Protein Expression Levels Of Bcl-2 Research Articles

Atractylenolide III Attenuates Apoptosis in H9c2 Cells by Inhibiting Endoplasmic Reticulum Stress through the GRP78/PERK/CHOP Signaling Pathway.

The objective of this study was to determine the effect of atractylenolide III (ATL-III) on endoplasmic reticulum stress (ERS) injury, H9c2 cardiomyocyte apoptosis induced by tunicamycin (TM), and the GRP78/PERK/CHOP signaling pathway. Molecular docking was applied to predict the binding affinity of ATL-III to the key proteins GRP78, PERK, IREα, and ATF6 in ERS. Then, in vitro experiments were used to verify the molecular docking results. ERS injury model of H9c2 cells was established by TM. Cell viability was detected by MTT assay, and apoptosis was detected by Hoechst/PI double staining and flow cytometry. Protein expression levels of GRP78, PERK, eIF2α, ATF4, CHOP, Bax, Bcl-2, and Caspase-3 were detected by Western blot. And mRNA levels of GRP78, CHOP, PERK, eIF2α, and ATF4 were detected by RT-qPCR. Moreover, the mechanism was further studied by using GRP78 inhibitor (4-phenylbutyric acid, 4-PBA), and PERK inhibitor (GSK2656157). The results showed that ATL-III had a good binding affinity with GRP78, and the best binding affinity was with PERK. ATL-III increased the viability of H9c2 cells, decreased the apoptosis rate, downregulated Bax and Caspase-3, and increased Bcl-2 compared with the model group. Moreover, ATL-III downregulated the protein and mRNA levels of GRP78, CHOP, PERK, eIF2α, and ATF4, consistent with the inhibition of 4-PBA. ATL-III also decreased the expression levels of PERK, eIF2α, ATF4, CHOP, Bax, and Caspase-3, while increasing the expression of Bcl-2, which is consistent with GSK2656157. Taken together, ATL-III could inhibit TM-induced ERS injury and H9c2 cardiomyocyte apoptosis by regulating the GRP78/PERK/CHOP signaling pathway and has myocardial protection.

A new approach on the regulation of NF-κB and Bax protein signaling pathway activation by royal jelly in fluoride-induced pancreas damage in rats

Forty-two healthy adult male rats (Wistar albino, n = 42, 8 weeks old, starting weights 200–250 g) employed in this study were subdivided into six groups randomly with seven rats per group as follows: (i) Control group: received standard diet; (ii) RJ group: received standard diet supplemented with royal jelly; (iii) F50 group: received standard diet supplemented with fluoride (50 mg/kg BW); (iv) F100 group: received standard diet supplemented with fluoride (100 mg/kg BW); (v) F50 +RJ group: received standard diet supplemented with fluoride (50 mg/kg BW) and royal jelly; (iv) F100 +RJ group: received standard diet supplemented with fluoride (100 mg/kg BW) and royal jelly. The study continued for a total of eight weeks. Western blot analysis was conducted to determine the post-translational expression levels of NF-κB, Bax, Bcl-2, TNF-α, Caspase-3 and Caspase-6 proteins in pancreas tissue. The pancreatic tissue was subjected to histopathological evaluation. Furthermore, MDA, GSH and CAT activities were examined by spectrophotometric analyzes. Our findings demonstrate that, compared to the control and RJ groups, Bcl-2 protein expression was augmented and, conversely, Caspase-6, Caspase-3 and Bax protein levels were decreased upon fluoride treatment. A statistically significant increase in TNF-α and NF-κB protein expressions was observed in the groups with fluoride-induced damage compared to the control and RJ groups. The MDA levels were increased in all fluoride-treated rats compared to those in the control and RJ groups, whereas the CAT and GSH activities were reduced in all rats with fluoride- induced damage. Although there was not a great difference between the groups regarding histopathological findings, there was a tendency to decrease in the rate of damage upon royal jelly treatment.

Curcumin alleviates aristolochic acid nephropathy based on SIRT1/Nrf2/HO-1 signaling pathway

