Calcitonin gene-related peptide inhibits neuronal apoptosis in heatstroke rats via PKA/p-CREB pathway / 中华创伤杂志(英文版)

PURPOSE@#The incidence of heatstroke (HS) is not particularly high; however, once it occurs, the consequences are serious. It is reported that calcitonin gene-related peptide (CGRP) is protective against brain injury in HS rats, but detailed molecular mechanisms need to be further investigated. In this study, we further explored whether CGRP inhibited neuronal apoptosis in HS rats via protein kinase A (PKA)/p-cAMP response element-binding protein (p-CREB) pathway.@*METHODS@#We established a HS rat model in a pre-warmed artificial climate chamber with a temperature of (35.5 ± 0.5) °C and a relative humidity of 60% ± 5%. Heatstress was stopped once core body temperature reaches above 41 °C. A total of 25 rats were randomly divided into 5 groups with 5 animals each: control group, HS group, HS+CGRP group, HS+CGRP antagonist (CGRP8-37) group, and HS+CGRP+PKA/p-CREB pathway blocker (H89) group. A bolus injection of CGRP was administered to each rat in HS+CGRP group, CGRP8-37 (antagonist of CGRP) in HS+CGRP8-37 group, and CGRP with H89 in HS+CGRP+H89 group. Electroencephalograms were recorded and the serum concentration of S100B, neuron-specific enolase (NSE), neuron apoptosis, activated caspase-3 and CGRP expression, as well as pathological morphology of brain tissue were detected at 2 h, 6 h, and 24 h after HS in vivo. The expression of PKA, p-CREB, and Bcl-2 in rat neurons were also detected at 2 h after HS in vitro. Exogenous CGRP, CGRP8-37, or H89 were used to determine whether CGRP plays a protective role in brain injury via PKA/p-CREB pathway. The unpaired t-test was used between the 2 samples, and the mean ± SD was used for multiple samples. Double-tailed p < 0.05 was considered statistically significant.@*RESULTS@#Electroencephalogram showed significant alteration of θ (54.50 ± 11.51 vs. 31.30 ± 8.71, F = 6.790, p = 0.005) and α wave (16.60 ± 3.21 vs. 35.40 ± 11.28, F = 4.549, p = 0.020) in HS group compared to the control group 2 h after HS. The results of triphosphate gap terminal labeling (TUNEL) showed that the neuronal apoptosis of HS rats was increased in the cortex (9.67 ± 3.16 vs. 1.80 ± 1.10, F = 11.002, p = 0.001) and hippocampus (15.73 ± 8.92 vs. 2.00 ± 1.00, F = 4.089, p = 0.028), the expression of activated caspase-3 was increased in the cortex (61.76 ± 25.13 vs. 19.57 ± 17.88, F = 5.695, p = 0.009) and hippocampus (58.60 ± 23.30 vs. 17.80 ± 17.62, F = 4.628, p = 0.019); meanwhile the expression of serum NSE (5.77 ± 1.78 vs. 2.35 ± 0.56, F = 5.174, p = 0.013) and S100B (2.86 ± 0.69 vs. 1.35 ± 0.34, F = 10.982, p = 0.001) were increased significantly under HS. Exogenous CGRP decreased the concentrations of NSE and S100B, and activated the expression of caspase-3 (0.41 ± 0.09 vs. 0.23 ± 0.04, F = 32.387, p < 0.001) under HS; while CGRP8-37 increased NSE (3.99 ± 0.47 vs. 2.40 ± 0.50, F = 11.991, p = 0.000) and S100B (2.19 ± 0.43 vs. 1.42 ± 0.30, F = 4.078, p = 0.025), and activated the expression caspase-3 (0.79 ± 0.10 vs. 0.23 ± 0.04, F = 32.387, p < 0.001). For the cell experiment, CGRP increased Bcl-2 (2.01 ± 0.73 vs. 2.15 ± 0.74, F = 8.993, p < 0.001), PKA (0.88 ± 0.08 vs. 0.37 ± 0.14, F = 20.370, p < 0.001), and p-CREB (0.87 ± 0.13 vs. 0.29 ± 0.10, F = 16.759, p < 0.001) levels; while H89, a blocker of the PKA/p-CREB pathway reversed the expression.@*CONCLUSIONS@#CGRP can protect against HS-induced neuron apoptosis via PKA/p-CREB pathway and reduce activation of caspase-3 by regulating Bcl-2. Thus CGRP may be a new target for the treatment of brain injury in HS.

Quercetin Alleviates Lipopolysaccharide-Induced Cardiac Inflammation via Inhibiting Autophagy and Programmed Cell Death / 生物医学与环境科学（英文）

OBJECTIVE@#The aim of this study is to explore the potential modulatory role of quercetin against Endotoxin or lipopolysaccharide (LPS) induced septic cardiac dysfunction.@*METHODS@#Specific pathogen-free chicken embryos ( n = 120) were allocated untreated control, phosphate buffer solution (PBS) vehicle, PBS with ethanol vehicle, LPS (500 ng/egg), LPS with quercetin treatment (10, 20, or 40 nmol/egg, respectively), Quercetin groups (10, 20, or 40 nmol/egg). Fifteen-day-old embryonated eggs were inoculated with abovementioned solutions via the allantoic cavity. At embryonic day 19, the hearts of the embryos were collected for histopathological examination, RNA extraction, real-time polymerase chain reaction, immunohistochemical investigations, and Western blotting.@*RESULTS@#They demonstrated that the heart presented inflammatory responses after LPS induction. The LPS-induced higher mRNA expressions of inflammation-related factors (TLR4, TNFα, MYD88, NF-κB1, IFNγ, IL-1β, IL-8, IL-6, IL-10, p38, MMP3, and MMP9) were blocked by quercetin with three dosages. Quercetin significantly decreased immunopositivity to TLR4 and MMP9 in the treatment group when compared with the LPS group. Quercetin significantly decreased protein expressions of TLR4, IFNγ, MMP3, and MMP9 when compared with the LPS group. Quercetin treatment prevented LPS-induced increase in the mRNA expression of Claudin 1 and ZO-1, and significantly decreased protein expression of claudin 1 when compared with the LPS group. Quercetin significantly downregulated autophagy-related gene expressions (PPARα, SGLT1, APOA4, AMPKα1, AMPKα2, ATG5, ATG7, Beclin-1, and LC3B) and programmed cell death (Fas, Bcl-2, CASP1, CASP12, CASP3, and RIPK1) after LPS induction. Quercetin significantly decreased immunopositivity to APOA4, AMPKα2, and LC3-II/LC3-I in the treatment group when compared with the LPS group. Quercetin significantly decreased protein expressions of AMPKα1, LC3-I, and LC3-II. Quercetin significantly decreased the protein expression to CASP1 and CASP3 by immunohistochemical investigation or Western blotting in treatment group when compared with LPS group.@*CONCLUSION@#Quercetin alleviates cardiac inflammation induced by LPS through modulating autophagy, programmed cell death, and myocardiocytes permeability.

