on a wide spectrum of evidences available from scientific literature which reflects nutritional and medicinal values of natural products such as plants and their extracts. Moringa oleifera is one such popular plant species amidst indigenous tribal communities which is frequently used to treat ailments such as piles, sore throat, eye and ear infections and even poisonous bites of tropical fauna such as insects or snakes. Furthermore decoction of leaf and bark was used to cure fever and cough. Evidences further reveal that Moringa oleifera L. (Family Moringaceae), is widely distributed not only over the Indian sub-continent, but also over Philippines, Central America, Saudi Arabia and the Caribbean Islands and have been traditionally used to treat cancers since ancient times. However, therapeutic effects of Moringa oleifera on Non-Hodgkin Lymphoma (NHL) are yet to be established. Aim of the studyThe study aims to investigate the anti-cancer effects of Moringa oleifera leaf extract against murine NHL Non-Hodgkin cells in vitro and in vivo. Material and methodsThe pharmacologically active compounds of Moringa oleifera leaf extract were identified by GC-HRMS analysis. Tests of Moringa oleifera leaf extract's cytotoxicity against DL cells were carried out using the MTT assay. Chromatin condensation along with other morphological alterations were visualized through Fluorescence microscopy. Changes in the mitochondrial membrane potential (ΔΨm), the cell cycle, and apoptosis were analysed through flow cytometer. We tried to identify proteins involved in apoptosis and cell cycle through Western blotting using BALB/c mice as a model organism. ResultsGC-HRMS study revealed that a methanol based leaf extract of Moringa oleifera (MOML) comprises of a variety of bioactive chemicals. Our results indicate that MOML successfully reduced the proliferation of DL cells by lowering ΔΨm, changing overall cell morphology. DL cells treated with MOML showed arrested cell cycle at the G2/M phase and substantially up-regulated the expression of p53 and p21. Elevated levels of Bax, Cyt-c, and Caspase-3 and lowered expression levels of Bcl-2 protein suggested induction of apoptosis. Mechanistically, the anticancer efficacy of MOML is attributed to MEK/ERK-mediated pathway inactivation in DL cells. It is also interesting to note that MOML-mediated inhibition of DL growth was accompanied by apoptosis induction and improvement in hematological parameters in DL-bearing mice. ConclusionOur finding suggested that MOML induces apoptosis and abrogates the growth of Dalton's lymphoma both in vitro and in vivo.

Xanthoceras sorbifolium Bunge flower extract inhibits benign prostatic hyperplasia in rats

To assess the inhibitory effect of the extract of Xanthoceras sorbifolium Bunge flower against benign prostatic hyperplasia (BPH) and explore its possible mechanism. MTT assay was used to examine the effect of the extract of Xanthoceras sorbifolium Bunge flower on proliferation of benign prostatic hyperplasia cells (BPH-1), and cell apoptosis and cell cycle changes following the treatment were analyzed using annexin V/PI double staining and flow cytometry. The protein expression levels of Bcl-2, Bax, caspase-3, PI3K and AKT in the treated cells were detected using Western blotting. A rat model of BPH established by subcutaneous injection of testosterone propionate was treated with the flower extract for 28 days, and pathological changes in the prostate tissue were observed with HE staining. The protein expression levels of Bcl-2, Bax, caspase3 and PI3K/AKT in the prostate tissue were detected with Western blotting. Within the concentration range of 125-1000 µg/mL, the flower extract of Xanthoceras sorbifolium Bunge significantly inhibited the proliferation of BPH-1 cells and caused obvious cell cycle arrest at G0/G1 phase; the apoptotic rate of the cells was positively correlated with the concentration of the flower extract (P < 0.05). Bcl-2, p-PI3K and p-AKT expression levels were significantly down-regulated and Bax and caspase-3 expression levels were significantly increased in the cells after treatment with the flowers extract (P < 0.05). In the rat models of BPH, the rats treated with the flowers extract at moderate and high doses showed obviously decreased expressions of p-AKT and Bcl-2 and an increased expression of Bax in the prostate tissue; a significantly lowered p-AKT expression was observed in the prostate tissue of rats receiving the low-dose treatment (P < 0.05). The flower extract of Xanthoceras sorbifolium Bunge has a inhibitory effect on BPH both in vitro and in rats, suggesting its potential value in the development of medicinal plant preparations for treatment of BPH.