Aristolochic acid I (AA-I), presenting in a variety of natural medicinal plants, which could cause tubular epithelial cell injury. Curcumin (CUR), a polyphenolic substance isolated from turmeric, is a natural antioxidant. The aim of this experiment was to investigate whether CUR attenuated AA-I-induced renal injury in rats through the SIRT1/Nrf2/HO-1 signaling pathway. SD rats were treated with AA-I (10 mg/kg) or/and CUR (200 mg/kg) for 28 days to assess the protective effect of CUR on AA-I-induced renal injury in vivo. NRK-52E cells were treated with AA-I (40 μ M) or/and CUR (20 μ M) for 24 h in vitro. The intervention pathway of CUR against oxidative stress injury induced by AA-I was assessed by observing pathological changes, oxidative stress status, apoptosis and the expression of SIRT1/Nrf2/HO-1 signaling pathway-related factors. The results showed that AA-I exposure increased the contents of BUN, Cr, KIM-1, NGAL, ALT and AST in serum. It increased the content of MDA, decreased the activities of SOD, GST, GSH and the content of ATP in renal tissue. Pathological changes such as inflammatory cell infiltration and mitochondrial injury occurred in renal tissue. AA-I exposure resulted in a substantial rise in the levels of BAX, Ccaspase-9, Cleaved Caspase-9, Caspase-3, Cleaved Caspase-3 and a significant decrease in mRNA and protein expression levels of Bcl-2, SIRT1, Nrf2, NQO1, HO-1 and Keap1. However, these changes were reversed by CUR intervention. In summary, AA-I exposure caused mitochondrial dysfunction and triggered apoptosis through the oxidative stress pathway. However, CUR could reduce AA-I-induced renal injury by activating the SIRT1/Nrf2/HO-1 signaling pathway.

Alleviating effect of quercetin on cadmium-induced oxidative damage and apoptosis by activating the Nrf2-keap1 pathway in BRL-3A cells.

Cadmium (Cd) is a toxic heavy metal extensively used in industrial and agricultural production. Among the main mechanisms of Cd-induced liver damage is oxidative stress. Quercetin (QE) is a natural antioxidant. Herein, the protective effect of QE on Cd-induced hepatocyte injury was investigated. BRL-3A cells were treated with 12.5 μmol/L CdCl2 and/or 5 μmol/L QE for 24 h. The cells and medium supernatant were collected, and the ALT, AST, and LDH contents of the medium supernatant were detected. The activities or contents of SOD, CAT, GSH, and MDA in cells were determined. Intracellular ROS levels were examined by flow cytometry. Apoptosis rate and mitochondrial-membrane potential (ΔΨm) were detected by Hoechst 33,258 and JC-1 methods, respectively. The mRNA and protein expression levels of Nrf2, NQO1, Keap1, CytC, caspase-9, caspase-3, Bax, and Bcl-2 were determined by real-time PCR (RT-PCR) and Western blot methods. Results showed that Cd exposure injured BRL-3A cells, the activity of antioxidant enzymes decreased and the cell ROS level increased, whereas the ΔΨm decreased, and the expression of apoptotic genes increased. Cd inhibited the Nrf2-Keap1 pathway, decreased Nrf2 and NQO1, or increased Keap1 mRNA and protein expression. Through the combined action of Cd and QE, QE activated the Nrf2-Keap1 pathway. Consequently, antioxidant-enzyme activity decreased, cellular ROS level decreased, ΔΨm increased, Cd-induced BRL-3A cell damage was alleviated, and cell apoptosis was inhibited. After the combined action of QE and Cd, Nrf2 and NQO1 mRNA and protein expression increased, Keap1 mRNA and protein expression decreased. Therefore, QE exerted an antioxidant effect by activating the Nrf2-Keap1 pathway in BRL-3A cells.

Synthesis of silver nanoparticles using Alpinia officinarum rhizome extract induces apoptosis through down-regulating Bcl-2 in human cancer cells.

In this study, silver nanoparticles were synthesized using Alpinia officinarum rhizome extract via an eco-friendly green synthesis method. The silver nanoparticles (AO-AgNPs) were characterized by UV-Vis spectrometry, scanning electron microscopy, energy-dispersive X-ray spectroscopy, and dynamic light scattering. Further, the cytotoxic and apoptotic effects of AO-AgNPs were investigated in human cancer cells with different tissue origins via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and flow cytometric analyses, respectively. The expression levels of anti-apoptotic Bcl-2 protein were evaluated via a real-time polymerase chain reaction. The synthesized AO-AgNPs induced a significant cytotoxic effect in all tested cancer cells but not in normal cells. AO-AgNPs induced the percentage of apoptotic cells and reduced the levels of anti-apoptotic Bcl-2 mRNA levels in cancer cells. These results demonstrate the potential application of AO-AgNPs in cancer treatment.

ROCK2 knockdown alleviates LPS-induced inflammatory injury and apoptosis of renal tubular epithelial cells via the NF-κB/NLRP3 signaling pathway.

