Human umbilical cord mesenchymal stem cells migrate to Raji cells and promote their proliferation

AIM：To investigate the interaction between human Burkitt lymphoma cell line Raji and human umbilical cord mesenchymal stem cells (hUC-MSC). METHODS：The direct and indirect effects of MSC on Raji cells were investigated. The viability of Raji cells was tested by CCK-8 assay, and the cell cycle was determined by flow cytometry. The importance of vascular endothelial growth factor (VEGF) in the migration of MSC to Raji cells was analyzed by blocking VEGF expression in Raji cells with small interfering RNA (siRNA). VEGF level in the supernatant was detected by ELISA, and the mRNA expression of VEGF was measured by quantitative real-time PCR (qRT-PCR). RESULTS：Both MSC and MSC-conditioned medium (MSC-CM) promoted the growth of Raji cells. The viability of Raji cells co-cultured with MSC-CM was 0.99±0.05 at 72 h, and that in control group was 0.71±0.07. Both direct co-culture with MSC and MSC-CM turned the Raji cells from G0/G1 phase to S phase. The number of Raji cells co-cultured with MSC-CM in S phase was increased from 16.33±1.37 to 28.50±1.41, and the number in G0/G1 phase was decreased from 77.70±1.57 to 54.40±1.57. The expression of VEGF was down-regulated either at mRNA or protein level after transfection with siRNA. The ability of MSC migrated to Raji cells was significantly declined (96.00±5.28 vs 143.00±7.20). CONCLUSION：Raji cells recruit MSC by secreting VEGF, and MSC promote the proliferation of Raji cells by turning the cells from G0/G1 phase to S phase.     

Effect of 27nt-miRNA on apoptosis of human umbilical vein endothelial cells induced by oxidized low-density lipoprotein

AIM To investigate the effect of 27nt-miRNA （27nt-miR） on apoptosis of human umbilical vein endothelial cells （HUVECs） induced by oxidized low-density lipoprotein （Ox-LDL） and its underlying mechanism. METHODS HUVECs were cultured in vitro and grouped as below： normal control group， Ox-LDL group， 27nt-miR+Ox-LDL group， anti-27nt-miR+Ox-LDL group and negative control+Ox-LDL group. The cells in Ox-LDL group were treated with Ox-LDL at 40 mg/L for 48 h， while those in normal control group were untreated but cultured normally. The cells in 27nt-miR+Ox-LDL group， anti-27nt-miR+Ox-LDL group and negative control+Ox-LDL group were transfected with their corresponding lentiviral vectors under the same procedure， followed by treatment with Ox-LDL at 40 mg/L for 48 h to induce apoptosis. The cell viability was measured by CCK-8 assay. The migration capacity was detected by scratch assay. The caspase-3 activity was measured by caspase-3 activity assay kit. The apoptotic rate was analyzed by Hoechst 33258 and flow cytometry. The mRNA and protein expression levels of Bcl-2， Bax and caspase-3 were determined by RT-qPCR and Western blot. RESUITS： Compared with negative control+Ox-LDL group， the cell viability and migration ability were significantly decreased by over-expression of 27nt-miR in the HUVECs （P<0.05）， while the activity of caspase-3 and apoptosis induced by Ox-LDL were significantly increased （P<0.05）. Furthermore， the mRNA and protein expression levels of Bax and caspase-3 were significantly up-regulated （P<0.05）， and the mRNA and protein expression level of Bcl-2 was down-regulated in 27nt-miR+Ox-LDL group （P<0.05）. Meanwhile， all the above indexes showed an opposite tendency in anti-27nt-miR+Ox-LDL group. CONCLUSION 27nt-miR promotes Ox-LDL-induced apoptosis and inhibits the viability and migration of HUVECs in vitro， possibly through regulating the expression of apoptotic/anti-apoptotic proteins such as Bax，caspase-3 and Bcl-2.     

Minocycline suppresses sodium nitroprusside-induced apoptosis in PC12 cells by regulating PI3K/Akt signaling

AIM：To explore the role of PI3K/Akt signaling in the anti-apoptotic effect of minocyline (MC) on the apoptosis of PC12 cells induced by sodium nitroprusside (SNP). METHODS：PC12 cells were divided into 4 groups: blank control group, SNP (500 μmol/L) group, MC (10 μmol/L)+SNP group and LY294002+MC+SNP group. The cell viability was observed by MTT assay. The expression of Akt and p-Akt was determined by Western blotting. RESULTS： The viability of the PC12 cells decreased after exposed to 500 μmol/L SNP for 24 h. Meanwhile, MC at concentration of 10 μmol/L significantly blocked the effect of SNP, such as decreasing the cell viability. Pretreatment with LY294002 for 60 min prior to exposure of the PC12 cells to MC and SNP down-regulated the expression of p-Akt induced by SNP. CONCLUSION：Minocycline regulates PI3K/Akt signaling pathway to restrain the apoptosis of PC12 cells induced by SNP.     