Flammulina velutipes stem regulates oxidative damage and synthesis of yolk precursors in aging laying hens by regulating the liver–blood–ovary axis

Egg production levels in late laying hens are negatively correlated with increasing age. Decreased liver and ovarian function in aging laying hens is accompanied by decreased antioxidant capacity, reproductive hormone levels, and follicular development, resulting in decreased synthesis of yolk precursors. The golden needle mushroom (Flammulina velutipes) has been reported to exhibit anti-inflammatory, antioxidant, and hypolipidemic properties. We aimed to reveal the therapeutic effects of F. velutipes stem (FVS) on liver-blood-ovary axis and investigate the underlying mechanisms. A total of 360 sixty-seven-wk-old laying hens were randomized into 4 treatment groups: 1) basal maize-soybean meal diet (CON); 2) basal maize+20 g/kg FVS (2% FVS); 3) basal maize+40 g/kg FVS (4% FVS); and 4) basal maize+60 g/kg FVS (6% FVS). FVS groups demonstrated significantly increased egg production and ovarian development compared with the CON group. The addition of FVS increased the levels of antioxidant enzymes (GSH-Px, T-SOD, and T-AOC) in the liver, serum, and ovaries and decreased malondialdehyde levels by regulating the expression of proteins related to the Keap1-Nrf2/ARE signaling pathway. Additionally, FVS significantly decreased ovarian apoptosis by regulating Bax, Bcl-2, and caspase3 mRNA and protein expression levels. FVS significantly increased the expression levels of estradiol, progesterone, luteinizing hormone, and follicle stimulating hormone and their respective receptors. With increased levels of estradiol transported to the liver through the bloodstream, targeted binding to estrogen receptor (ER)-α and ER-β led to significant increases in ApoVLDL II, ApoB, and VTG II mRNA expression associated with yolk precursor synthesis. FVS decreased the levels of triglyceride and total cholesterol and significantly increased the expression of lipid metabolism, and transport-related mRNAs (FAS, PPAR-a/γ, and MTTP) in the liver. Therefore, the dietary supplementation of FVS can maintain the productive performance of aging laying hens by alleviating the degree of oxidative stress and regulating the transport of functional substances along the liver-blood-ovary axis, thereby improving the synthesis of yolk precursors.

Isolongifolene alleviates liver ischemia/reperfusion injury by regulating AMPK-PGC1α signaling pathway-mediated inflammation, apoptosis, and oxidative stress

Isolongifolene (ISO) has antioxidant, anti-inflammatory, anticancer, and neuroprotective effects; however, it is unclear whether ISO has a protective effects against liver ischemia/reperfusion (I/R) injury. In this study, a mouse liver I/R injury model and a mouse AML12 cell Hypoxia reoxygenation (H/R) model were established after pretreatment with different concentrations of ISO. Serum transaminase levels, necrotic liver area, cell viability, inflammation response, oxidative stress, and apoptosis were used to evaluate the effect of ISO on liver I/R or cell H/R injury. Western blotting was used to detect Bax, Bcl-2, C-Caspase3, AMPK, P-AMPK, and PGC1α protein expression levels. The AMPK inhibitor, compound C, was used to inhibit the AMPK expression. The results showed that ISO reduced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, liver necrosis, inflammatory factors IL-1β, IL-6, MCP-1, and TNF-α expression, MPO+ inflammatory cell infiltration, MDA content, TUNEL-positive cell number, cell apoptosis rate, and the expression of pro-apoptotic proteins Bax and C-Caspase3, while increasing cell viability, SOD and GSH activity, and the expression of anti-apoptotic protein Bcl-2. Moreover, Western blotting results showed that ISO could increase the protein expression of P-AMPK and PGC1α. Following the addition of compound C, the protective effect of ISO was significantly weakened. Therefore, our results suggest that ISO alleviates liver I/R injury by regulating AMPK-PGC1α signaling pathway-mediated anti-inflammatory, and antioxidant and anti-apoptotic effects.