Rho-associated protein kinase 2 (ROCK2) is an important regulator of the inflammatory response and has been reported to serve a role in sepsis. The present study aimed to investigate whether ROCK2 served a role in sepsis-associated acute kidney injury (S-AKI). HK-2 cells were stimulated with lipopolysaccharide (LPS) to simulate S-AKI in vitro. Subsequently, the change in ROCK2 expression levels were determined. ROCK2 in LPS-induced HK-2 cells was knocked down using short hairpin RNA-ROCK2, in the absence or presence of phorbol 12-myristate 13-acetate (PMA), an activator of NF-κB. Cell viability, cytotoxicity, inflammation and apoptosis were assessed using MTT, lactate dehydrogenase (LDH) release, reverse transcription-quantitative PCR, ELISA, TUNEL and western blotting assays. The protein expression levels of proteins involved in the NF-κB/NLR family pyrin domain containing 3 (NLRP3) signaling pathway were also assessed using western blotting. The results demonstrated that ROCK2 was upregulated in HK-2 cells upon LPS treatment. LPS also reduced cell viability, promoted LDH activity and increased TNF-α, IL-6 and IL-1β mRNA expression levels and concentrations. Apoptosis was also induced by LPS as indicated by an increase in the proportion of TUNEL-positive cells, decreased Bcl-2 protein expression levels and increased cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase protein expression levels. However, ROCK2 knockdown in LPS-induced HK-2 cells reversed cell viability damage and inhibited LDH activity, the generation of pro-inflammatory cytokines and apoptosis caused by LPS. Furthermore, ROCK2 knockdown inhibited the LPS-induced expression of phosphorylated-NF-κB p65, NLRP3, apoptosis-associated speck-like protein containing a CARD and caspase-1 p20. PMA treatment reversed all the aforementioned effects of ROCK2 knockdown on LPS-treated HK-2 cells. Therefore, ROCK2 knockdown may alleviate LPS-induced HK-2 cell injury via the inactivation of the NF-κB/NLRP3 signaling pathway.

Synthesis and Biological Evaluation of Novel Allobetulon/Allobetulin-Nucleoside Conjugates as AntitumorAgents.

Allobetulin is structurally similar tobetulinic acid, inducing the apoptosis of cancer cells with low toxicity. However, both of them exhibited weak antiproliferation against several tumor cell lines. Therefore, the new series of allobetulon/allobetulin–nucleoside conjugates 9a–10i were designed and synthesized for potency improvement. Compounds 9b, 9e, 10a, and 10d showed promising antiproliferative activity toward six tested cell lines, compared to zidovudine, cisplatin, and oxaliplatin based on their antitumor activity results. Among them, compound 10d exhibited much more potent antiproliferative activity against SMMC-7721, HepG2, MNK-45, SW620, and A549 human cancer cell lines than cisplatin and oxaliplatin. In the preliminary study for the mechanism of action, compound 10d induced cell apoptosis and autophagy in SMMC cells, resulting in antiproliferation and G0/G1 cell cycle arrest by regulating protein expression levels of Bax, Bcl-2, and LC3. Consequently, the nucleoside-conjugated allobetulin (10d) evidenced that nucleoside substitution was a viable strategy to improve allobetulin/allobetulon’s antitumor activity based on our present study.

MTHFD1L knockdown diminished cells growth in papillary thyroid cancer

Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1 like (MTHFD1L) is a mitochondrial enzyme involved in the synthesis of tetrahydrofolate (THF). This study aimed to investigate the effect of MTHFD1L in papillary thyroid cancer (PTC). Tumor tissues and adjacent tissues from 11 patients with PTC were collected, the expression level of MTHFD1L mRNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The cancer genome atlas (TCGA) database was used for analysis MTHFD1L differentially expressed between tumor tissue and adjacent tissues. MTHFD1L was knocked down by a lentivirus-based system and CRISPR-Cas9. Affymetrix genechip human transcriptome array 2.0 was used to assess gene expression. Cell growth and motility were evaluated in vivo and in vitro. Cell apoptosis and cell cycle were investigated by flow cytometry assay. The expression levels of proteins were detected by western blotting. MTHFD1L mRNA and protein expression levels significantly increased in tumor tissues and CAL-62, K1 and TPC-1 cell lines. After knockdown MTHFD1L, the growth of cells were reduced while cell apoptosis was increased. In addition, tumor growth was inhibited after MTHFD1L knockdown in nude mice. Affymetrix genechip human transcriptome array 2.0 was founded that MTHFD1L knockdown can inhibit the expression levels of CCND1 and Notch2. Furthermore, we identified that MTHFD1L knockdown inhibited cells growth and induced cell apoptosis in PTC. Importantly, MTHFD1L knockdown decreased the expression levels of Notch2, Hes1 CCND1, Bcl-2, and PCNA protein, whereas the level of Bax increased. Our study suggested MTHFD1L knockdown could diminished PTC cell proliferation. MTHFD1L serves as a valuable therapeutic target.

Glaucocalyxin A Inhibits the Malignant Progression of Epithelial Ovarian Cancer by Affecting the MicroRNA-374b-5p/HMGB3/Wnt-β-Catenin Pathway Axis.

