Mechanism of Fufang Congrong Yizhi Capsules in treatment of mild cognitive impairment based on network pharmacology / 中国药理学通报

Aim To predict the mechanism of Fufang Congrong Yizhi Capsules (FCYC) in the treatment of mild cognitive impairment (MCI) by network pharmacology method, and further validate it in combination with cellular experiments. Methods TCMSP, Gene-Cards, OMIM and TTD databases, Chinese Pharmacopoeia and related literature were used to screen the active ingredients of FCYC and the targets of MCI treatment. The TCM-compound-target-disease network and PPI of intersection targets were constructed, and the GO and KEGG analysis were performed by the Ehamb bioinformation platform. GO and KEGG analysis were performed through Yihanbo biological information platform. Cell model of MCI was established by PC-12 injury induced by Aβ

Salvianolic Acid F Regulates Bax/Caspase-3/GSDME Signaling Pathway to Inhibit Pyroptosis of HK-2 Cells / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of salvianolic acid F （Sal F） in repairing the high glucose-induced injury in human kidney-2 （HK-2） cells via the B-cell lymphoma-2 （Bcl-2）-associated X protein （Bax）/cysteinyl aspartate-specific proteinase 3 （Caspase-3）/gasdermin-E （GSDME） pathway. MethodThe cell counting kit-8 （CCK-8） was used to measure the relative viability of HK-2 cells exposed to high glucose and different concentrations （2.5， 5， 10， 20 μmol·L-1） of Sal F and the relative viability of HK-2 cells treated with Sal F for different time periods. The levels of lactate dehydrogenase （LDH） and interleukin-1β （IL-1β） in the supernatant of the cell culture were measured by the LDH assay kit and enzyme-linked immunosorbent assay （ELISA） kit， respectively. Flow cytometry combined with Annexin V-FITC/propidium iodide （PI） and Hoechst 33342/PI staining was employed to reveal the proportion of PI-positive HK-2 cells exposed to high glucose. Western blotting was employed to determine the protein levels of Bax， Bcl-2， cytochrome C， cysteinyl aspartate-specific proteinase （Caspase）-9， Caspase-3， and GSDME in the HK-2 cells exposed to high glucose and treated with Sal F. The 2，7-dichlorodihydrofluorescein diacetate fluorescence probe （DCFH-DA） and mitochondrial membrane potential assay kit （JC-1） were used to determine the production of reactive oxygen species （ROS） and the mitochondrial membrane potential in the HK-2 cells exposed to high glucose and treated with Sal F. ResultCompared with the blank group， the model group showed decreased cell viability （P<0.01）， elevated levels LDH and IL-1β， increased proportion of PI-positive cells （P<0.01）， up-regulated protein levels of Bax， cytochrome C， Caspase-9， Caspase-3， and GSDME （P<0.01）， down-regulated protein level of Bcl-2 （P<0.01）， decreased mitochondrial membrane potential， and excessive ROS accumulation. Compared with the model group， Sal F repaired the high glucose-induced injury in HK-2 cells （P<0.05）， lowered the levels of LDH and IL-1β （P<0.05， P<0.01）， and decreased the proportion of PI-positive cells （P<0.01）. In addition， Sal F down-regulated the protein levels of Bax， cytochrome C， Caspase-9， Caspase-3， and GSDME and up-regulated the protein level of Bcl-2 （P<0.05， P<0.01）， increased the mitochondrial membrane potential， and decreased the accumulation of ROS in HK-2 cells. ConclusionSal F can reduce the production of ROS， restore the balance of mitochondrial membrane potential， and inhibit pyroptosis via the Bax/Caspase-3/GSDME signaling pathway to repair the high glucose-induced injury in HK-2 cells.

Análise da imunoexpressão de BAX e MMP-9 no líquen plano oral / Analysis of BAX and MMP-9 immunoexpression in oral lichen planus

Introdução: O líquen plano oral é uma doença crônica imunologicamente mediada relativamente comum, que acomete a mucosa oral. Clinicamente, o LPO é classificado em seis padrões bem identificados: placa, reticular, bolhoso, atrófico, papular e erosivo.Sendo os mais comuns oos tipos reticulares e erosivos. A ativação dos linfócitos TCD4+ no LPO, pode induzir os ceratinócitos ao processo de apoptose através da respostaimunológica citotóxica. A proteína Bax desempenha uma função relevante para o processo apoptótico. Deste modo, a presente pesquisa consistiu em um estudo transversal retrospectivo, descritivo, quantitativo e comparativo. Objetivo: Avaliar a expressão imuno-histoquímica das proteínas MMP9 e Bax no LPO. Método: Foram utilizados 43 casos de LPO para análise da imunoexpressão de Bax e MMP-9. Os resultados foram analisados através dos testes estatísticos apropriados e serão considerados significativos, valores onde p<0,05. Resultado: A imunoexpressão de MMP9 foi significativamente maior nos ceratinócitos e quando analisados os subtipos de líquen plano oral, não foram observados diferenças estatísticas entre os tipos reticulares e erosivos para as proteínas analisadas. Conclusões: Com essas observações, infere-se que a alteração na expressão das proteínas estudadas sugere um distúrbio nos mecanismos apoptóticos, os quais estão associados às lesões de LPO, e podemos concluir também que as imunoexpressões dessas proteínas não apresentaram diferença, quando relacionada ao tipo clínico reticular ou erosivo. Com esse resultado pode-se contribuir para um maior entendimento sobre os possíveis mecanismos celulares envolvidos na etiopatogenia dessa lesão (AU).