Polysaccharide of Alocasia cucullata Exerts Antitumor Effect by Regulating Bcl-2, Caspase-3 and ERK1/2 Expressions during Long-Time Administration / 中国结合医学杂志

OBJECTIVE@#To study the in vitro and in vivo antitumor effects of the polysaccharide of Alocasia cucullata (PAC) and the underlying mechanism.@*METHODS@#B16F10 and 4T1 cells were cultured with PAC of 40 µg/mL, and PAC was withdrawn after 40 days of administration. The cell viability was detected by cell counting kit-8. The expression of Bcl-2 and Caspase-3 proteins were detected by Western blot and the expressions of ERK1/2 mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR). A mouse melanoma model was established to study the effect of PAC during long-time administration. Mice were divided into 3 treatment groups: control group treated with saline water, positive control group (LNT group) treated with lentinan at 100 mg/(kg·d), and PAC group treated with PAC at 120 mg/(kg·d). The pathological changes of tumor tissues were observed by hematoxylin-eosin staining. The apoptosis of tumor tissues was detected by TUNEL staining. Bcl-2 and Caspase-3 protein expressions were detected by immunohistochemistry, and the expressions of ERK1/2, JNK1 and p38 mRNA were detected by qRT-PCR.@*RESULTS@#In vitro, no strong inhibitory effects of PAC were found in various tumor cells after 48 or 72 h of administration. Interestingly however, after 40 days of cultivation under PAC, an inhibitory effect on B16F10 cells was found. Correspondingly, the long-time administration of PAC led to downregulation of Bcl-2 protein (P<0.05), up-regulation of Caspase-3 protein (P<0.05) and ERK1 mRNA (P<0.05) in B16F10 cells. The above results were verified by in vivo experiments. In addition, viability of B16F10 cells under long-time administration culture in vitro decreased after drug withdrawal, and similar results were also observed in 4T1 cells.@*CONCLUSIONS@#Long-time administration of PAC can significantly inhibit viability and promote apoptosis of tumor cells, and had obvious antitumor effect in tumor-bearing mice.

Asperuloside Promotes Apoptosis of Cervical Cancer Cells through Endoplasmic Reticulum Stress-Mitochondrial Pathway / 中国结合医学杂志

OBJECTIVE@#To investigate the effects of asperuloside on cervical cancer based on endoplasmic reticulum (ER) stress and mitochondrial pathway.@*METHODS@#Different doses (12.5-800 µg/mL) of asperuloside were used to treat cervical cancer cell lines Hela and CaSki to calculate the half maximal inhibitory concentration (IC50) of asperuloside. The cell proliferation was analyzed by clone formation assay. Cell apoptosis, intracellular reactive oxygen species (ROS) and mitochondrial membrane potential were determined by flow cytometry. The protein expressions of cleaved-caspase-3, Bcl-2, Bax, Cyt-c, cleaved-caspase-4 and glucose-regulated protein 78 (GRP78) were analyzed by Western blot. And the inhibitor of ER stress, 4-phenyl butyric acid (4-PBA) was used to treat cervical cancer cells to further verify the role of ER stress in the apoptosis of cervical cancer cells induced by asperuloside.@*RESULTS@#Asperuloside of 325, 650, and 1300 µg/mL significantly inhibited the proliferation and promoted apoptosis of Hela and CaSki cells (P<0.01). All doses of asperuloside significantly increased intracellular ROS levels, reduced mitochondrial membrane potential, significantly reduced Bcl-2 protein expression level, and increased Bax, Cyt-c, GRP78 and cleaved-caspase-4 expressions (P<0.01). In addition, 10 mmol/L 4-PBA treatment significantly promoted cell proliferation and reduced apoptosis (P<0.05), and 650 µg/mL asperuloside could reverse 4-PBA-induced increased cell proliferation, decreased apoptosis and cleaved-caspase-3, -4 and GRP78 protein expressions (P<0.05).@*CONCLUSION@#Our study revealed the role of asperuloside in cervical cancer, suggesting that asperuloside promotes apoptosis of cervical cancer cells through ER stress-mitochondrial pathway.

Effect of Survivin Down-Regulation by Egr1-Survivin shRNA Combined with Radiotherapy on Radiosensitivity of Esophageal Squamous Carcinoma / Efecto de la Regulación Negativa de Survivina por shRNA de Egr1-Survivina Combinado con Radioterapia sobre la Radiosensibilidad del Carcinoma Escamoso de Esófago

SUMMARY: This study is to investigate the effect of survivin down-regulation by Egr1-survivin shRNA combined with radiotherapy on the apoptosis and radiosensitivity of esophageal squamous cell carcinoma ECA109 and KYSE150 cells. ECA109 and KYSE150 cells were transfected with Egr1-survivin shRNA, and then treated with radiotherapy. After 24 h, the mRNA and protein levels of Egr1-survivin were detected by qPCR and Western-Blot. Cell cycle and apoptosis were detected by flow cytometry. Western blot also detected levels of cleavaged Caspase 3 and Caspase 9. YM155 was used as a positive control to inhibit survivin expression. The levels of survivin mRNA and protein in ECA109 and KYSE150 cells treated with Egr1-survivin shRNA combined with radiotherapy were significantly lower than those of the blank control group, the empty vector control group, and, the YM155 + radiotherapy group (P<0.05). Meanwhile, after survivin down-regulation, the ratio of G2 to S phase of ECA109 and KYSE150 cells increased significantly, leading to significant G2 and S phase arrest. Additionally, apoptosis of ECA109 and KYSE150 cells increased significantly (P <0.01). Further, protein levels of cleavaged Caspase 3 and Caspase 9 significantly increased in Egr1-survivin shRNA combined with radiotherapy group. Egr1-survivin shRNA combined with radiotherapy can down-regulate survivin expression, which further increases the apoptosis, and enhances the radiosensitivity of ECA109 and KYSE150 cells.

Este estudio tuvo como objetivo investigar el efecto de la regulación negativa de survivina por el shRNA de Egr1-survivina combinado con radioterapia sobre la apoptosis y la radiosensibilidad del carcinoma de células escamosas de esófago Células ECA109 y KYSE150. Las células ECA109 y KYSE150 se transfectaron con shRNA de survivina Egr1 y luego se trataron con radioterapia. Después de 24 h, los niveles de ARNm y proteína de Egr1-survivina se detectaron mediante qPCR y Western-Blot. El ciclo celular y la apoptosis se detectaron mediante citometría de flujo. La transferencia Western también detectó niveles de Caspasa 3 y Caspasa 9 escindidas. Se usó YM155 como control positivo para inhibir la expresión de survivina. Los niveles de ARNm y proteína de survivina en células ECA109 y KYSE150 tratadas con shRNA de survivina Egr1 combinado con radioterapia fueron significativamente más bajos que los del grupo control en blanco, el grupo control de vector vacío y el grupo de radioterapia YM155 + (P <0,05). Mientras tanto, después de la regulación negativa de survivina, la proporción entre las fases G2 y S de las células ECA109 y KYSE150 aumentó significativamente, lo que llevó a una detención significativa de las fases G2 y S. Además, la apoptosis de las células ECA109 y KYSE150 aumentó significativamente (P <0,01). Además, los niveles de proteína de Caspasa 3 y Caspasa 9 escindidas aumentaron significativamente en el shRNA de Egr1- survivina combinado con el grupo de radioterapia. El shRNA de survivina de Egr1 combinado con radioterapia puede regular negativamente la expresión de survivina, lo que aumenta aún más la apoptosis y mejora la radiosensibilidad de las células ECA109 y KYSE150.