ObjectiveGlaucocalyxin A (GLA) is an ent-kaurene diterpenoid from Rabdosia japonica var possessing anti-tumor activity. This study aimed to investigate effects of GLA on epithelial ovarian cancer (EOC) and elucidate underlying mechanisms.MethodsThe expression of HMGB3 in EOC tissues was analyzed by GEPIA and immunohistochemistry. Cell proliferation was determined using CCK-8 and colony formation assays. Cell invasion, migration, and apoptosis were detected using Transwell, wound healing, and flow cytometry assays, respectively. Interactions between HMGB3 and miRNAs were predicted using ENCORI and validated using a dual-luciferase assay. mRNA expression levels of HMGB3 and miRNAs were measured using qPCR. Protein expression levels of HMGB3, E-cadherin, N-cadherin, Wnt3a,β-catenin, Bcl-2, and Bax were measured by western blotting. A tumor xenograft model was established to validate the efficacy and mechanism of GLA in vivo.ResultsHMGB3 was upregulated in EOC tissues and cells. GLA dose-dependently inhibited EOC cell proliferation and epithelial-mesenchymal transition (EMT). HMGB3 overexpression promoted proliferation, invasion, migration, and EMT, and suppressed the apoptosis of EOC cells. In addition, miR-374b-5p was targeted by HMGB3, and its overexpression hindered malignant characteristics of EOC cells. HMGB3 overexpression weakened antitumor effects of GLA and miR-374b-5p in EOC cells. Moreover, the Wnt-β-catenin pathway was inhibited by the GLA-mediated miR-374b-5p/HMGB3 axis. In vivo experiments showed that GLA inhibited EOC tumor growth, meanwhile, upregulated the miR-374b-5p level and downregulated the expression of HMGB3, Wnt3a, and β-catenin in tumor tissues.ConclusionsGLA suppressed the malignant progression of EOC by regulating the miR-374b-5p/HMGB3/Wnt-β-catenin pathway axis.

Effects of radiofrequency radiation on apoptotic and antiapoptotic factors in colorectal cancer cells

ABSTRACT In this study, it is aimed to investigate the effect of radiofrequency radiation (RFR) on apoptotic and antiapoptotic factors under different exposure conditions in human colonic adenocarcinoma cells (Caco-2). We analyzed the effects of 2.5 GHz continuous wave and 3 GPP modulated radiofrequency radiation exposure (15 min on, 15 min off) for 1 h and (1 h on, 1 h off) for 3 hours on Caco-2 cell lines. The cell viability of Caco-2 cells was determined by XTT method. Then, the cells were analyzed by flow cytometry to determine the effects on apoptosis staining with AnnexinV-FITC and PI. Protein expression levels of Bcl-2, Bax, Caspase-3 and Survivin were subsequently analyzed by using flow cytometric methods. Bax, Caspase 8, and Survivin protein levels were also analyzed by western blot. The cell viability rates were not significantly different after 2.5 GHz of RFR exposure for 1 h, but RFR exposure for 3 h at 2.5 GHz frequencies caused a decrease on cell viability of Caco-2 cells. RFR exposure for 1 and 3 hours at 2.5 GHz frequencies resulted in an apoptotic response. Protein analyses of Bcl-2, Bax, Survivin, Caspase-3, and Caspase-8 showed that RFR led to increase the levels of proapoptotic Bax, Caspase-3, and Caspase 8 in Caco-2 cells under different exposure conditions. However, 3-h exposure caused a decrease in antiapoptotic survivin levels. The results of our study indicate that RFR exposure affects the cell death mechanism due to apoptotic pathway.

Exploring the protective mechanisms of total tannins from Geum japonicum var. chinense F.Bolle in mice with hematopoietic dysfunction via the JAK2/STAT3/5 signaling pathway

Ethnopharmacological relevanceGeum japonicum var. chinense F.Bolle (Rnglish name Gei herba, GH), a traditional Miao medicine, promotes hematopoiesis. Emerging evidence shows that total tannins of GH (TGH) can treat ischemic diseases.Aim of the study: To explore the protective mechanism of TGH in hematopoietic dysfunction (HD) mice. Materials and methodForty-eight female mice were randomly assigned to 6 groups: control, model, Zhenqi Fuzheng positive, and three doses TGH. Cyclophosphamide was injected in mice to establish an HD model. Spleen tissue was examined histomorphologically, peripheral hemograms and organ index were calculated, and serum hematopoietic factor levels were determined. The expression of proteins in the Janus kinase 2 (JAK2)/transcription 3/5 (STAT3/5) pathway, as well as upstream and downstream proteins, was examined using western blot to elucidate the underlying protective mechanisms of TGH. ResultsTGH could effectively alleviate spleen tissue damage in HD mice, improve peripheral hemogram and antagonize organ atrophy, and increase levels of Granulocyte-macrophage Colony Stimulating Factor (GM-CSF) and Erythropoietin (EPO) in HD mouse serum. Furthermore, after TGH treatment, the protein expression levels of P-JAK2, P-STAT3, P-STAT5, M-CSF, G-CSF, Bcl-2, and Bcl-xL were significantly higher than in the model group. At the same time, following TGH treatment, the protein expression levels of LC3 A/B, Beclin1, ATG5, and ATG7 were significantly lower than in the model group. ConclusionsTGH has been shown to protect HD mice through a mechanism linked to the activation of the JAK2/STAT3/5 pathway, as well as autophagy inhibition and apoptosis activation.