Background: Oral lichen planus is a relatively common immune-mediated chronic disease that affects the oral mucosa. Clinically, OLP is classified into six well-identified patterns: plaque, reticular, bullous, atrophic, papular, and erosive. The most common being the reticular and erosive types. The activation of TCD4+ lymphocytes in the OLP can induce keratinocytes to the process of apoptosis through the cytotoxic immune response. Thus, the present research consisted of a retrospective, descriptive, quantitative and comparative crosssectional study. Objective: to evaluate the immunohistochemical expression of MMP-9 and Bax proteins in OLP. Methods: We used 20 cases of Inflammatory Fibrous Hyperplasia as control. The results were analyzed through the appropriate statistical tests and will be considered significant, values where p<0.05. Results: The immunoexpression of MMP-9 was significantly higher in keratinocytes and when the subtypes of oral lichen planus were analyzed, no statistical differences were observed between the reticular and erosive types for the proteins analyzed. Conclusions: With these observations, it is inferred that the alteration in the expression of the studied proteins suggests a disturbance in the proliferative and apoptotic mechanisms, which are associated with a pathological behavior of the oral mucosa, and consequently with a repercussion on the lesions of OLP, and we can also conclude that the immunoexpression of these proteins had no difference, when related to the reticular or erosive clinical type. This research aims to contribute to a greater understanding of the possible cellular mechanisms involved in the etiopathogenesis of this lesion, thus enabling the understanding of the clinical aspects of the pathology (AU).

Research progress on islet protection and functional maintenance / 器官移植

Islet transplantation is a promising treatment of diabetes mellitus and its complications. Nevertheless, dysfunction post-transplantation, rejection and shortage of donors are the bottleneck issues in the field of islet transplantation. Optimizing the preservation method of pancreas plays a positive role in obtaining a sufficient quantity of effective islets and maintaining their functions. During the culture stage, anti-rejection and anti-apoptosis treatment of islets, including mesenchymal stem cell (MSC), MSC-derived exosomes, anti-apoptosis drugs and gene modification, may become major approaches for islet protection and functional maintenance in clinical islet transplantation. Use of anti-instant blood-mediated inflammatory reaction (IBMIR) drugs after islet transplantation also plays a critical role in protecting islet function. In this article, the whole process from islet preparation to islet transplantation was illustrated, and relevant strategies of islet protection and functional maintenance were reviewed, aiming to provide reference for improving the quality of donors to compensate for the shortage of absolute quantity of donors and elevating the efficiency of islet transplantation.

Effect of Bushen Huoxuetang on Expression of Bax/Bcl-2 and Cleaved Caspase-3 in Nude Mice with Bone Metastasis of Breast Cancer / 中国实验方剂学杂志

ObjectiveTo study the effect of Bushen Huoxuetang on the apoptosis and the expression of B-cell lymphoma （Bcl-2）-associated X protein （Bax）/ Bcl-2 and cleaved cysteine-containing aspartate proteolytic enzyme-3 （cleaved Caspase-3） in the nude mouse model of bone metastasis of breast cancer， and explore the mechanism of Bushen Huoxuetang in inhibiting bone destruction. MethodThirty BALB/c female nude mice were randomly assigned into blank group （n=6） and model group （n=24）. The suspension of 4T1 breast cancer cells was injected into the tibia of mouse right lower limb to establish model of bone metastasis of breast cancer. The successfully modeled nude mice were randomly assigned into model group， Bushen Huoxuetang group， zoledronic acid group， and combined drug group， with 6 mice in each group. Bushen Huoxuetang was administrated at a dose of 36.67 g·kg-1， once a day， and zoledronic acid was administrated by subcutaneous injection at a dose of 100 μg·kg-1， twice a week. The combined drug group was administrated with the same doses of Bushen Huoxuetang group by gavage and zoledronic acid by subcutaneous injection. The mice in the blank group and the model group were administrated with the same volume of distilled water by gavage for 14 days. On the next day at the end of drug administration， the mice were sacrificed by cervical dislocation. The general situation and weight changes of the mice were examined. The right lower limb was collected， and X-ray scanning and hematoxylin-eosin （HE） staining methods were used for observation of pathological changes in the bone. The terminal deoxynucleotidyl transferase dUTP nick-end labeling （TUNEL） was employed to detect the apoptosis of bone tissue in nude mice， and Western blot to determine the expression of Bax/Bcl-2 and cleaved Caspase-3 in the bone tissue. ResultCompared with the blank group， the modeling reduced the body weight （P<0.01） and increased the right lower limb weight of the nude mice （P<0.01）. Compared with the model group， Bushen Huoxuetang， zoledronic acid， and their combination increased the body weight （P<0.01） and decreased the right lower limb weight （P<0.01）. Compared with the blank group， the other groups showed obvious tumor cell atypia， deep nuclear staining， and clear bone metastasis， and the model group showed obvious osteolytic damage in right lower limb and loss of proximal tibia and knee joint. Compared with the model group， Bushen Huoxuetang， zoledronic acid， and their combination reduced the osteolytic lesions in the right lower limb and recovered part of the bone structure， demonstrating an inhibitory effect on bone destruction. The TUNEL assay showed that the model group had lower apoptosis rate of bone metastatic tumor cells than the blank group， Bushen Huoxuetang group， zoledronic acid group， and combined drug group （P<0.01）. Compared with the blank group， the modeling down-regulated the expression of Bax and cleaved Caspase-3 （P<0.01） and up-regulated the expression of Bcl-2 （P<0.01）. Compared with the model group， Bushen Huoxuetang， zoledronic acid， and their combination up-regulated the expression of Bax （P<0.01） and cleaved Caspase-3 （P<0.05， P<0.01） and down-regulated the expression of Bcl-2 （P<0.05， P<0.01）. ConclusionBushen Huoxuetang may inhibit bone destruction in the nude mouse model of bone metastasis of breast cancer by up-regulating the expression of Bax， down-regulating the expression of Bcl-2， activating cleaved Caspase-3， and further inducing apoptosis.