Zarzio Ameliorates Non-alcoholic Fatty Liver Induced By a High Fat Diet in a Rat Experimental Model: A Histological and Immunohistochemical Study

SUMMARY: The therapeutic effect of a granulocyte-colony stimulating factor (G-CSF) biosimilar drug, zarzio, on non-alcoholic fatty liver disease (NAFLD) in a rat model was investigated in this study. Thirty-two rats were randomly divided into four groups. Groups I and II were fed a standard laboratory diet, whereas groups III and IV were fed a high fat diet (HFD) for 14 weeks. After 12 weeks of feeding, groups I and III were administered normal saline, and groups II and IV were intraperitoneally administered zarzio (200 mg/kg/day) for two consecutive weeks. Hematoxylin-eosin (H&E) staining was used to assess hepatic and pancreatic morphology in all groups, oil red O (ORO) staining for lipid accumulation, Masson's staining for fibrosis, and immunohistochemistry assay for hepatic protein expression of insulin receptor substrate 1 (IRS1), nuclear factor erythroid 2-related factor 2 (Nrf2), tumour necrosis factor alpha (TNF-α) and pancreatic caspase-3. The NAFLD rats (group III) developed hepatic steatosis with increased lipid accumulation, perisinusoidal fibrosis, upregulated IRS1, TNF-α (all P<0.05) without a significant increase in Nrf2 protein expression compared with normal control. In comparison, model rats treated with zarzio (group IV) showed significant rejuvenation of the hepatic architecture, reduction of fat accumulation, and fibrosis. This was accompanied by the upregulation of Nrf2, downregulation of IRS1 and TNF-α protein expression (all P<0.05). No correlation was detected between NAFLD and non-alcoholic fatty pancreas disease (NAFPD). However, the pancreatic β-cells in group III showed increased caspase-3 expression, which was decreased (P<0.05) in group IV. In conclusion, zarzio ameliorates NAFLD by improving the antioxidant capacity of liver cells, reducing hepatic IRS1, TNF-α protein expression and pancreatic β-cells apoptosis, suggesting that zarzio could be used as a potential therapy for NAFLD.

En este estudio se investigó el efecto terapéutico de un fármaco biosimilar del factor estimulante de colonias de granulocitos (G-CSF), zarzio, sobre la enfermedaddel hígado graso no alcohólico (NAFLD) en un modelo de rata. Treinta y dos ratas se dividieron aleatoriamente en cuatro grupos. Los grupos I y II fueron alimentados con una dieta estándar de laboratorio, mientras que los grupos III y IV fueron alimentados con una dieta alta en grasas (HFD) durante 14 semanas. Después de 12 semanas de alimentación, a los grupos I y III se les administró solución salina normal, y a los grupos II y IV se les administró zarzio por vía intraperitoneal (200 mg/kg/ día) durante dos semanas consecutivas. Se utilizó tinción de hematoxilina-eosina (H&E) para evaluar la morfología hepática y pancreática en todos los grupos, tinción con rojo aceite O (ORO) para la acumulación de lípidos, tinción de Masson para la fibrosis y ensayo de inmunohistoquímica para la expresión de la proteína hepática del sustrato 1 del receptor de insulina (IRS1), factor nuclear eritroide 2 relacionado con el factor 2 (Nrf2), factor de necrosis tumoral alfa (TNF-α) y caspasa-3 pancreática. Las ratas NAFLD (grupo III) desarrollaron esteatosis hepática con aumento de la acumulación de lípidos, fibrosis perisinusoidal, IRS1 y TNF-α regulados positivamente (todos P <0,05) sin un aumento significativo en la expresión de la proteína Nrf2 en comparación con el control normal. En comparación, las ratas modelo tratadas con zarzio (grupo IV) mostraron un rejuvenecimiento significativo de la arquitectura hepática, una reducción de la acumulación de grasa y fibrosis. Esto estuvo acompañado por la regulación positiva de Nrf2, la regulación negativa de la expresión de la proteína IRS1 y TNF-α (todas P <0,05). No se detectó correlación entre NAFLD y la enfermedad del páncreas graso no alcohólico (NAFPD). Sin embargo, las células β pancreáticas en el grupo III mostraron una mayor expresión de caspasa-3, que disminuyó (P <0,05) en el grupo IV. En conclusión, zarzio mejora la NAFLD al mejorar la capacidad antioxidante de las células hepáticas, reduciendo el IRS1 hepático, la expresión de la proteína TNF-α y la apoptosis de las células β pancreáticas, lo que sugiere que zarzio podría usarse como una terapia potencial para la NAFLD.

Protective effect of linoleic acid on liver toxicity induced by methotrexate / Efecto protector del ácido linoleico sobre la toxicidad hepática inducida por metotrexato

SUMMARY: We aimed to investigate the protective effect of linoleic acid on liver toxicity induced by methotrexate. The study was carried out in partnership with the Department of Anatomy and Department of Medical Pharmacology of Çukurova University Faculty of Medicine, using the laboratory facilities of the Department of Medical Pharmacology. Human hepatocyte cell line (CRL- 11233) cells obtained from the American Type Culture Collection Organization (ATCC) were used. Expressions of apoptotic pathway markers, apoptosis inducing factor (AIF), BAX, BCL 2, GADD 153, 78-kDa glucose-regulated protein (GRP78), and CASPASE-3 were evaluated. All analyzes were examined in four groups (Group 1; control, Group 2; linoleic acid given, Group 3; methotrexate given and Group 4; linoleic acid and methotrexate given). The mean ± standard error values of the obtained results as nanogram / milliliter (ng / ml) are in Group I, Group II, Group III and Group IV, respectively; AIF values, 0.4150 ± 0.1208, 0.3633 ± 0.2389, 1.792 ± 0.3611 and 1.077 ± 0.1646, BAX values, 0.900 ± 0.1864, 1.002 ± 0.2098, 8.352 ± 1.467 and 4.295 ± 1.522, BCL 2 values, 13.93 ± 1.198, 13.92 ± 1.739, 2.938 ± 1.059 and 9.250 ± 1.492, GADD 153, 0.7333 ± 0.1751, 0.7067 ± 0.2115, 1.650 ± 0.2950 and 1.237 ± 0.1805, GRP78, 0.4767 ± 0.1804, 0.5233 ± 0.1590, 2.183 ± 0.2639 and 1.112 ± 0.2693, CASPASE-3 values , 1.127 ± 0.2033, 0.8317 ± 0.3392, 13.50 ± 1.871 and 8.183 ± 1.030. It was determined that linoleic acid has a protective effect on methotrexate-induced liver toxicity.