In Vitro Differential Sensitivity of Head and Neck Squamous Cell Carcinoma to Cisplatin, Silver Nanoparticles, and Photodynamic Therapy.

The clinical effect of photodynamic therapy (PDT) may be correlated with the degree of dysplasia of cancer tissues. The aim of this study was to compare the effects of cisplatin, silver nanoparticles (AgNps), and photodynamic therapy (PDT) using methylene blue (MB) photosensitizer on Head and Neck squamous cell carcinoma - cell line (HNSCC), Hep-2, through genes expression. Hep-2 cells were divided into four groups: group I as control and without any treatment, group II and III were treated by cisplatin and AgNps, respectively, and group IV were incubated with MB for four minutes followed by PDT using laser irradiation at 650 nm for 8 minutes. The resulting toxicity was assessed in cell lines using MTT cytotoxicity assay. Further, apoptosis and the response to treatment was examined via RT-qPCR. MB-PDT inhibited the proliferation of Hep-2 cells. Following PDT, compared with AgNps cells and via MTT assay, a highly significant decrease was observed in cell proliferation in Cancer cells treated with AgNps and MB- PDT groups compared to cancer group cells and cancer cells treated with Cisplatin (p value< 0.001). Mechanistically, both the mRNA and protein expression levels of Bcl-2, Caspase-3, Cyclin-D, HIF-1, IL-8, MAPK-38, and ROS were found to be down regulated in Hep-2 cell line after MB-PDT. MB-PDT effectively killed Hep-2 cells in vitro, however, under the same conditions, the susceptibilities of the cell line to cisplatin, AgNps, and MB-PDT were different. Further studies are necessary to confirm whether this difference is present in clinical oral cancer lesions.

Effects of miR-124-3p Silencing on Neuronal Damage in the Hippocampus of Depression Rats by Regulating STAT3 Gene.

Objective A large amount of evidence shows that the abnormal expression of miRNA plays an important role in the development of depression. Therefore, we investigated the effect of miR-124-3p on neuronal damage in the hippocampus of depression rats. Methods The target genes of miR-124-3p were predicted by the database; the depression model was prepared by subcutaneous injection of corticosterone (CORT), and LV-miR-124-3p asponge lentiviral suspension was given to determine the weight of rats and open-field test, sugar preference experiment, Serum CORT, 5-HT, DA, and NE were measured, observe and record the behavior of rats, including behavior, diet, and hair. The expression of miR-124-3p, STAT3, Bcl-2, and Bax in rat hippocampus was measured. The rat hippocampal neuron cells were extracted and transfected with miR-124-3p inhibitor; the cells were cultured with CORT, and the cell survival rate was evaluated by MTT experiment, and the expressions of miR-124-3p, STAT3, Bcl-2, and Bax in the cells were detected. Luciferase reporter gene verifies the targeted regulation of miR-124-3p on STAT3. Results Compared with depression rats, silencing miR-124-3p increased the weight of the rats, increased the number of open-field activities, and significantly improved the general state and pathological state of the rats. The sugar water preference rate was significantly increased, the CORT content in the serum of rats decreased significantly, and the levels of 5-HT, DA, and NE increased significantly. After the treatment of silencing miR-124-3p, the expression level of miR-124-3p was decreased, while the STAT3 mRNA and protein expression levels were increased. And the protein and mRNA expression levels of Bcl-2 were increased, and the Bax protein and mRNA expression were decreased. Cell experiments verified that silencing miR-124-3p increased cell survival, the expression level of miR-124-3p decreased remarkably, while the expression levels of STAT3 mRNA and protein increased significantly. Silencing miR-124-3p reversed the effects of CORT treatment on miR-124-3p and STAT3 in neuronal cells. The luciferase reporter gene experiment confirmed that miR-124-3p targets and regulates STAT3 expression. Conclusion Silencing miR-124-3p may protect hippocampal neurons from damage in depression rats by upregulating STAT3 gene.