Geju Hugan Tablets Ameliorate Alcohol-induced Liver Injury in Mice by Regulating NF-κB and Bcl-2/Bax Signaling Pathways / 中国实验方剂学杂志

ObjectiveTo investigate the protective effect of Geju Hugan tablets on the liver of mice with alcohol-induced liver injury， and explore the underlying mechanism based on nuclear factor-κB p65 （NF-κB p65） and B-cell lymphoma-2 （Bcl-2）/Bcl-2-associated X protein （Bax） signaling pathways. MethodAccording to the body weight， 60 SPF-grade male ICR mice were randomized into normal， model， Compound Yiganling tablets （0.16 g·kg-1）， and low-， medium-， and high-dose （0.2， 0.4， 0.8 g·kg-1， respectively） Geju Hugan tablets groups. The drugs were administrated at the corresponding doses by gavage， and the normal and model groups with equal volume of pure water once a day for 28 consecutive days. On day 29， the mice in other groups except the normal group were administrated with liquor （53% Vol） by gavage twice a day at the doses of 20， 10 mL·kg-1 and with the interval of 6 h. Samples were harvested on day 30. The histopathological changes in the liver were observed by hematoxylin-eosin （HE） staining， and the ultrastructural changes in hepatocytes were observed by transmission electron microscopy. The enzyme-linked immunosorbent assay was employed to measure the levels of malonaldehyde （MDA）， reduced glutathione （GSH）， and triglycerides （TG） in the liver tissue and the levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in the serum. Western blotting was employed to determine the protein levels of NF-κB p65， phosphorylated p-inhibitor kappa B alpha （p-IκBα）， Bcl-2， and Bax in the liver tissue. ResultCompared with the normal group， the model group showed increases in the ALT， AST， MDA， and TG levels， a decrease in the GSH level， and increases in the liver injury scores evaluated based on the HE， oil red O， and transmission electron microscopy （P<0.01）. Moreover， the model group showed up-regulated expression of NF-κB， p-IκBα， and Bax （P<0.05， P<0.01） and down-regulated expression of Bcl-2 （P<0.05） in the liver tissue. Compared with the model group， Geju Hugan tablets of all the doses lowered the ALT， AST， MDA， and TG levels and elevated the GSH level （P<0.01）. The liver injury scores assessed based on HE staining and transmission electron microscopy in the medium- and high-dose Geju Hugan tablets groups were lower than those in the model group （P<0.01）. Compared with the model group， medium- and high-dose Geju Hugan tablets down-regulated the protein levels of NF-κB， p-IκBα， and Bax （P<0.01） and all doses of Geju Hugan tablets up-regulated the protein level of Bcl-2 （P<0.01）. ConclusionGeju Hugan tablets protect mice from alcohol-induced liver injury by down-regulating NF-κB signaling pathway to alleviate inflammation in the liver tissue and down-regulating the expression of Bax and up-regulating the expression of Bcl-2 to inhibit hepatocyte apoptosis.

Study on mechanism of inhibition effect of N 6-methyladenosine methyltransferase-like 3 on vascular calcification in chronic kidney disease through Bax/Bcl-2 / 中华肾脏病杂志

Objective:To investigate the role and mechanism of N 6-methyladenosine (m 6A) methyltransferase-like 3 (METTL3) in vascular calcification (VC) of chronic kidney disease (CKD) through apoptosis-associated protein. Methods:(1) Real-time fluorescence quantitative PCR was used to test METTL3 mRNA in serum of maintenance hemodialysis (MHD) patients. (2) Western blotting was used to detect the expression of METTL3 protein in high-phosphorus stimulated vascular smooth muscle cells (VSMCs), and immunofluorescence double lable was used to observe the distribution of METTL3 and Runt-related transcription factor 2 (Runx2). The METTL3 overexpressed and knockdown plasmids were constructed and transfected into VSMCs. Alizarin red staining was used to detect calcification degree. Western blotting was used to detect the expressions of osteogenic markers [Runx2, bone morphogenetic protein-2(BMP-2), collagen Ⅰ] and apoptosis- related proteins Bax and Bcl-2. (3) SD rats were randomly divided into control group, CKD-VC group and S-adenosylhomocysteine (SAH) intervention group. The calcification of thoracic aorta was evaluated by von Kossa staining, and the protein expressions of Runx2, Bax and Bcl-2 were detected by immunohistochemistry and Western blotting.Results:(1) METTL3 mRNA expression in MHD patients with VC was significantly lower than that in non-VC patients ( P<0.05), and was negatively correlated with coronary artery calcium score ( r=-0.65, P<0.001). (2) The expression of METTL3 in VSMCs stimulated by high phosphorus was decreased and showed a time dependence. Immunofluorescence double label showed that METTL3 and Runx2 were co-expressed in the nucleus. METTL3 was overexpressed in high-phosphorus induced VSMCs, and the expressions of Runx2, collagen I and BMP-2 were significantly decreased, accompanied by the decrease of calcified nodules and Bax/Bcl-2 ratio (all P<0.05). Conversely, METTL3 knockdown aggravated VSMCs calcification by inducing apoptosis. (3) Furthermore, METTL3 inhibitor SAH was administered in vivo, and it was found that inhibition of METTL3 expression significantly increased the calcification of rat thoracic aorta, and the Bax/Bcl-2 ratio and Runx2 expression were up-regulated. Conclusions:Serum METTL3 level is reduced in MHD patients with VC. In vivo and in vitro studies demonstrate that METTL3 inhibits VC in CKD by mediating the apoptosis-related protein Bax/Bcl-2.