Pathogenesis and Diagnostic Significance of EBV-miR-BARTs in Nasopharyngeal Carcinoma.

Objective Examining the role of EBV-miR-BARTs in nasopharyngeal cancer etiology and diagnosis. Method As the subjects of this study, nasopharyngeal cancer cell lines were chosen and then randomly assigned to one of four groups: the control group, EBV-miR-BART5-3p NC, EBV-miR-BART5-3p mimics, and EBV-miR-BART5-3p inhibitor groups. Utilizing reverse transcription polymerase chain reaction, we determined the levels of gene expression in nasopharyngeal cancer cells that had been treated with EBV-miR-BART5-3p (RT-PCR). The MTT, Transwell, and scratch tests were used to determine the degree to which cells underwent apoptosis, invasion, and migration. The Western blotting method was used in order to examine the protein expression. Result Compared with normal nasopharyngeal cells, P 0.05 showed that nasopharyngeal cancer cells had greater EBV-miR-BART5-3p expressions and proliferation rates in the control, EBV-miR-BART5-3p NC, and EBV-miR-BART5-3p No statistically significant differences were seen between the mimic groups (P > 0.05); compared with the control group, the proliferation rate of the EBV-miR-BART5-3p inhibitor group was lower with P < 0.05. At a significance threshold of P 0.05, there was no difference in the rates of apoptosis between the control group and the EBV-miR- BART5-3p NC group. Comparing the control group to the EBV-miR-BART5-3p mimics group and the EBV-miR-BART5-3p inhibitors group revealed that the rate of apoptosis was dramatically enhanced in the EBV-miR-BART5-3p inhibitors group but significantly decreased in the control group (P 0.05). When comparing the control group to the EBV-miR-BART5-3p NC group, there was no statistically significant change in the total number of invasive cells (P > 0.05). When comparing the EBV-miR-BART5-3p mimics group to the control group, we found a statistically significant increase in the former and a decrease in the latter (P 0.05). The migration rates of the control group, the EBV-miR-BART5-3p NC group, and the EBV-miR-BART5-3p mimics group did not vary from one another in a way that was statistically significant (P > 0.05). When compared to the control group, the migration rate was considerably (P 0.05) lower in the EBV-miR-BART5-3p inhibitor group. There were no discernible changes identified (P > 0.05) in the levels of Bcl-2 protein expression in the control group, the EBV-miR-BART5-3p NC group, and the EBV-miR-BART5-3p mimic group in a research that compared these three groups. Protein levels of BCL-2 were significantly decreased (P 0.05) in the EBV-miR-BART5-3p inhibitor group, in comparison to the control group. When comparing the control and EBV- miR-BART5-3p NC groups, we found no statistically significant differences in Bax and Caspase-3 protein expression levels (P > 0.05). The protein expressions of Bax and Caspase-3 were statistically significantly greater in the EBV-miR-BART5-3p contrast between the inhibitor and control groups. When comparing the protein expressions of MMP-2 and MMP-9 between the control group, the EBV-miR-BART5-3p NC group, and the EBV-miR-BART5-3p mimics group, there was no statistically significant change (P > 0.05). Protein levels of MMP-2 and MMP-9 were inhibited by EBV-miR-BART5-3p to a greater extent (P 0.05) in the experimental group compared to the control group. Conclusion The understanding that inhibiting expression of EBV-miR-BART5-3p might reduce the risk of developing nasopharyngeal cancer may help direct clinical treatment for the condition.

Expression of miR-126 in Diffuse Large B-Cell Lymphoma and Its Biological Function