Effects of Regular and Continuous Exercise on Oxidative Stress and Apoptosis

In this study, it was aimed to investigate the effects of intense acute exercise and regular and continuous exercise on oxidative stress parameters and apoptosis-related Bax and Bcl-2 protein levels in rats. In the study, 1 control and 4 experimental groups were formed from 35 male Wistar-Albino rats, 7 in each group. The control group was not exercised, the other four groups exercised at a speed of 1.5 km/h for 1 hour on a 20 degree incline treadmill every day. Control and 1st group at the end of the 1st day, 2nd group 7th day, 3rd group 15th day and 4th group 30th day were sacrificed under anesthesia. Malondialdehyde (MDA) as an indicator of lipid peroxidation, and Glutathione (GSH), Catalase (CAT), Glutathione Peroxidase (GSH-Px) levels as an antioxidant indicator in muscle and plasma levels were measured. Bax and Bcl-2 protein expression levels were also checked for apoptosis in skeletal muscle. According to the results obtained, skeletal muscle and plasma MDA values increased after acute exercise (P&amp;lt;0.05), while skeletal muscle and plasma GSH-Px, CAT values increased significantly after continuing regular and continuous exercise (P&amp;lt;0.01). It was observed that Bax protein expression level increased, Bcl-2 protein expression level decreased and Bax/Bcl-2 ratio increased in acute exercise (p&amp;lt;0.05). As a result, it was concluded that regular and continuous exercise has a protective effect against oxidative stress and apoptosis triggered by acute exercise can be suppressed by regular and continuous exercise.

Network pharmacology identification and in Vivo validation of key pharmacological pathways of Phyllanthus reticulatus (Euphorbiaceae) leaf extract in liver cancer treatment

Ethnopharmacological relevancePhyllanthus reticulatus (Euphorbiaceae) is a medicinal plant that has been used in Zhuang medicine since ancient times. Traditionally, it has the effect of removing toxins and detumescence and can be used to treat hepatitis in China and India. Our previous studies have proved that the ethyl acetate extract of its leaves (PRPE) has an anti-hepatoma effect. Aim of the studyTo predict targets of an ethyl acetate extract of Phyllanthus reticulatus leaves (PRPE) in hepatoma treatment via network pharmacology and verify the predictions in a mouse model of liver cancer. Materials and methodsChemical constituents and therapeutic targets of P. reticulatus (PRP) were searched and predicted via public databases. A protein-protein interaction network comprising common targets was constructed, and the key gene targets were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used for biological function and pathway enrichment analyses. The effects of PRP on BEL-7404 and HepG2 cells were determined by MTT assay, apoptosis was measured by flow cytometry and hoechst44432/PI. And a nude mouse xenograft model was established to verify the anti-tumour effect in vivo. The histopathology of tumours was observed by staining with haematoxylin and eosin (H&E). Reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry were used to determine the gene and protein expression levels of phosphoinositide 3-kinase (PI3K), Akt1, p53, caspase-3, Bcl-2 and Bax, respectively. ResultsTwenty-seven chemical components and 567 potential therapeutic targets of PRP were identified. GO analysis indicated that these targets are mainly associated with peptidyl-tyrosine phosphorylation and steroid metabolic process. KEGG analysis showed that the targets are mainly located in the PI3K/Akt, apoptosis, mitogen-activated protein kinase (MAPK), Ras and vascular endothelial growth factor (VEGF) signalling pathways. According to the p-adjust value, the PI3K/Akt pathway is the core pathway. In vitro, PRPE could inhibit proliferation and induce apoptosis in hepatoma cells. IC50 values of PRPE were 2.48 and 6.34 mg/mL for BEL-7404 and hepG2 cells, respectively. PRPE significantly reduced tumour volume and weight. H&E results showed that PRPE repaired necrotic areas in hepatoma cells. PRPE reduced the protein expression of PI3K, Akt1 and Bcl-2 and increased the protein expression of p53 and Bax. Meanwhile, PRPE reduced the mRNA expression of PI3K, AKT1 and BCL2 and increased the mRNA expression of TP53, CASP3 and BAX. ConclusionThe targets of PRPE are the PI3K/Akt, apoptosis, MAPK, Ras and VEGF signalling pathways. Passing through the PI3K/Akt pathway to induce apoptosis is the main mechanism of PRPE.

Identification of Key Genes in Severe Burns by Using Weighted Gene Coexpression Network Analysis.