Gabapentin effectively alleviates pain sensitization and regulates CACNA2D1 expression in postherpetic neuralgia rats / 中国药理学通报

Aim To identify the molecular target of gabapentin in the treatment of postherpetic neuralgia(PHN). Methods The molecular target of gabapentin for PHN was analyzed by network pharmacology and molecular docking and confirmed by coprecipitation test. Rats were randomly divided into control group, model group, model+50 mg·kg-1 gabapentin group, model+100 mg·kg-1 gabapentin group, and model+200 mg·kg-1 gabapentin group, with nine rats in each group. The pain-related behaviors of the rats were measured at different time points. The mRNA and protein expressions of CACNA2D1, Bax, and Bcl-2 in rat spinal cord were determined by immunofluorescence, Western blot, and qPCR. Results CACNA2D1 was the target gene of gabapentin that determined via network pharmacology, molecular docking, and co-precipitation tests. After modeling, mechanical pain threshold and thermal pain threshold significantly decreased, and the number of apoptotic GABA cells significantly increased. However, after intraperitoneal injection of 50, 100, and 200 mg·kg-1 gabapentin, mechanical pain threshold and thermal pain threshold significantly increased(P<0.05), and the number of apoptotic GABA cells significantly decreased(P<0.01). Immunofluorescence and Western blot results showed that compared with the model group, with the increase of gabapentin concentration, the positive expression rate of Bax significantly decreased, and the positive expression rate of Bcl-2 and CACNA2D1 significantly increased. The mRNA expression levels of Bax, Bcl-2 and CACNA2D1 detected by qPCR were consistent with the results of immunofluorescence and Western blot. Conclusions Gabapentin up-regulates the expression of target protein CACNA2D1, inhibits the proapoptotic protein Bax, and promotes the expression of apoptotic inhibitor Bcl-2.

The protective effect of trigonelline on H / 中国药理学通报

Aim To study the protective effect of trigonelline on H

Mechanism of action of Shen-Fu decoction on cardiogenic shock based on network pharmacology and experimental validation / 中国药理学通报

Aim To investigate the mechanism of action of Shen-Fu decoction in the prevention and treatment of cardiogenic shock based on network pharmacology and animal experiments. Methods The relevant targets and signaling pathways of cardiogenic shock of Shen-Fu decoction were predicted by network pharmacology, and a cardiogenic shock rat model was created by coronary artery ligation. Before modeling, rats were given the appropriate dose of Shen-Fu decoction or saline by gavage for 14 days according to the group, and real-time mean arterial pressure (MAP) changes were recorded after successful modeling. HE method was used to detect the myocardial histopathological changes of cardiogenic shock. TUNEL method was employed to detect rat myocardial cell apoptosis, and Western blotting was applied to determine the expression levels of rat myocardial Bax, Bcl-2, caspase-3, cleaved caspase-3 proteins. Results A total of 51 potential active ingredients of Shen-Fu decoction were screened out by network pharmacology, 80 targets of co-action with cardiogenic shock, and 43 core targets of close relationship between proteins, and GO enrichment analysis revealed that the core proteins were involved in the biology process (BP), mainly involving positive regulation of apoptotic process. KEGG enrichment analysis showed signaling pathways involving atherosclerosis-related, apoptosis and other signaling pathways. The results of animal model validation showed that Shen-Fu decoction could increase the shock blood pressure of rats with cardiogenic shock and alleviate the pathological changes of myocardial tissue, reduce the degree of apoptosis of rat cardiomyocytes, reduce the expression level of caspase-3, cleaved caspase-3 and Bax protein in rat myocardial tissue, and improve the expression level of Bcl-2 protein in myocardial tissue of rats. Conclusions The potential active ingredient of Shen-Fu decoction may play a role in the prevention and control of cardiogenic shock rats by acting on the target Bax, Bcl-2 to regulate the apoptosis signaling pathway of cardiomyocytes.

Effect of SMAC Gene on Sensitivity of Lung Adenocarcinoma Cells to Paclitaxel and Cell Viability Based on caspase-3/Bcl-2/Bax Signaling Pathway / 肿瘤防治研究