Nuestro objetivo fue investigar el efecto protector del ácido linoleico sobre la toxicidad hepática inducida por metotrexato. El estudio se llevó a cabo en colaboración con el Departamento de Anatomía y el Departamento de Farmacología Médica de la Facultad de Medicina de la Universidad de Çukurova, utilizando las instalaciones del laboratorio del Departamento de Farmacología Médica. Se usaron células de la línea celular de hepatocitos humanos (CRL-11233) obtenidas de la American Type Culture Collection Organisation (ATCC). Se evaluaron las expresiones de marcadores de vías apoptóticas, factor inductor de apoptosis (AIF), BAX, BCL 2, GADD 153, proteína regulada por glucosa de 78 kDa (GRP78) y CASPASE-3. Todos los análisis se examinaron en cuatro grupos (Grupo 1; control, Grupo 2; se administró ácido linoleico, Grupo 3; se administró metotrexato y Grupo 4; se administró ácido linoleico y metotrexato). Los valores medios ± error estándar de los resultados obtenidos como nanogramo/mililitro (ng/ml) se encuentran en el Grupo I, Grupo II, Grupo III y Grupo IV, respectivamente; Valores de AIF, 0,4150 ± 0,1208, 0,3633 ± 0,2389, 1,792 ± 0,3611 y 1,077 ± 0,1646, valores de Bax, 0,900 ± 0,1864, 1,002 ± 0,2098, 8,352 ± 1,467 y 4,295 ± 1,522, BCL 2 valores, 13,93 ± 1,199. 2,938 ± 1,059 y 9,250 ± 1,492, GADD 153, 0,7333 ± 0,1751, 0,7067 ± 0,2115, 1,650 ± 0,2950 y 1,237 ± 0,1805, Grp78, 0,4767 ± 0,1804, 0,5233 ± 0,1590, 2,183, ± 1,263. 1,127 ± 0,2033, 0,8317 ± 0,3392, 13,50 ± 1,871 y 8,183 ± 1,030. Se determinó que el ácido linoleico tiene un efecto protector sobre la toxicidad hepática inducida por metotrexato.

Effect of Long Non-Coding RNA LINC01268 on the Malignant Biological Behaviors of Acute Myeloid Leukemia Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of long non-coding RNA LINC01268 on apoptosis of acute myeloid leukemia (AML) cells and related mechanisms.@*METHODS@#The expression levels of LINC01268 and miR-217 in peripheral blood samples from AML patients and AML cell lines HL-60 and KG-1 were detected by qRT-PCR. HL-60 cells were divided into pcDNA3.1-NC, pcDNA3.1-LINC01268, si-NC, si-LINC01268, miR-NC, miR-217 mimics, si-LINC01268 + inhibitor-NC and si-LINC01268+ miR-217 inhibitor groups. The mRNA expressions of LINC01268 and miR-217 were detected by qRT-PCR. The targeting relationship between LINC01268 and miR-217 was detected by dual-luciferase reporter assay. Cell viability was detected by CCK-8 assay. Cell cycle distribution and apoptosis were detected by flow cytometry. The expression of cell cycle and apoptosis-related proteins p21, Bcl-2, Bax, caspase-3 and PI3K/AKT signaling pathway-related proteins were detected by Western blot.@*RESULTS@#The expression of LINC01268 in peripheral blood samples of AML patients and AML cell lines HL-60 and KG-1 was increased (P < 0.05), and the expression of miR-217 was decreased (P < 0.05). Compared with si-NC group and miR-NC group, the viability of HL-60 cells was decreased in si-LINC01268 group and miR-217 mimics group (P < 0.05), the proportion of cells in G1 phase and apoptosis rate were increased (P < 0.05), the protein expression levels of p21, Bax and caspase-3 were increased (P < 0.05), while the protein expression level of Bcl-2 was decreased (P < 0.05). LINC01268 targeted and negatively regulated the expression of miR-217, and inhibiting the expression of miR-217 partially reversed the effects of LINC01268 interference on the viability, cell cycle and apoptosis of HL-60 cells. Interference with LINC01268 could inhibit the activity of PI3K/AKT signaling pathway. Inhibiting the expression of miR-217 could partially reverse the inhibition of LINC01268 interference on PI3K/AKT signaling pathway.@*CONCLUSION@#LINC01268 is highly expressed and miR-217 is lowly expressed in AML cells. LINC01268 can promote the activity of PI3K/AKT signaling pathway, increase the survival rate and inhibit the apoptosis of AML cells by targeting miR-217 expression.

Mechanism of Proliferation and Apoptosis of Acute Promyelocytic Leukemia Cell Line NB4 Induced by TPA / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of phorbol-12-myristate-13-ace-tate (TPA) on the proliferation and apoptosis of acute promyelocytic leukemia cell line NB4 and its molecular mechanism.@*METHODS@#The effect of different concentrations of TPA on the proliferation of NB4 cells at different time points was detected by CCK-8 assay. The morphological changes of NB4 cells were observed by Wright-Giemsa staining. The cell cycle and apoptosis of NB4 cells after TPA treatment were detected by flow cytometry. The mRNA expressions of NB4 cells after TPA treatment were analyzed by high-throughput microarray analysis and real-time quantitative PCR. Western blot was used to detect the protein expression of CDKN1A, CDKN1B, CCND1, MYC, Bax, Bcl-2, c-Caspase 3, c-Caspase 9, PIK3R6, AKT and p-AKT.@*RESULTS@#Compared with the control group, TPA could inhibit the proliferation of NB4 cells, induce the cells to become mature granulocyte-monocyte differentiation, and also induce cell G1 phase arrest and apoptosis. Differentially expressed mRNAs were significantly enriched in PI3K/AKT pathway. TPA treatment could increase the mRNA levels of CCND1, CCNA1, and CDKN1A, while decrease the mRNA level of MYC. It could also up-regulate the protein levels of CDKN1A, CDKN1B, CCND1, Bax, c-Caspase 3, c-Caspase 9, and PIK3R6, while down-regulate MYC, Bcl-2, and p-AKT in NB4 cells.@*CONCLUSION@#TPA induces NB4 cell cycle arrest in G1 phase and promotes its apoptosis by regulating PIK3/AKT signaling pathway.