To investigate the expression of miR-126 in diffuse large B-cell lymphoma (DLBCL) tissues and its biological function. The lymphoma tissues of 46 DLBCL patients in our hospital were selected as the research object, and the lymph node hyperplasia tissue of 31 patients with reactive hyperplasia were selected as controls. The expression level of miR-126 in the patients' tissues was detected by real-time fluorescent quantitative PCR (RT-qPCR), and the correlation of miR-126 expression with the pathological characteristics and prognosis of the patients was analyzed. The DLBCL cell line SU-DHL-4 was transfected with miR-126 inhibitor and its negative control (NC inhibitor) or miR-126 mimics and its negative control (NC mimics). RT-qPCR assay was used to detect the expression level of miR-126 in cells; MTT method was used to detect cell proliferation activity; single clone formation test was used to detect cells colony-forming ability; Annexin V/PI double staining assay was used to detect cell apoptosis; Transwell test was used to detect cell migration and invasion ability; the expression levels of apoptosis-related proteins cleaved-Caspase-3, Bcl-2 and Bax were detected by Western blot. miR-126 was highly expressed in lymphoma tissues of DLBCL patients, and its expression level was significantly correlated with Hans type, IPI score and Ann-Arbor stage of DLBCL patients (P<0.05). Kaplan-Meier survival analysis showed that the survival rate of DLBCL patients with high expression of miR-126 was significantly lower than that of patients with low expression (P<0.05). Compared with the NC mimics group, the miR-126 expression level, cell proliferation rate, number of colony-forming units, migration and invasion ability, and Bcl-2 protein expression level in the miR-126 mimics group were significantly increased (P<0.05), but the cells apoptotic rate, cleaved-Caspase-3 and Bax protein expression levels were significantly reduced (P<0.05). Compared with the NC inhibitor group, the miR-126 expression level, cell proliferation rate, number of colony-forming units, migration and invasion ability, and Bcl-2 protein expression level in the miR-126 inhibitor group were significantly reduced (P<0.05), but the cells apoptosis rate, cleaved-Caspase-3 and Bax protein expression levels were significantly increased (P<0.05). miR-126 is highly expressed in lymphoma tissues of DLBCL patients and its expression level is related to the poor prognosis of patients. miR-126 can promote DLBCL cell proliferation, invasion and migration, and inhibit cell apoptosis.

Protective effect of Tongqiao Huoxue Decoction containing cerebrospinal fluid on OGD/R-damaged HT22 cells via regulation of ASK1/MKK4/JNK signaling pathway

To investigate the protective effect of Tongqiao Huoxue Decoction containing cerebrospinal fluid(TQHXD-CSF) on HT22 cells damaged by oxygen-glucose deprivation/reoxygenation(OGD/R) and whether the mechanism is related to the regulation of ASK1/MKK4/JNK signaling pathway. HT22 cells were subjected to OGD/R to simulate cerebral ischemia-reperfusion injury(CIRI). Then the cells were randomly divided into five groups: blank cerebrospinal fluid(control group), OGD/R group, TQHXD-CSF group, Z-VAD-FMK group(20 μmol·L~(-1)) and TQHXD-CSF+Z-VAD-FMK group. Except the control group, cells in the other groups were reoxygenated for 12 h after 6 h of oxygen and glucose deprivation for modeling OGD/R, and group administration was performed. Cell viability and cytotoxicity were detected by CCK8 and LDH assay kit, respectively and the morphology of HT22 cells was observed by inverted microscope. Western blot and qRT-PCR were employed to detect the protein and mRNA expression levels of Bax, Bcl-2 and caspase-3, respectively. Then HT22 cells were assigned into the control group, OGD/R group, si-NC group, si-ASK1 group, TQHXD-CSF group and TQHXD-CSF+si-ASK1 group. Cell viability, proliferation and apoptosis were determined by CCK8, electric cell-substrate impedance sensing(ECIS), and Hoechst staining and flow cytometry, respectively. The protein expression of MKK4, p-MKK4, JNK, p-JNK, c-Jun, p-c-Jun, Cyt C, Bax, Bcl-2 and caspase-3 was tested by Western blot. The results showed that compared with OGD/R group, TQHXD-CSF significantly enhanced cell viability, improved cell morphology and reduced the protein and mRNA expression levels of Bax, Bcl-2 and caspase-3. In addition, when ASK1 was silenced, compared with OGD/R group, TQHXD-CSF remarkably improved cell viability, and decreased apoptosis rate and the protein expression levels of p-MKK4, p-JNK, p-c-Jun, Cyt C, Bax/Bcl-2 and caspase-3, but the effect was not as good as that of TQHXD-CSF+si-ASK1 group. In conclusion, TQHXD-CSF can inhibit apoptosis mediated by ASK1/MKK4/JNK signaling pathway in OGD/R-damaged HT22 cells, and has protective effect on ischemia-reperfusion injury.