Objective To investigate the effect of the SMAC gene on paclitaxel sensitivity and cellular activity in lung adenocarcinoma cells based on the caspase-3/Bcl-2/Bax signaling pathway. Methods A paclitaxel-resistant cell line A549/Taxol was established for lung adenocarcinoma, and the cells were divided into four following groups: pcDNA-NC (transfected with pcDNA-NC blank vector), pcDNA-SMAC (transfected with pcDNA-SMAC vector), siRNA-NC (transfected with siRNA-NC empty virus vector), and siRNA-SMAC groups (transfected with siRNA-SMAC lentiviral vector). The SMAC mRNA expression in cells was detected by qRT-PCR; cell sensitivity was detected by MTT; cell proliferation ability was detected by cloning assay; cell invasion ability was detected by Transwell; apoptosis ability was detected by flow cytometry assay; and caspase-3, Bcl-2 and Bax protein expression in cells were detected by Western blot analysis. Results The SMAC mRNA expression was significantly lower in A549 cells compared with BEAS-2B cells (P < 0.05). The SMAC mRNA expression was significantly higher in the pcDNA-SMAC group than that in the pcDNA-NC group cells (P < 0.05). The SMAC mRNA expression was significantly lower in the cells of the siRNA-SMAC group (P < 0.05) than that in the siRNA-NC group. The SMAC mRNA expression was significantly lower in the cells of the siRNA-SMAC group (P < 0.05) than in the siRNA-NC group. Compared with the pcDNA-NC group, the cell IC50, cell clone number, cell invasion ability, and Bcl-2 protein and Bcl-2/Bax ratio were significantly lower in the pcDNA-SMAC group, the cell resistance index reversal was 2.51-fold, and the apoptosis ability and caspase-3, as well as Bax protein expression, were significantly higher (P < 0.05). Compared with the siRNA-NC group, cell IC50, cell clone number, cell invasion ability, and Bcl-2 protein and Bcl-2/Bax ratio were significantly higher in the siRNA-SMAC group, and apoptosis ability and caspase-3 and Bax protein expression were significantly lower (P < 0.05). Conclusion High expression of SMAC increases paclitaxel sensitivity, inhibits cell growth and invasion, promotes apoptosis in lung adenocarcinoma cells, and has a regulatory effect on the caspase-3/Bcl-2/Bax signaling pathway.

Mechanism of Ethyl Acetate Extract of Gentianopsis paludosa in Treatment of Ulcerative Colitis Based on Bcl-2/Bax Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of ethyl acetate extract of Tibetan medicine dampness bud Gentianopsis paludosa in the prevention and treatment of recurrent ulcerative colitis （UC） in rats with dampness-heat in large intestine syndrome based on the apoptotic pathway mediated by the B-cell lymphoma-2 （Bcl-2） and Bcl-2-associated X protein （Bax）. MethodUsing the disease-syndrome combination method， a recurrent UC model of dampness-heat in large intestine syndrome was constructed in rats. Seventy SPF-grade male SD rats were randomly divided into control group， model group， high-， medium-， and low-dose ethyl acetate of G.paludosa groups （150， 75， 37.5 mg·kg-1）， and mesalazine group （135 mg·kg-1）. The rats were orally administered with respective drugs for 14 days. The general conditions of the rats were recorded， and colon length and mucosal damage were observed. The colon wet weight index and organ coefficients of the liver， spleen， and thymus were calculated. Enzyme-linked immunosorbent assay （ELISA） was used to measure the levels of interleukin-6 （IL-6） and interleukin-1β （IL-1β） in the serum of each group. Hematoxylin-eosin （HE） staining was performed to observe pathological changes in the colon. Terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） was used to detect apoptosis in colonic epithelial cells. Western blot was used to measure the expression levels of Bcl-2， Bax， Caspase-3， Caspase-9， Zona Occludens-1 （ZO-1）， Claudin3， and Occludin in colonic tissue. Immunohistochemistry （IHC） was used to observe the expression of Bax and Caspase-3 in colonic epithelial cells. ResultCompared with the control group， the model group showed significant increases in the disease activity index （DAI） score， colonic mucosal damage index （CMDI）， intestinal epithelial apoptosis， liver and spleen indexes， and levels of inflammatory factors IL-1β and IL-6 in the serum （P<0.01）， decreased expression of intestinal mucosal protective proteins ZO-1， Claudin3， and Occluding （P<0.01）， increased expression of pro-apoptotic proteins Bax， Caspase-3， and Caspase-9 （P<0.01）， and decreased expression of anti-apoptotic protein Bcl-2 （P<0.01）. Compared with the model group， the high-， medium-， and low-dose ethyl acetate of G.paludosa groups all significantly improved the general condition of the rats， reduced colonic lesions， decreased intestinal epithelial cell apoptosis， reduced liver and spleen indexes， upregulated the expression of ZO-1， Claudin3， Occludin， and Bcl-2 proteins， and downregulated the expression of Bax， Caspase-3， and Caspase-9 proteins， with the high- and medium-dose ethyl acetate of G.paludosa groups showing the superior effects （P<0.05， P<0.01）. ConclusionEthyl acetate of G.paludosa can alleviate colonic mucosal damage and exert a therapeutic effect on UC by regulating the Bcl-2/Bax signaling pathway.

Capacidad pro-apoptótica in vitro de Valeriana rígida y Valeriana decusata sobre una línea celular de cáncer de mama / In vitro pro-apoptotic capacity of Valerian rigid and Valerian decussata in cancer cells

Resumen El cáncer representa un problema de salud pública a nivel mundial, con altas tasas de incidencia y mortalidad en países desarrollados y no desarrollados. En la actualidad se están evaluando alternativas terapéuticas de origen natural, con el propósito de establecer tratamientos más eficientes y menos invasivos. Dado que la apoptosis es el tipo de muerte programada que experimentan las células cancerosas por los tratamientos con los fármacos antineoplásicos,el objetivo de esta investigación, fue evaluar in vitro la capacidad pro-apoptótica y citotóxica de los extractos de valeriana, sobre una línea celular de cáncer de mama (MCF-7). En este estudio las células MCF7 se cultivaron y trataron con diferentes concentraciones de los extractos de la raíz, hojas y tallos de Valeriana rígida y Valeriana decussata. La viabilidad celular se evaluó mediante el ensayo MTT. Para la determinación de la expresión génica de las proteínas anti y pro-apoptóticas (Bax, Bcl-2 y p53), se usó el ensayo de la PCR cuantitativa de transcripción inversa. Las diferentes concentraciones de los extractos (10-8 a 10-1 mg/mL) disminuyeron la viabilidad (proliferación) celular en concentraciones dependientes. Estos extractos indujeron la expresión génica de las proteínas Bax y Bcl-2, pero no de p53. La expresión de Bax fue mayor que la de Bcl-2 e indujo un elevado índice Bax/Bcl-2 (condición proapoptotica). En conclusión, se determinó que los extractos de Valeriana decussata y Valeriana rígida poseen efecto reductor de la viabilidad (proliferación) de la línea celular de cáncer de mama MCF-7, probablemente mediado por la alteración de la relación de las proteínas Bax y Bcl-2 vinculadas a la apoptosis.