Effect of LINC00174 on the Malignant Proliferation of Multiple Myeloma Cells and Its Related Mechanism / 中国实验血液学杂志

OBJECTIVE@#To explore the biological function of LINC00174 in multiple myeloma (MM).@*METHODS@#Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expressions of LINC00174 and miR-150 in peripheral blood of MM patients and MM cell lines. EdU staining and flow cytometry were used to detect the effects of LINC00174 and miR-150 on the proliferation and apoptosis of MM cells. Western blot was used to detect the expressions of proliferation marker nuclear-related antigen Ki67, apoptosis-related protein cleaved caspase-3 and transcription factor forkhead box protein P1 (FOXP1). Bioinformatics and dual-luciferase reporter assay were used to verify the targeting relationship between LINC00174 and miR-150 and the targeting relationship between miR-150 and FOXP1.@*RESULTS@#The level of LINC00174 was significantly increased in peripheral blood of MM patients and MM cell lines (P <0.05). Compared with NC-siRNA group, the expression of LINC00174 was significantly reduced in LINC00174-siRNA group, the proliferation of U266 cells was reduced, the apoptosis rate was significantly increased, the level of Ki67 protein was reduced, and the level of cleaved caspase-3 protein was increased (all P <0.05). LINC00174 targeted regulation of the expression of miR-150. Compared with LINC00174-siRNA+NC inhibitor group, the expression of miR-150 in U266 cells in LINC00174-siRNA+miR-150 inhibitor group was significantly reduced, the cell proliferation was enhanced, the apoptosis rate was reduced, the level of Ki67 protein was increased, and the level of cleaved caspase-3 was decreased (all P <0.05). FOXP1 is the target gene of miR-150. Compared with NC mimic group, the expression of FOXP1 protein in miR-150 mimic group was significantly reduced, the cell proliferation was reduced, the apoptosis rate was significantly increased, Ki67 protein level was decreased, and the level of cleaved caspase-3 was increased. Compared with miR-150 mimic + vector group, the expression of FOXP1 protein in miR-150 mimic + pcDNA-FOXP1 group was significantly increased, the cell proliferation was enhanced, the apoptosis rate was reduced, the level of Ki67 protein was increased, and the level of cleaved caspase-3 was decreased (all P <0.05).@*CONCLUSION@#LINC00174 promotes the proliferation of MM cells and inhibits cell apoptosis by regulating the miR-150/ FOXP1 axis.

Role and mechanisms of CHI3L1 in coronary artery lesions in a mouse model of Kawasaki disease-like vasculitis / 中国当代儿科杂志

OBJECTIVES@#To explore the role and potential mechanisms of chitinase-3-like protein 1 (CHI3L1) in coronary artery lesions in a mouse model of Kawasaki disease (KD)-like vasculitis.@*METHODS@#Four-week-old male SPF-grade C57BL/6 mice were randomly divided into a control group and a model group, with 10 mice in each group. The model group mice were intraperitoneally injected with 0.5 mL of lactobacillus casei cell wall extract (LCWE) to establish a mouse model of KD-like vasculitis, while the control group mice were injected with an equal volume of normal saline. The general conditions of the mice were observed on the 3rd, 7th, and 14th day after injection. Changes in coronary artery tissue pathology were observed using hematoxylin-eosin staining. The level of CHI3L1 in mouse serum was measured by enzyme-linked immunosorbent assay. Immunofluorescence staining was used to detect the expression and localization of CHI3L1, von Willebrand factor (vWF), and α-smooth muscle actin (α-SMA) in coronary artery tissue. Western blot analysis was used to detect the expression of CHI3L1, vWF, vascular endothelial cadherin (VE cadherin), Caspase-3, B cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), nuclear factor κB (NF-κB), and phosphorylated NF-κB (p-NF-κB) in coronary artery tissue.@*RESULTS@#The serum level of CHI3L1 in the model group was significantly higher than that in the control group (P<0.05). Compared to the control group, the expression of CHI3L1 in the coronary artery tissue was higher, while the expression of vWF was lower in the model group. The relative expression levels of CHI3L1, Bax, Caspase-3, NF-κB, and p-NF-κB were significantly higher in the model group than in the control group (P<0.05). The relative expression levels of vWF, VE cadherin, and Bcl-2 were lower in the model group than in the control group (P<0.05).@*CONCLUSIONS@#In the LCWE-induced mouse model of KD-like vasculitis, the expression levels of CHI3L1 in serum and coronary arteries increase, and it may play a role in coronary artery lesions through endothelial cell apoptosis mediated by inflammatory reactions.

Knock-down of ROCK2 gene improves cognitive function and reduces neuronal apoptosis in AD mice by promoting mitochondrial fusion and inhibiting its division / 细胞与分子免疫学杂志

Objective To explore the effect of knocking down Rho-associated coiled-coil kinase (ROCK2) gene on the cognitive function of amyloid precursor protein/presenilin-1 (APP/PS1) double transgenic mice and its mechanism. Methods APP/PS1 double transgenic mice were randomly divided into AD model group (AD group), ROCK2 gene knock-down group (shROCK2 group), ROCK2 gene knock-down control group (shNCgroup), and wild-type C57BL/6 mice of the same age served as the wild-type control (WT group). Morris water maze and Y maze were employed to test the cognitive function of mice. Neuron morphology was detected by Nissl staining. Immunofluorescence histochemical staining was used to detect the expression of phosphorylated dynamin-related protein 1 (p-Drp1) and mitochondrial fusion 1 (Mfn1). Western blot analysis was used to detect the expression ROCK2, cleaved-caspase-3 (c-caspase-3), B-cell lymphoma 2 (Bcl2), Bcl2-related protein X (BAX), p-Drp1, mitochondrial fission 1 (Fis1), optic atrophy 1 (OPA1), Mfn1 and Mfn2. Results Compared with AD group mice, the expression of ROCK2 in shROCK2 group mice was significantly reduced; the cognitive function was significantly improved with the number of neurons in the hippocampal CA3 and DG areas increasing, and nissl bodies were deeply stained; the expression of c-caspase-3 and BAX was decreased, while the expression of Bcl2 was increased; the expression of mitochondrial division related proteins p-Drp1 and Fis1 were decreased, while the expression of mitochondrial fusion-related proteins OPA1, Mfn1 and Mfn2 were increased. Conclusion Knock-down of ROCK2 gene can significantly improve the cognitive function and inhibit the apoptosis of nerve cells of APP/PS1 mice. The mechanism may be related to promoting mitochondrial fusion and inhibiting its division.

Effect and mechanism of Poria cocos polysaccharides on myocardial cell apoptosis in rats with myocardial ischemia-reperfusion injury by regulating Rho-ROCK signaling pathway / 中国中药杂志