Mechanism of Dexmedetomidine Intervention on Neurogenic Inflammation in Cognitive Impairment Rats after Partial Hepatectomy.

Objective To study the effect of dexmedetomidine on cognitive function in rats with cognitive impairment after partial hepatectomy and its mechanism. Methods 60 SD rats were randomly divided into 4 groups (n = 15): blank control group (CG group), sham operation group (Sham group), cognitive impairment model group (POCD group), and dexmedetomidine + cognitive impairment model group (DEX group). Rats in the POCD group underwent left lobe hepatectomy and intraperitoneal injection of the same amount of normal saline after resuscitation. Rats in the DEX group underwent left lobe hepatectomy and intraperitoneal injection of dexmedetomidine 50 μg/kg. Group CG was not operated on and the same amount of normal saline was injected intraperitoneally. In the Sham group, liver resection was not allowed after the abdominal incision, and normal saline was injected intraperitoneally. Rats were injected every 24 hours for 5 consecutive days. Morris water maze (MWM) were used to evaluate the effects of dexmedetomidine on learning and memory ability of POCD rats. TUNEL method was used to detect apoptotic neurons in the hippocampus. INOS, Arg-1, IL-6, and TNF-αexpression levels were detected. Western blot detects the expression level of TNF-α, Bcl-2, and NF-κB protein. Result Compared with the CG group, the escape latency of the other three groups was prolonged on the 5th day after the operation, and the number of crossing the platform was reduced. Compared with the Sham group, the escape latency of the POCD group and DEX group was significantly prolonged, and the number of crossing the platform was significantly reduced on day 5 (P < 0.05). Compared with the POCD group, the DEX group shortened the escape latency and increased the number of crossing the platform on the 5th day (P < 0.05). It shows that the spatial learning and memory function of rats has been restored to a certain extent.The number of iNOS and Arg-1 positive cells in the POCD group and DEX group was higher than that in the control group, and the number of Arg-1 positive cells in the DEX group was higher than that in the POCD group (P < 0.05). Western blot results the expression of Bcl-2 and NF-κB protein in POCD group, and DEX group was higher than that of the sham group (P < 0.05). The expression of Bcl-2 and NF-κB protein was the most in POCD group. The expression of Bcl-2 and NF-κB protein in DEX group was lower than that in POCD group (P < 0.05). Conclusion Behavioral results showed that the learning and cognitive ability of POCD model rats after hepatectomy was impaired, and inflammatory factors and activated microglia were found in the hippocampus of POCD rats. Dexmedetomidine may improve the brain function of POCD rats by inhibiting neuronal apoptosis,partly through NF-κB apoptosis pathway.

Atlas of metabolism reveals palmitic acid results in mitochondrial dysfunction and cell apoptosis by inhibiting fatty acid β-oxidation in Sertoli cells.

In recent years, the impact of lipotoxicity on male fertility has received extensive attention, especially on Sertoli cells (SCs). In SCs, energy metabolism is important as disorders of energy metabolism result in infertility eventually. However, the underlying mechanism of lipotoxicity on energy metabolism in SCs remains unknown. Advances in high-throughput metabolomics and lipidomics measurement platforms provide powerful tools to gain insights into complex biological systems. Here, we aimed to explore the potential molecular mechanisms of palmitic acid (PA) regulating energy metabolism in SCs based on metabolomics and lipidomics. The results showed that glucose metabolism-related metabolites were not significantly changed, which suggested that PA treatment had little effect on glucose metabolism and may not influence the normal energy supply from SCs to germ cells. However, fatty acid β-oxidation was inhibited according to accumulation of medium- and long-chain acylcarnitines in cells. In addition, the pool of amino acids and the levels of most individual amino acids involved in the tricarboxylic acid (TCA) cycle were not changed after PA treatment in SCs. Moreover, PA treatment of SCs significantly altered the lipidome, including significant decreases in cardiolipin and glycolipids as well as remarkable increases in ceramide and lysophospholipids, which indicated that mitochondrial function was affected and apoptosis was triggered. The increased apoptosis rate of SCs was verified by elevated protein expression levels of Cleaved Caspase-3 and Bax as well as decreased Bcl-2 protein expression level. Together, these findings indicated that PA may result in mitochondrial dysfunction and increased apoptosis by inhibiting fatty acid β-oxidation of SCs.