Abstract Cancer represents a worldwide public health problem, with high incidence and mortality rates in developed and undeveloped countries. Currently, therapeutic alternatives of natural origin are being evaluated with the purpose of establishing more efficient and less invasive treatments. Apoptosis is the type of programmed death cancer cells undergo during treatment with anti-neoplastic drugs. Therefore, the aim of this research was to evaluate in vitro the pro-apoptotic and cytotoxic capacity of valerian extracts on a breast cancer cell line (MCF-7). In this study, MCF7 cells were cultured and treated with different concentrations of the extracts of the root, leaves and stems of Valeriana rígida and Valeriana decussata. Cell viability was assessed by the MTT assay. Quantitative reverse transcription PCR assays were used for the determination of gene expression of anti- and proapoptotic proteins (Bax, Bcl-2, p53). Different concentrations of the extracts (10-8 to 10-1 mg/mL) decreased cell viability (proliferation) in a concentration-dependent manner. These extracts induced gene expression of Bax and Bcl-2 proteins but not of p53. The expression of Bax was higher than that of Bcl-2, causing an elevated Bax/Bcl-2 ratio (proapoptotic condition). In conclusion, it was determined that Valeriana decussata and Valeriana rígida extracts have a viability (proliferation) reducing effect on the MCF-7 breast cancer cell line, probably mediated by altering the ratio of Bax and Bcl-2 proteins linked to apoptosis.

Study on the apoptosis of human retinal pigment epithelium cells induced by EGCG / 国际眼科杂志(Guoji Yanke Zazhi)

AIM:To investigate the effect of epigallocatechin gallate(EGCG)on the apoptosis of human retinal pigment epithelium(ARPE-19)cells and its mechanism. METHODS:The ARPE-19 cells were cultured in vitro and treated with 0,40,80 and 160 μg/mL EGCG, respectively. At the proposed time of treatment the morphological changes were detected by hoechst 33258 staining. The apoptosis rate was detected by flow cytometry. The expression of apoptosis-related factors B lymphocytoma-2 gene(bcl-2), BCL2-Associated X protein(Bax),caspase-3 and p53 were detected by quantitative RT-PCR and Western blotting.RESULTS: Hoechst 33258 staining showed that the ARPE-19 cells with the increase of EGCG drug concentration, the number of apoptotic cells gradually increased and the apoptotic bodies were observed. Flow cytometry showed that the apoptosis rate increased gradually with the increase of EGCG drug concentration. The apoptosis rates at 40, 80 and 160 μg/mL were 4.95%±0.071%, 11.75%±0.075% and 21.25%±0.919% respectively, which was significantly different compared with the control group(2.8%±1.556%)(P<0.01), presented with a drug concentration-dependent. The results of quantitative PCR and Western blotting showed that EGCG could significantly up-regulate the expression of apoptosis-promoting factors Bax, caspase-3 and the mRNA and protein expression of p53, and down-regulate the apoptosis-inhibiting factor bcl-2, all of these showed concentration-dependent effects.CONCLUSION:EGCG can obviously induce the apoptosis of ARPE-19 cells. The mechanism is related with the inhibition of bcl-2 and increase the expression of Bax, caspase-3 and p53.

Bax inhibitor 1 inhibits vascular calcification in mice by activating optic atrophy 1 expression / 南方医科大学学报

OBJECTIVE@#To investigate the effects of Bax inhibitor 1 (BI- 1) and optic atrophy protein 1 (OPA1) on vascular calcification (VC).@*METHODS@#Mouse models of VC were established in ApoE-deficient (ApoE-/-) diabetic mice by high-fat diet feeding for 12 weeks followed by intraperitoneal injections with Nε-carboxymethyl-lysine for 16 weeks. ApoE-/- mice (control group), ApoE-/- diabetic mice (VC group), ApoE-/- diabetic mice with BI-1 overexpression (VC + BI-1TG group), and ApoE-/- diabetic mice with BI-1 overexpression and OPA1 knockout (VC+BI-1TG+OPA1-/- group) were obtained for examination of the degree of aortic calcification using von Kossa staining. The changes in calcium content in the aorta were analyzed using ELISA. The expressions of Runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 2 (BMP-2) were detected using immunohistochemistry, and the expression of cleaved caspase-3 was determined using Western blotting. Cultured mouse aortic smooth muscle cells were treated with 10 mmol/L β-glycerophosphate for 14 days to induce calcification, and the changes in BI-1 and OPA1 protein expressions were examined using Western blotting and cell apoptosis was detected using TUNEL staining.@*RESULTS@#ApoE-/- mice with VC showed significantly decreased expressions of BI-1 and OPA1 proteins in the aorta (P=0.0044) with obviously increased calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 (P= 0.0041). Overexpression of BI-1 significantly promoted OPA1 protein expression and reduced calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 (P=0.0006). OPA1 knockdown significantly increased calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 in the aorta (P=0.0007).@*CONCLUSION@#BI-1 inhibits VC possibly by promoting the expression of OPA1, reducing calcium deposition and inhibiting osteogenic differentiation and apoptosis of the vascular smooth muscle cells.