This study aimed to investigate the effect and underlying mechanism of Poria cocos polysaccharides(PCP) on myocardial cell apoptosis in the rat model of myocardial ischemia-reperfusion injury(MI/RI). Male SPF-grade SD rats were randomly divided into a sham group(saline), a model group(saline), low-and high-dose PCP groups(100 and 200 mg·kg~(-1)), and a fasudil group(10 mg·kg~(-1)), with 16 rats in each group. Except for the sham group, the other four groups underwent left anterior descending coronary artery ligation for 30 min followed by reperfusion for 2 h to establish the MI/RI model. The myocardial infarct area was assessed by TTC staining. Histological changes were observed through HE staining. Myocardial cell apoptosis was evaluated using TUNEL staining. Serum lactate dehydrogenase(LDH), creatine kinase MB(CK-MB), interleukin-1β(IL-1β) and IL-18 levels, myocardial superoxide dismutase(SOD) activity and malondialdehyde(MDA) levels were detected by ELISA. Protein expression of B-cell lymphoma 2(Bcl-2), Bcl-2 associated X protein(Bax), cleaved caspase-3, Ras homolog gene A(RhoA), myosin phosphatase target subunit 1(MYPT-1), phosphorylated MYPT-1(p-MYPT-1), and Rho-associated coiled-coil forming kinase 1(ROCK 1) were measured by Western blot. Pathological staining of myocardial tissue revealed that in the model group, there was focal necrosis of myocardial tissue, myocardial cell swelling, unclear boundaries, and neutrophil infiltration. These pathological changes were alleviated in the low-and high-dose PCP groups and the fasudil group. Compared with the model group, the low-and high-dose PCP groups and the fasudil group showed significantly reduced myocardial infarct area and myocardial cell apoptosis rate. Compared with the sham group, the model group exhibited elevated serum LDH, CK-MB, IL-1β and IL-18 levels, increased MDA levels, relative protein expression of Bax, cleaved caspase-3, RhoA, ROCK1 and p-MYPT-1, and decreased myocardial SOD levels and Bcl-2 protein expression. Compared with the model group, the PCP groups and the fasudil group showed lowered serum LDH, CK-MB, IL-1β and IL-18 levels, decreased MDA levels, relative protein expression of Bax, cleaved caspase-3, RhoA, ROCK1 and p-MYPT-1, and increased myocardial SOD levels and Bcl-2 protein expression. PCP exhibited a certain preventive effect on myocardial tissue pathological damage and myocardial cell apoptosis in MI/RI rats, possibly related to the inhibition of the Rho-ROCK signaling pathway activation, thereby reducing oxidative stress and inflammatory responses.

Effect and mechanism of Danggui Buxue Decoction-containing serum in mitigating H9c2 cell injury caused by exposure to intermittent low oxygen / 中国中药杂志

This study aims to explore the effect and mechanism of Danggui Buxue Decoction(DBD)-containing serum in alleviating the H9c2 cell injury caused by the exposure to intermittent low oxygen. H9c2 cells were assigned into five groups: control(CON) group, intermittent low oxygen(IH) group, intermittent low oxygen plus DBD-containing serum(IH+DBD) group, intermittent low oxygen plus the autophagy enhancer rapamycin(IH+RAPA) group, and intermittent low oxygen plus DBD-containing serum and the autophagy inhibitor 3-methyladenine(IH+DBD+3-MA) group. Monodansylcadaverine(MDC) staining was employed to detect the changes of autophagosomes. Cell counting kit-8(CCK-8) assay was employed to determine the activity of myocardial cells, and lactate dehydrogenase(LDH) and creatine kinase(CK) kits were used to measure the LDH and CK levels in the cell culture, which would reflect the degree of cell damage. TdT-mediated dUTP nick-end labeling(TUNEL) staining was used to detect the apoptosis of myocardial cells, and JC-1 fluorescence probe to detect the changes in mitochondrial membrane potential. Western blot was employed to determine the expression levels of the autophagy-related proteins microtubule-associated proteins light chain 3Ⅱ(LC3Ⅱ), microtubule-associated proteins light chain 3Ⅰ(LC3Ⅰ), P62, Parkin and apoptosis related proteins pro caspase-3, caspase-3, B-cell lymphoma-2(Bcl-2), Bcl-2-associated X(Bax). The results showed that compared with the CON group, the IH group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated expression of P62. In addition, the IH group showed decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, and decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. Compared with the IH group, the IH+DBD and IH+RAPA groups showed increased fluorescence intensity of MDC staining, increased LC3Ⅱ/LC3Ⅰ ratio, up-regulated Parkin expression, and down-regulated P62 expression. In addition, the two groups showed increased cell survival rate, reduced content of LDH and CK in the culture medium, decreased number of TUNEL positive cells, and increased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. The IH+DBD+3-MA and IH groups showed no significant differences in the above indicators. Compared with the IH+DBD group, the IH+DBD+3-MA group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated P62 expression. In addition, the group had decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios, and declined mitochon-drial membrane potential. To sum up, DBD could promote the mitophagy, inhibit the apoptosis, and alleviated the injury of H9c2 cells exposed to low oxygen.

Mechanism of Gegen Qinlian Decoction in improving glucose metabolism in vitro and in vivo by alleviating hepatic endoplasmic reticulum stress / 中国中药杂志

This study investigated the mechanism of Gegen Qinlian Decoction(GQD) in improving glucose metabolism in vitro and in vivo by alleviating endoplasmic reticulum stress(ERS). Molecular docking was used to predict the binding affinity between the main effective plasma components of GQD and ERS-related targets. Liver tissue samples were obtained from normal rats, high-fat-induced diabetic rats, rats treated with metformin, and rats treated with GQD. RNA and protein were extracted. qPCR was used to measure the mRNA expression of ERS marker glucose-regulated protein 78(GRP78), and unfolded protein response(UPR) genes inositol requiring enzyme 1(Ire1), activating transcription factor 6(Atf6), Atf4, C/EBP-homologous protein(Chop), and caspase-12. Western blot was used to detect the protein expression of GRP78, IRE1, protein kinase R-like ER kinase(PERK), ATF6, X-box binding protein 1(XBP1), ATF4, CHOP, caspase-12, caspase-9, and caspase-3. The calcium ion content in liver tissues was determined by the colorimetric assay. The ERS-HepG2 cell model was established in vitro by inducing with tunicamycin for 6 hours, and 2.5%, 5%, and 10% GQD-containing serum were administered for 9 hours. The glucose oxidase method was used to measure extracellular glucose levels, flow cytometry to detect cell apoptosis, glycogen staining to measure cellular glycogen content, and immunofluorescence to detect the expression of GRP78. The intracellular calcium ion content was measured by the colorimetric assay. Whereas Western blot was used to detect GRP78 and ERS-induced IRE1, PERK, ATF6, and eukaryotic translation initiation factor 2α(eIF2α) phosphorylation. Additionally, the phosphorylation levels of insulin receptor substrate 1(IRS1), phosphatidylinositol 3-kinase regulatory subunit p85(PI3Kp85), and protein kinase B(Akt), which were involved in the insulin signaling pathway, were also measured. In addition, the phosphorylation levels of c-Jun N-terminal kinases(JNKs), which were involved in both the ERS and insulin signaling pathways, were measured by Western blot. Molecular docking results showed that GRP78, IRE1, PERK, ATF4, and various compounds such as baicalein, berberine, daidzein, jateorhizine, liquiritin, palmatine, puerarin and wogonoside had strong binding affinities, indicating that GQD might interfere with ERS-induced UPR. In vivo results showed that GQD down-regulated the mRNA transcription of Ire1, Atf6, Atf4, Grp78, caspase-12, and Chop in diabetic rats, and down-regulated GRP78, IRE1, PERK, as well as ERS-induced apoptotic factors ATF4 and CHOP, caspase-12, caspase-9, and caspase-3, while up-regulating XBP1 to enhance adaptive UPR. In addition, GQD increased the calcium ion content in liver tissues, which facilitated correct protein folding. In vitro results showed that GQD increased glucose consumption in ERS-induced HepG2 cells without significantly affecting cell viability, increased liver glycogen synthesis, down-regulated ATF6 and p-eIF2α(Ser51), and down-regulated IRE1, PERK, and GRP78, as well as p-IRS1(Ser312) and p-JNKs(Thr183/Tyr185), while up-regulating p-PI3Kp85(Tyr607) and p-Akt(Ser473). These findings suggested that GQD alleviates excessive ERS in the liver, reduces insulin resistance, and improves hepatic glucose metabolism in vivo and in vitro.