The aims of this work were to explore the use of weighted gene coexpression network analysis (WGCNA) for identifying the key genes in severe burns and to provide a reference for finding therapeutic targets for burn wounds. The GSE8056 dataset was selected from the gene expression database of the US National Center for Biotechnology Information for analysis, and a WGCNA network was constructed to screen differentially expressed genes (DEGs). Gene Ontology and pathway enrichment of DGEs were analyzed, and protein interaction network was constructed. A burn mouse model was constructed, and the burn tissue was taken to identify the expression levels of differentially expressed genes. The results showed that the optimal soft threshold for constructing the WGCNA network was 9. 10 coexpressed gene modules were identified, among which the green, brown, and gray modules had the largest number of burn-related genes. The DEGs were mainly related to immune cell activation, inflammatory response, and immune response, and they were enriched in PD-1/PD-L1, Toll-like receptor, p53, and nuclear factor-kappa B (NF-κB) signaling pathways. 5 DEGs were screened and identified, namely, Jun protooncogene (JUN), signal transducer and activator of transcription 1 (STAT1), BCL2 apoptosis regulator (Bcl2), matrix metallopeptidase 9 (MMP9), and Toll-like receptor 2 (TLR2). Compared with skin tissue of normal mouse, the messenger ribose nucleic acid (mRNA) and protein expression levels (PEL) of STAT1 and Bcl2 in burn tissue were greatly decreased, while those of JUN, MMP9, and TLR2 were increased obviously (p < 0.05). In conclusion, STAT1, Bcl2, JUN, MMP9, and TLR2 can be potential biological targets for the treatment of severe burn wounds.

The Combination of Salidroside and Hedysari Radix Polysaccharide Inhibits Mitochondrial Damage and Apoptosis via the PKC/ERK Pathway

Background Beta-amyloid (Aβ) peptide is a widely recognized pathological marker of Alzheimer's disease (AD). Salidroside and Hedysari Radix polysaccharide (HRP) were extracted from Chinese herb medicine Rhodiola rosea L and Hedysarum polybotrys Hand-Mazz, respectively. The neuroprotective effects and mechanisms of the combination of salidroside and Hedysari Radix polysaccharide (CSH) against Aβ25–35 induced neurotoxicity remain unclear. Objective This study aims to investigate the neuroprotective effects and pharmacological mechanisms of CSH on Aβ25–35-induced HT22 cells. Materials and Methods HT22 cells were pretreated with various concentrations of salidroside or HRP for 24 h, followed by exposed to 20 μm Aβ25–35 in the presence of salidroside or RHP for another 24 h. In a CSH protective assay, HT22 cells were pretreated with 40 μm salidroside and 20 μg/mL HRP for 24 h. The cell viability assay, cell morphology observation, determination of mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and cell apoptosis rate were performed. The mRNA expression of protein kinase C-beta (PKCβ), Bax, and Bcl-2 were measured by qRT-PCR. The protein expression levels of cleaved caspase-3, Cyt-C, PKCβ, phospho-ERK1/2, Bax, and Bcl-2 were measured by Western blot. Results CSH treatment increased cell viability, MMP, and decreased ROS generation in Aβ25–35-induced HT22 cells. PKCβ and Bcl-2 mRNA expression were elevated by CSH while Bax was decreased. CSH increased the protein expression levels of PKCβ, Bcl-2, and phospho-ERK1/2, and decreased those of Bax, Cyt-C, and cleaved caspase-3. Conclusions CSH treatment have protective effects against Aβ25–35-induced cytotoxicity through decreasing ROS levels, increasing MMP, inhibiting early apoptosis, and regulating PKC/ERK pathway in HT22 cells. CSH may be a potential therapeutic agent for treating or preventing neurodegenerative diseases.

Jujuboside A Ameliorates Myocardial Apoptosis and Inflammation in Rats with Coronary Heart Disease by Inhibiting PPAR-α Signaling Pathway.

Background Coronary heart disease (CHD) is a chronic disease caused by atherosclerosis (AS), which can cause myocardial ischemia, hypoxia, or necrosis, seriously threatening human health. There is an urgent need for effective treatments and drugs to reduce the various risk factors for coronary heart disease and relieve symptoms of angina pectoris and myocardial infarction in patients. Jujuboside A (JuA) is a triterpenoid saponin extracted from jujube seeds, which has various biological activities such as antioxidant, anti-inflammatory, antiapoptotic, and neuroprotective effects. We study the function of JuA in myocardial injury, dyslipidemia, and inflammation in the CHD rat model, to explore its potential mechanism of improving CHD. Methods A rat model of CHD was established by feeding a high-fat diet. The rats were randomly divided into 5 groups (n = 6): control group, CHD group, JuA 25 mg/kg group, JuA 50 mg/kg group, and JuA 75 mg/kg group. Echocardiography was used to detect the cardiac function parameters of rats in each group, and then, hematoxylin and eosin staining was used to assess the histopathological injury in myocardial tissues. Levels of blood lipids, myocardial injury indexes, and inflammatory factors of rats in each group were measured by biochemical tests and enzyme linked immunosorbent assay, and the levels of Bax, Bcl-2, c-caspase-3, PPAR-α, p65, p-p65, IκBα, and p-IκBα protein expression in myocardial tissues were detected by western blot. Results Compared with the CHD group, JuA therapy significantly improved injury in myocardial tissue and endothelial tissue. It also strengthened cardiac function, while decreasing total cholesterol, triacylglycerol, and low-density lipoprotein cholesterol levels in the serum and increasing high-density lipoprotein cholesterol levels. In addition, JuA also restrained cardiomyocytes apoptosis and inhibited the inflammatory reaction by reducing TNF-α, IL-1β, and IL-6 expression in myocardial tissues. Furthermore, administration of JuA inhibited the activation of PPAR-α pathway by preventing the phosphorylation of p65 and IκBα in myocardial tissues of CHD rats. Conclusion JuA may improve cardiac function, alleviate myocardial and endothelial injury, and also ameliorate dyslipidemia and inflammatory reaction in rats with CHD, where JuA probably plays a protective role by inhibiting the activation of PPAR-α pathway.