Effect of Hypoxia Inducible Factor-1α on Chemosensitivity of B-ALL Cells to Vincristine and Its Mechanism / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of hypoxia on the chemosensitivity of B-acute lymphoblastic leukemia （B-ALL） cells to Vincristine (VCR) and the mechanisms.@*METHODS@#B-ALL cells SUP-B15, Nalm-6 and RS4;11 were selected as the research objects. The cells were divided into the control group and the hypoxia mimic group (CoCl2 pretreatment). The two groups were treated with VCR at different concentrations for 24 hours, CCK-8 was used to detect cell viability, flow cytometry was used to detect cell apoptosis, and Western bolt method was used to detect hypoxia inducible factor (HIF-1α), BAX, Bcl-2 and β-actin protein expression. Quantitative real-time fluorescent PCR (qRT-PCR) was used to detect BAX and β-actin mRNA levels.@*RESULTS@#CoCl2 could simulate hypoxic environment to induce the expression of HIF-1α. The cells SUP-B15 and RS4;11 of the hypoxia mimic group were lower sensitivity to VCR as compared with the control group; the apoptosis rate of the hypoxia mimic group was lower than that of the control group after 80 nmol/L VCR treatment. The expression levels of BAX protein and mRNA in the hypoxia mimic group were lower than those of the control group, and there was no significant difference in the expression levels of Bcl-2 protein between two groups.@*CONCLUSION@#Under hypoxic conditions, HIF-1α may mediate VCR resistance in B-ALL cells by downregulating the pro-apoptotic protein BAX.

Potentiality of curcumin on ISHAK scoring system and the expressions of BAX, IL-17A, and EGF in the treatment of Schistosoma mansoni infection using Swiss albino mice

@#The present study evaluated the antiparasitic effect of curcumin extract on Schistosoma mansoni in Swiss albino mice. The experimental design included four groups of S. mansoniinfected mice; without treatment (controls), curcumin-treated, Praziquantel (PZQ)-treated, and PZQ +curcumin treated mice. The results showed that curcumin improved ISHAK confluent necrosis score up to zero. PZQ +curcumin showed a significant reduction in portal inflammation. Both activity and fibrosis demonstrated lower scores in all treated groups, however, PZQ revealed a marked increase in confluent necrosis and interface hepatitis. Besides, the lobular inflammation revealed worsening in the overall ISHAK score in all treated groups compared with the control. Few periocular granulomas were recovered by PZQ +curcumin treatment at day 35 post-treatment (6±1.2), P-value <0.05. Curcumin revealed a mild reduction (60±7.376). Curcumin-treated groups, with and without PZQ, resulted in higher significant Immunoreactivity score (IRS) for Bcl-2-associated X (BAX) and lower Interleukine17A (IL-17A), and Human epidermal growth factor (EGF), compared to the control. However, PZQ revealed a lower mean IRS value in BAX, higher IL-17A and EGF in the periovulatory granuloma. It was concluded that PZQ +curcumin treatment had a potent synergistic outcome through lessening the number of granulomas, the inflammatory events, and the expression of EGF, and amelioration of apoptosis in the periovulatory granulomas if compared with either PZQ or curcumin alone.

Study on Anti-colorectal Cancer Mechanism of Gallic Acid Based on JAK/STAT Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effects of gallic acid （GA） on human colon cancer HCT-116 and Caco-2 cell activities， intracellular Janus kinase （JAK）/signal transducer and activator of transcription factor （STAT） signaling pathway， and the expression of anti-apoptotic protein B-cell lymphoma-2 （Bcl-2） and pro-apoptotic protein B-cell lymphoma-2-associated X protein （Bax）， so as to explore its underlying molecular mechanism. MethodFollowing the classification of cells into GA group， blank group， and 5-fluorouracil （5-FU， 0.05 g·L-1） group， the HCT-116 and Caco-2 cells were treated with GA （0.02， 0.05， 0.1， 0.15， 0.2 g·L-1） for 12， 24， 48， and 72 h， respectively， and the cell proliferation inhibition rats were determined by cell counting kit-8 （CCK-8） assay to select the GA concentration that effectively inhibited proliferation. The colony formation ability was detected by crystal violet staining and the migration of cells by scratch test. The level of reactive oxygen species （ROS） was measured using a fluorescent probe （DCFH-DA）. The expression of interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） in cell supernatant were determined using the enzyme-linked immunosorbent assay （ELISA） kits. The expression levels of JAK2， phosphorylated （p）-JAK2， STAT3， p-STAT3， Bcl-2， and Bax were assayed by Western blot. ResultCCK-8 assay showed that after 12， 24， 48， and 72 h of treatment， GA （0.02， 0.05， 0.1， 0.15， 0.2 g·L-1） inhibited the proliferation of HCT-116 and Caco-2 cells in a dose- and time-dependent manner， and the inhibition rates were higher than those in the blank control group. Compared with the 5-FU group， GA （0.2 g·L-1） enhanced the inhibition of cell proliferation in a time-dependent manner. Compared with the blank control group， GA （0.1， 0.15， and 0.2 g·L-1） significantly decreased the number of cell colonies （P<0.01）， increased the inhibition rate of cell colony formation （P<0.01）， diminished the scratch healing rate （P<0.05， P<0.01）， elevated the fluorescence intensity of intracellular ROS （P<0.01）， and down-regulated the expression of IL-6 and TNF-α in the supernatant （P<0.01） in a dose-dependent manner. Compared with the 5-FU group， GA （0.2 g·L-1） decreased the scratch healing rate （P<0.01）， enhanced the fluorescence intensity of intracellular ROS （P<0.01）， and down-regulated the levels of IL-6 and TNF-α in cell supernatant （P<0.01）. According to Western blot analysis， compared with the blank control group， GA （0.1， 0.15， 0.2 g·L-1） obviously lowered the expression of p-JAK2， p-STAT3， Bcl-2， p-JAK2/JAK2， p-STAT3/STAT3， and Bcl-2/Bax （P<0.01） and raised Bax protein expression （P<0.05， P<0.01） in a dose-dependent manner. Compared with the 5-FU group， GA （0.2 g·L-1） down-regulated the expression of p-JAK2， p-STAT3， Bcl-2， p-JAK2/JAK2， p-STAT3/STAT3， and Bcl-2/Bax （P<0.05， P<0.01） and up-regulated the expression of Bax protein （P<0.05， P<0.01）. ConclusionGA significantly inhibits the proliferation of HCT-116 and Caco-2 cells， which may be related to the increased accumulation of intracellular ROS， down-regulation of inflammatory factors IL-6 and TNF-α， p-JAK2 and p-STAT3 protein expression in JAK/STAT signaling pathway， and Bcl-2， and up-regulation of Bax.