Effect of Shionone on Neuron Apoptosis After Spinal Cord Injury in Mice / 中国医学科学院学报

Objective To explore the effect of shionone(SHI)on motor function in the mouse model of spinal cord injury(SCI)and probe into the underlying molecular mechanism.Methods C57BL/6 mice were treated to induce the SCI model and then assigned into a model group(SCI group),a SCI+SHI group,and a sham surgery(control)group.The Basso mouse scale(BMS)score was determined to evaluate the recovery of motor function in SCI mice.Hematoxylin-eosin(HE)staining,Nissl staining,and immunofluorescence staining were employed to examine the fibrosis,morphological changes of neurons,and neuron apoptosis in the spinal cord tissue of SCI mice,respectively.The mouse hippocampal neuronal cell line HT22 was cultured in vitro and then classified into tumor necrosis factor α(TNF-α)induction and SHI groups.Western blotting was employed to determine the expression of apoptosis-associated proteins.Network pharmacology,gene ontology annotation,and Kyoto Encyclopedia of Genes and Genomes pathway enrichment were employed to predict the possible molecular targets and signaling pathways of SHI in promoting functional recovery from SCI.Furthermore,the prediction results were verified by in vitro and in vivo experiments.Results Compared with the SCI group,the SCI+SHI group showed increased BMS score on days 21,28,35,and 42(P=0.003,P=0.004,P=0.023,and P=0.007,respectively),reduced area of spinal cord fibrosis(P=0.021),increased neurons survived(P=0.001),and down-regulated expression of cleaved cysteine aspastic acid-specific protease 3(cleaved Caspase-3)(P=0.017).Compared with the TNF-α group,the SHI group presented down-regulated expression levels of cleaved Caspase-3 and Bax(P=0.010,P=0.001)and up-regulated expression level of Bcl-2(P=0.001).The results of bioinformatics analysis showed that SHI might improve the motor function of SCI mice via the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway.The results of in vivo and in vitro experiments showed that SHI inhibited the phosphorylation of PI3K and Akt in SCI mice or HT22 cells exposed to TNF-α(all P<0.05).The number of apoptotic HT22 cells after treatment with insulin-like growth factor 1 was higher than that in the SHI group(P=0.003).Conclusion SHI may inhibit neuron apoptosis via the PI3K/Akt signaling pathway,thereby promoting the recovery of motor function in SCI mice.

Panax notoginseng saponins improve monocrotaline-induced pulmonary arterial hypertension in rats by inhibiting ADAM10/Notch3 signaling pathway / 生理学报

In this study, we investigated the effects of Panax notoginseng saponins (PNS) on pulmonary vascular remodeling and ADAM10/Notch3 pathway in pulmonary arterial hypertension (PAH). PAH rat model was established, and male Sprague Dawley (SD) rats were randomly divided into control group, monocrotaline (MCT) group and MCT+PNS group, with 10 rats in each group. Rats in the control group were intraperitoneally injected with equal volume of normal saline. Rats in the MCT group was injected intraperitoneally with 60 mg/kg MCT on the first day, and then with the same volume of normal saline every day. Rats in the MCT+PNS group was injected intraperitoneally with 60 mg/kg MCT on the first day, and then with 50 mg/kg PNS every day. The modeling time of each group lasted for 21 days. After the model was established, the mean pulmonary artery pressure (mPAP) was measured by right heart catheterization technique, the right ventricular hypertrophy index (RVHI) was calculated, the microscopic morphology and changes of pulmonary vascular wall were observed by HE and Masson staining, and the expressions of ADAM10, Notch3, Hes-1, P27, PCNA, Caspase-3 proteins and mRNA in pulmonary vascular tissue of rats were detected by Western blot and qPCR. The expression and localization of Notch3 and α-SMA were detected by immunofluorescence staining. The protein expression of ADAM10 was detected by immunohistochemical staining. The results showed that compared with the control group, mPAP, RVHI, pulmonary vessels and collagen fibers in the MCT group were significantly increased, the expressions of ADAM10, Notch3, Hes-1, and PCNA protein and mRNA were significantly increased, while the expressions of P27 and Caspase-3 protein and mRNA were decreased significantly. Compared with the MCT group, mPAP and RVHI were significantly decreased, pulmonary vessels were significantly improved and collagen fibers were significantly reduced, the expressions of protein and mRNA of ADAM10, Notch3, Hes-1, and PCNA were decreased in MCT+PNS group, but the expressions of protein and mRNA of P27 and Caspase-3 were increased slightly. The results of immunofluorescence showed that Notch3 and α-SMA staining could overlap, which proved that Notch3 was expressed in smooth muscle cells. The expression of Notch3 in the MCT group was increased significantly compared with that in the control group, while PNS intervention decreased the expression of Notch3. Immunohistochemical staining showed that compared with the control group, the amount of ADAM10 in the MCT group was increased significantly, and the expression of ADAM10 in the MCT+PNS group was decreased compared with the MCT group. These results indicate that PNS can improve the PAH induced by MCT in rats by inhibiting ADAM10/Notch3 signaling pathway.

Baicalin attenuates dexamethasone-induced apoptosis of bone marrow mesenchymal stem cells by activating the hedgehog signaling pathway / 中华医学杂志(英文版)