Resveratrol protects against sepsis induced acute kidney injury in mice by inducing Klotho mediated apoptosis inhibition

Purpose: To investigate the mechanism of resveratrol protection against sepsis-induced acute kidney injury in mice.&#x0D; Methods: A sepsis-induced acute kidney injury model was established in mice by cecal ligation and puncture (CLP). Sixty healthy male ICR mice were randomly divided into the sham operation (sham) group, sepsis-induced acute kidney injury model (CLP) group, CLP + low-dose (20 mg/kg) resveratrol treatment (CLP + ResL) group, CLP + high-dose (40 mg/kg) resveratrol treatment (CLP + ResH) group and CLP + Klotho (0.01 mg/kg) treatment (CLP + Klotho) group. All mice were administered treatment on the day after surgery and once every 24 h for 3 days. Various serum biochemical parameters and protein expressions were evaluated.&#x0D; Results: After CLP, the levels of serum creatinine (Scr) and blood urea nitrogen (BUN) increased and the pathology was exacerbated. The protein and mRNA expression levels of Klotho and Bcl-2 decreased, while those of Bax and Caspase-3 increased (p &lt; 0.05). After resveratrol and Klotho protein intervention, Scr and BUN levels recovered, and pathological changes were alleviated. The protein and mRNA expression levels of Klotho and Bcl-2 increased, while those of Bax and Caspase-3 decreased. The conditions of the mice in CLP + ResH group and the CLP + Klotho group improved more significantly than those of the mice in the CLP + ResL group (p &lt; 0.05).&#x0D; Conclusion: Resveratrol upregulates the expression of endogenous Klotho to exert its antiapoptotic effects, which can protect the kidneys of mice against sepsis-induced acute kidney injury. Thus, the compound has potentials for development for protection against acute kidney injury.

MiR-99a alleviates apoptosis and extracellular matrix degradation in experimentally induced spine osteoarthritis by targeting FZD8

BackgroundOur previous study identified miR-99a as a negative regulator of early chondrogenic differentiation. However, the functional role of miR-99a in the pathogenesis of osteoarthritis (OA) remains unclear.MethodsWe examined the levels of miR-99a and Frizzled 8 (FZD8) expression in tissue specimens. Human SW1353 chondrosarcoma cells were stimulated with IL-6 and TNF-α to construct an in vitro OA environment. A luciferase reporter assay was performed to analyze the relationship between miR-99a and FZD8. CCK-8 assays, flow cytometry, and ELISA assays were used to assess cell viability, apoptosis, and inflammatory molecule expression, respectively. Percutaneous intra-spinal injections of papain mixed solution were performed to create an OA Sprague–Dawley rat model. Alcian Blue staining, Safranin O Fast Green staining, and Toluidine Blue O staining were performed to detect the degrees of cartilage injury.ResultsMiR-99a expression was downregulated in the severe spine OA patients when compared with the mild spine OA patients, and was also decreased in the experimentally induced in vitro OA environment when compared with the control environment. Functionally, overexpression of miR-99a significantly suppressed cell apoptosis and extracellular matrix degradation stimulated by IL-6 and TNF-α. FZD8 was identified as a target gene of miR-99a. Furthermore, the suppressive effects of miR-99a on cell injury induced by IL-6 and TNF-α were reversed by FZD8 overexpression. Moreover, the levels of miR-99a expression were also reduced in the induced OA model rats, and miR-99a agomir injection relieved the cartilage damage. At the molecular level, miR-99a overexpression downregulated the levels of MMP13, β-catenin, Bax, and caspase-3 protein expression and upregulated the levels of COL2A1 and Bcl-2 protein expression in the in vitro OA-like chondrocyte model and also in the experimental OA model rats.ConclusionsOur data showed that miR-99a alleviated apoptosis and extracellular matrix degradation by targeting FZD8, and thereby suppressed the development and progression of experimentally induced spine osteoarthritis.