Effect of Hedysarum Polysaccharides on Bcl-2/Caspase-3 Signaling Pathway of Schwann Cells Cultured in High Glucose / 中国实验方剂学杂志

ObjectiveTo observe the effects of Hedysarum polysaccharides（HPS）on the signaling pathways of B-cell lymphoma 2 （Bcl-2）， cysteinyl aspartate-specific protease 3 （Caspase-3）， and Bcl-2-associated X protein （Bax） in Schwann cells（SCs）cultured in high glucose，and explore the possible mechanism of HPS against diabetic peripheral neuropathy（DPN）. MethodFour SD suckling mice aged 5-7 days were randomly divided into a normal group，a high-glucose group，an HPS + high-glucose group，and an α-lipoic acid（α-LA）+ high-glucose group. SCs were extracted from the sciatic nerve and cultured in a 37 ℃，5% CO2 incubator. After the cells reached 80% confluence，Cell Counting Kit-8（CCK-8）was used to screen the experimental concentrations suitable for high glucose，HPS， and α-LA interventions. Western blot and Real-time polymerase chain reaction （Real-time PCR）were used to detect the protein and mRNA expression of Bcl-2，Bax，and Caspase-3. The apoptosis rate of SCs was detected by flow cytometry using Annexin V-fluorescein isothiocyanate （FITC）/propidium iodide （PI）. ResultAs revealed by Western blot and real-time PCR，compared with the normal group，the high-glucose group showed reduced protein and mRNA expression of Bcl-2 and increased protein and mRNA expression of Bax and Caspase-3（P<0.01）. Compared with the high-glucose group，the HPS + high-glucose group and the α-LA + high-glucose group showed increased protein and mRNA expression of Bcl-2 and decreased protein and mRNA expression of Bax and Caspase-3（P<0.01）. As displayed by the results of flow cytometry using Annexin V/PI， compared with the normal group，the high-glucose group showed increased apoptosis rate；compared with the high-glucose group，the HPS + high-glucose group and the α-LA + high-glucose group showed reduced apoptosis rate（P<0.01）. ConclusionHPS can alleviate the apoptotic response of SCs，and its mechanism may be related to the inhibition of the activation of the Bcl-2/Caspase-3 signaling pathway.

Mechanism of Sinitang against Myocardial Ischemia-reperfusion Injury： Prediction Based on Network Pharmacology and Verification by Cellular Experiments / 中国实验方剂学杂志

ObjectiveTo predict the mechanism of Sinitang in treating myocardial ischemia-reperfusion injury （MI/RI） based on network pharmacology and verify the prediction results by cellular experiments. MethodThe traditional Chinese medicine system pharmacology database and analysis platform （TCMSP） was employed for retrieval of the main components and potential targets of Sinitang. Online Mendelian Inheritance in Man （OMIM） and GeneCards were employed to obtain the targets of Sinitang in treating MI/RI. STRING was employed to construct the protein-protein interaction （PPI） network， and DAVID to perform gene ontology （GO） annotation and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment. Finally， cellular experiments were carried out to verify the predicted anti-MI/RI mechanism of Sinitang. ResultA total of 105 active ingredients and 234 targets of Sinitang were screened out， among which 116 targets were predicted to be involved in the treatment of MI/RI. The GO annotation gave 587 entries， including 417 biological process entries， 101 cell component entries， and 69 molecular function entries. The KEGG analysis enriched 125 signaling pathways， involving vascular endothelial growth factor （VEGF）， phosphoinositide 3-kinase/protein kinase B （PI3K/Akt）， forkhead box transcription factor O （FoxO）， hypoxia-inducible factor-1 （HIF-1） apoptosis and other signaling pathways. The results of cell viability assay showed that Sinitang increased the survival rate of H9C2 cells damaged by hypoxia/reoxygenation （H/R）. Sinitang decreased the levels of tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， and creatine kinase-MB （CK-MB） in H9C2 cells damaged by H/R. The results of flow cytometry demonstrated that Sinitang decreased the apoptosis rate of H9C2 cells damaged by H/R. Western blot showed that Sinitang down-regulated the expression of Bcl-2 related X protein （Bax） and up-regulated that of B-cell lymphoma-2 （Bcl-2） in H/R-injured H9C2 cells. ConclusionSinitang treats MI/RI in a multi-target and multi-pathway manner， which involves the signaling pathways associated with apoptosis.