BACKGROUND@#Perturbations in bone marrow mesenchymal stem cell (BMSC) differentiation play an important role in steroid-induced osteonecrosis of the femoral head (SONFH). At present, studies on SONFH concentrate upon the balance within BMSC osteogenic and adipogenic differentiation. However, BMSC apoptosis as well as proliferation are important prerequisites in their differentiation. The hedgehog (HH) signaling pathway regulates bone cell apoptosis. Baicalin (BA), a well-known compound in traditional Chinese medicine, can affect the proliferation and apoptosis of numerous cell types via HH signaling. However, the potential role and mechanisms of BA on BMSCs are unclear. Thus, we aimed to explore the role of BA in dexamethasone (Dex)-induced BMSC apoptosis in this study.@*METHODS@#Primary BMSCs were treated with 10 -6 mol/L Dex alone or with 5.0 μmol/L, 10.0 μmol/L, or 50.0 μmol/L BA for 24 hours followed by co-treatment with 5.0 μmol/L, 10.0 μmol/L, or 50.0 μmol/L BA and 10 -6 mol/L Dex. Cell viability was assayed through the Cell Counting Kit-8 (CCK-8). Cell apoptosis was evaluated using Annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining followed by flow cytometry. The imaging and counting, respectively, of Hochest 33342/PI-stained cells were used to assess the morphological characteristics and proportion of apoptotic cells. To quantify the apoptosis-related proteins (e.g., apoptosis regulator BAX [Bax], B-cell lymphoma 2 [Bcl-2], caspase-3, and cleaved caspase-3) and HH signaling pathway proteins, western blotting was used. A HH-signaling pathway inhibitor was used to demonstrate that BA exerts its anti-apoptotic effects via the HH signaling pathway.@*RESULTS@#The results of CCK-8, Hoechst 33342/PI-staining, and flow cytometry showed that BA did not significantly promote cell proliferation (CCK-8: 0 μmol/L, 100%; 2.5 μmol/L, 98.58%; 5.0 μmol/L, 95.18%; 10.0 μmol/L, 98.11%; 50.0 μmol/L, 99.38%, F   =  2.33, P   >  0.05), but it did attenuate the effect of Dex on apoptosis (Hoechst 33342/PI-staining: Dex+ 50.0 μmol/L BA, 12.27% vs. Dex, 39.27%, t  = 20.62; flow cytometry: Dex + 50.0 μmol/L BA, 12.68% vs. Dex, 37.43%, t  = 11.56; Both P  < 0.05). The results of western blotting analysis showed that BA reversed Dex-induced apoptosis by activating the HH signaling pathway, which down-regulated the expression of Bax, cleaved-caspase 3, and suppressor of fused (SUFU) while up-regulating Bcl-2, sonic hedgehog (SHH), and zinc finger protein GLI-1 (GLI-1) expression (Bax/Bcl-2: Dex+ 50.0 μmol/L BA, 1.09 vs. Dex, 2.76, t  = 35.12; cleaved caspase-3/caspase-3: Dex + 50.0 μmol/L BA, 0.38 vs . Dex, 0.73, t  = 10.62; SHH: Dex + 50.0 μmol/L BA, 0.50 vs . Dex, 0.12, t  = 34.01; SUFU: Dex+ 50.0 μmol/L BA, 0.75 vs . Dex, 1.19, t  = 10.78; GLI-1: Dex+ 50.0 μmol/L BA, 0.40 vs . Dex, 0.11, t  = 30.68. All P  < 0.05).@*CONCLUSIONS@#BA antagonizes Dex-induced apoptosis of human BMSCs by activating the HH signaling pathway. It is a potential candidate for preventing SONFH.

Astragali Radix-Curcumae Rhizoma combination inhibits proliferation, migration, and invasion of colon cancer HT-29 cells by regulating EMT / 中国中药杂志

This study aims to investigate the effect of Astragali Radix-Curcumae Rhizoma(AC) combination on the proliferation, migration, and invasion of colon cancer HT-29 cells based on epithelial-mesenchymal transition(EMT). HT-29 cells were respectively treated with 0, 3, 6 and 12 g·kg~(-1) AC-containing serum for 48 h. The survival and growth of cells were measured by thiazole blue(MTT) colorimetry, and the proliferation, migration, and invasion of cells were detected by 5-ethynyl-2'-deoxyuridine(EdU) test and Transwell assay. Cell apoptosis was examined by flow cytometry. The BALB/c nude mouse model of subcutaneous colon cancer xenograft was established, and then model mice were classified into blank control group, 6 g·kg~(-1) AC group, and 12 g·kg~(-1) AC group. The tumor weight and volume of mice were recorded, and the histopathological morphology of the tumor was observed based on hematoxylin-eosin(HE) staining. The expression of apoptosis-associated proteins B-cell lymphoma-2-associated X protein(Bax), cysteine-aspartic acid protease-3(caspase-3), and cleaved caspase-3, and EMT-associated proteins E-cadherin, MMP9, MMP2 and vimentin in HT-29 cells and mouse tumor tissues after the treatment of AC was determined by Western blot. The results showed that cell survival rate and the number of cells at proliferation stage decreased compared with those in the blank control group. The number of migrating and invading cells reduced and the number of apoptotic cells increased in the administration groups compared with those in the blank control group. As for the in vivo experiment, compared with the blank control group, the administration groups had small tumors with low mass and shrinkage of cells and karyopycnosis in the tumor tissue, indicating that the AC combination may improve EMT. In addition, the expression of Bcl2 and E-cadherin increased and the expression of Bax, caspase-3, cleaved caspase-3, MMP9, MMP2, and vimentin decreased in HT-29 cells and tumor tissues in each administration group. In summary, the AC combination can significantly inhibit the proliferation, invasion, migration, and EMT of HT-29 cells in vivo and in vitro and promote the apoptosis of colon cancer cells.

Mechanism of total flavonoids of Rhododendra simsii in alleviating ischemic brain injury / 中国中药杂志

This study explores the effect of total flavonoids of Rhododendra simsii(TFR) on middle cerebral artery occlusion(MCAO)-induced cerebral injury in rats and oxygen-glucose deprivation/reoxygenation(OGD/R) injury in PC12 cells and the underlying mechanism. The MCAO method was used to induce focal ischemic cerebral injury in rats. Male SD rats were randomized into sham group, model group, and TFR group. After MCAO, TFR(60 mg·kg~(-1)) was administered for 3 days. The content of tumor necrosis factor-α(TNF-α), interleukin-1(IL-1), and interleukin-6(IL-6) in serum was detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes of brain tissue and cerebral infarction were observed based on hematoxylin and eosin(HE) staining and 2,3,5-triphenyltetrazolium chloride(TTC) staining. RT-qPCR and Western blot were used to detect the mRNA and protein levels of calcium release-activated calcium channel modulator 1(ORAI1), stromal interaction molecule 1(STIM1), stromal intera-ction molecule 2(STIM2), protein kinase B(PKB), and cysteinyl aspartate specific proteinase 3(caspase-3) in brain tissues. The OGD/R method was employed to induce injury in PC12 cells. Cells were randomized into the normal group, model group, gene silencing group, TFR(30 μg·mL~(-1)) group, and TFR(30 μg·mL~(-1))+gene overexpression plasmid group. Intracellular Ca~(2+) concentration and apoptosis rate of PC12 cells were measured by laser scanning confocal microscopy and flow cytometry. The effect of STIM-ORAI-regulated store-operated calcium entry(SOCE) pathway on TFR was explored based on gene silencing and gene overexpression techniques. The results showed that TFR significantly alleviated the histopathological damage of brains in MCAO rats after 3 days of admini-stration, reduced the contents of TNF-α, IL-1, and IL-6 in the serum, down-regulated the expression of ORAI1, STIM1, STIM2, and caspase-3 genes, and up-regulated the expression of PKB gene in brain tissues of MCAO rats. TFR significantly decreased OGD/R induced Ca~(2+) overload and apoptosis in PC12 cells. However, it induced TFR-like effect by ORAI1, STIM1 and STIM2 genes silencing. However, overexpression of these genes significantly blocked the effect of TFR in reducing Ca~(2+) overload and apoptosis in PC12 cells. In summary, in the early stage of focal cerebral ischemia-reperfusion injury and OGD/R-induced injury in PC12 cells TFR attenuates ischemic brain injury by inhibiting the STIM-ORAI-regulated SOCE pathway and reducing Ca~(2+) overload and inflammatory factor expression, and apoptosis.