Protective effect of royal jelly on fluoride-induced nephrotoxicity in rats via the some protein biomarkers signalling pathways: a new approach for kidney damage

Introduction Protective effect of royal jelly (RJ) on fluoride-induced nephrotoxicity was investigated in this study. Methods 42 healthy male Wistar rats (n = 42, 8 weeks of age) were divided equally into 6 groups with 7 rats in each; (1) Group-1: Controls fed with standard diet; (2) Group-2: RJ [100 mg/kg] bw (body weight), by oral gavage; (3) Group-3: Fluoride [50 mg/kg] bw, in drinking water; (4) Group-4: Fluoride [100 mg/kg] bw, in drinking water; (5) Group-5: RJ [100 mg/kg] bw, by oral gavage + Fluoride [50 mg/kg] bw, in drinking water; (6) Group-6: RJ [100 mg/kg] bw, by oral gavage + Fluoride [100 mg/kg] bw, in drinking water. After 8 weeks, all rats were decapitated and their kidney tissues were removed for further analysis. The protein expression levels of caspase-3, caspase-6, caspase-9, Bcl-2, Bax, VEGF, GSK-3, BDNF, COX-2 and TNF-α proteins in kidney tissue were analysed by western blotting technique Results RJ increased Bcl-2, COX-2, GSK-3, TNF-α and VEGF protein levels and a decreased caspase-3, caspase −6, caspase-9, Bax and BDNF protein levels in fluoride-treated rats. Conclusion RJ application may have a promising therapeutical potential in the treatment of many diseases in the future by reducing kidney damage.

High CBX8 Expression Leads to Poor Prognosis in Laryngeal Squamous Cell Carcinoma by Inducing EMT by Activating the Wnt/β-Catenin Signaling Pathway.

BackgroundIn this study, we detected the expression of chromobox protein homolog 8 (CBX8) in laryngeal squamous cell carcinoma (LSCC) and its influence on the occurrence and progression of LSCC.MethodsPancancer analysis of CBX8 was analyzed by TCGA database and its expression in LSCC.The expression of CBX8 in 30 pairs of LSCC and adjacent tissues was analyzed by quantitative real-time PCR(qRT-PCR)and immunohistochemical assays, and its association with the prognosis and clinicopathological features of LSCC was further evaluated. A CBX8 knockdown model was constructed in AMC-HN-8 and Hep2 cell lines. The effects of CBX8 on LSCC cell proliferation, migration, invasion and apoptosis were detected by CCK8,EdU,wound healing, Transwell and flow cytometry assays. Levels of apoptosis-related protein, WNT/β-catenin signaling pathway and epithelial to mesenchymal transition (EMT) proteins, including Bax, Bcl2, β-catenin, DKK1, GSK3β, N-cadherin, E-cadherin and Snail1, in LSCC cells were detected by Western blotting.ResultsCBX8 was overexpressed in LSCC. High expression of CBX8 in LSCC patients led to shorter overall survival and correlated with tumor stage and lymphatic metastasis. After CBX8 knockdown, the proliferation of AMC-HN-8 and Hep2 cells slowed, and the number of EdU-positive cells decreased. Wound healing slowed down, and the number of Transwell invading cells decreased. The percentage of apoptotic cells increased. The expression levels of Bcl2, β-catenin, N-cadherin and Snail11 proteins were significantly reduced in the CBX8 knockdown cells, while Bax, DKK1, GSK3β and E-cadherin significantly increased with their corresponding controls.ConclusionCBX8 is highly expressed in LSCC and induces the EMT process by activating the Wnt/β-catenin signaling pathway to promote LSCC cell proliferation and migration and inhibit apoptosis, resulting in poor prognosis.