Indirubin relieves inflammatory injury of chondrocytes in a mouse model of osteoarthritis

To investigate the effect of indirubin for relieving joint inflammation and injury in a rat model of osteoarthritis. Articular cartilage chondrocytes were isolated from adult rat knee joint and cultured in the presence of interleukin-1β (IL-1β) and 0.1, 0.5, 1.0, or 2.0 μmol/L indirubin. The cells were transfected with NPAS2 siRNA or a non-specific siRNA, and the cell proliferation and apoptosis were evaluated using tetramethylthiazole blue staining and flow cytometry. The protein expression levels of Bax, Bcl-2, ACAN, COL2A1, MMP-13 and NPAS2 were detected with Western blotting, and the levels of NO, PGE2 and TNF-α in the culture supernatant were determined with ELISA. The mRNA expression levels of NPAS2, ACAN, COL2A1 and MMP-13 were detected using fluorescence quantitative PCR. In a C57BL/6 mouse model of osteoarthritis, the effect of indirubin on BAX, Bcl-2, ACAN and MMP-13 protein expressions in the bone and joint tissues were evaluated with Western blotting. Treatment with 0.1 μmol/L indirubin produced no significant changes in chondrocyte proliferation, apoptosis, caspase-3 activity, or BAX and Bcl-2 protein expressions. At higher doses (0.5, 1.0 and 2.0 μmol/L), indirubin significantly promoted cell proliferation, increased Bcl-2 protein expression, and lowered cell apoptosis rate, caspase-3 activity and Bax protein expression (P < 0.05). Indirubin treatment at 0.5 μmol/L up-regulated the protein and mRNA expressions of NPAS2, ACAN and COL2A1, and down-regulated the expressions of MMP-13, NO, PGE2 and TNF-α (P < 0.05). Interference of NPAS2 expression significantly attenuated the protective effect of 0.5 μmol/L indirubin against IL-1β-induced chondrocyte injury. The mouse model of osteoarthritis showed obviously increased protein levels of BAX and MMP-13 (P < 0.01) and decreased levels of Bcl-2 (P < 0.05) and ACAN (P < 0.01) in the knee joint, and indirubin treatment of the mouse models significantly inhibited the increase of BAX and MMP-13 protein expressions (P < 0.01) and up-regulated the protein expressions of Bcl-2 and ACAN (P < 0.05). Indirubin has a protective effect on osteoarthritis tissue and alleviates inflammation and damage of osteoarthritis chondrocytes possibly through NPAS2.

Wharton jelly-derived mesenchymal stem cell exosomes induce apoptosis and suppress EMT signaling in cervical cancer cells as an effective drug carrier system of paclitaxel.

Mesenchymal stem cells can be obtained and multiplied from various sources and have a very high capacity to release exosomes. Exosomes are nano-sized extracellular vesicles containing biological signaling molecules. This study aimed to determine the effect of MSC-derived exosomes as a drug delivery system for paclitaxel in cervical cancer cells. In this study, human MSC were isolated from wharton jelly of umbilical cord tissue (WJ-MSC), and cells were characterized by CD44, CD90, CD105, and CD34 staining. Exosomes were released in WJ-MSC cells with serum-starved conditions for 48 hours, and particle sizes and structures were examined with zeta-sizer and TEM. In addition, exosomes CD9, CD63, and CD81 markers were checked by western blot. Paclitaxel was loaded into exosomes (Exo-PAC) by electroporation and then incubated with Hela cervical cancer cells for 24 hours. TGF-β, SMAD, Snail, Slug, β-catenin, Notch, Caspase-3, Caspase-9, Bax, Bcl-2 protein and gene expression levels were analyzed in Hela cells. As a result, low concentration Exo-PAC induced apoptosis, and suppressed epithelial-mesenchymal transition proteins in Hela cells. In this study, it has been demonstrated that WJ-MSCs can be used as drug delivery systems for cervical cancer if exosomes are produced scalably in the future.