Exogenous ATP induces formation of membrane pore in PC12 cells

AIM：To investigate the formation of membrane pore in PC12 cells induced by exogenous adenosine triphosphate (ATP) and to identify the key molecular targets. METHODS：PC12 cells were treated with different concentrations of ATP to establish the injury model. The morphological change was observed under an inverted phase-contrast microscope. The viability of the PC12 cells was measured by CCK-8 assay. Fluorescent dye YO-PRO-1 was used to detect the membrane permeability. The expression of P2X7 receptor and pannexin 1 (Panx1) at mRNA and protein levels was assessed by real-time PCR and Western blotting. RESULTS：After exposed to ATP (1 mmol/L, 3 mmol/L and 5 mmol/L) for 3 h, the PC12 cells became edematous, and the number of adherent cells decreased gradually in a dose-dependent manner. The cell viabilities in 3 mmol/L ATP group and 5 mmol/L ATP group were significantly decreased compared with control group (P<0.05). YO-PRO-1 uptake in the PC12 cells exposed to ATP (0, 1, 3 and 5 mmol/L) for 15 min, 30 min and 60 min increased in a dose-dependent and time-dependent manner. The cell viability increased and the intracellular fluorescence intensity induced by ATP were significantly antagonized in brilliant blue G (a P2X7 receptor inhibitor) pretreatment group (P<0.05), whereas it did not change in carbenoxolone (a Panx1 inhibitor) pretreatment group (P>0.05). The expression of P2X7 receptor at mRNA and protein levels was significantly increased (P<0.05), but the expression of Panx1 was not changed (P>0.05) when PC12 cells were exposed to ATP for 3 h. CONCLUSION：Extracellular ATP at high concentration may induce membrane pore formation with the expression and activation of P2X7 receptor in PC12 cells.     

miR-155-specific siRNA enhances chemosensitivity of Burkitt lymphoma Raji cells to cytosine arabinoside by inducing apoptosis

AIM：To investigate the effect of miR-155-specific siRNA alone or in combination with cytosine arabinoside (Ara-C) on the growth and apoptosis of Burkitt lymphoma Raji cells. METHODS：miR-155-specific siRNA and/or Ara-C were used to treat the cells. Quantitative real-time polymerase chain reaction was used to detect the expression of miR-155. The growth of the cells was analyzed by CKK-8 assay. The cell apoptosis was determined by flow cytometry. RESULTS：The miR-155 expression level of the cells transfected with miR-155 siRNA was significantly lower than that in the 2 control groups. Ara-C or miR-155 siRNA alone inhibited the growth of Raji cells in a dose-depend manner. miR-155 siRNA combined with Ara-C produced more inhibition of cell proliferation (P<0.05). After treatment for 48 h, the apoptotic rate of Raji cells in miR-155 siRNA+Ara-C group ［(38.4±1.4)%］ was higher than that in Ara-C group ［(16.5±0.3)%］ and miR-155 siRNA group ［(14.6±0.3)%］, with statistically significant difference (P<0.05). The expression of caspase-3 in Ara-C+miR-155 siRNA group was increased significantly as compared with Ara-C group and miR-155 siRNA group. CONCLUSION：miR-155-specific siRNA enhances the chemosensitivity of Raji cells to Ara-C by inducing apoptosis through the caspase-3 pathway.     

Cholestane-3β, 5α, 6β-triol induces apoptosis of malignant glioma cells

AIM：To investigate the effect of cholestane-3β, 5α, 6β-triol (Triol) on apoptosis of malignant glioma cells. METHODS：C6 cells and A172 cells were incubated with Triol at different concentrations for different time durations. MTT assay was used to detect the cell viability. Hoechst 3f3342 staining and TUNEL assay were used to analyze the cell apoptosis. The caspase activity was measured. The expression of apoptosis-related proteins, Bcl-2 family members, was determined by Western blotting. RESULTS：Triol decreased the cell viability of C6 and A172 cells in a dose- and time-dependent manner and the IC50 values were (17.8±0.6)μmol/L and (20.6±0.2) μmol/L, respectively. Visible nuclei with apoptotic characteristics, significant increase in TUNEL-positive cells, and the activation of apoptotic execution enzyme caspase-3 indicated that cell apoptosis was induced by Triol in both cell lines. After C6 cells were exposed to Triol for 12 h, 24 h and 48 h, the activity of caspase-8 in extrinsic apoptotic pathway and caspase-9 in intrinsic apoptotic pathway was increased time-dependently. Meanwhile, the levels of anti-apoptotic proteins, Bcl-2 and Bcl-xL, was down-regulated, while pro-apoptotic protein Bak was up-regulated in a time-dependent manner. CONCLUSION：Triol induces apoptosis of malignant glioma cells by activating intrinsic and extrinsic apoptotic pathways, and Bcl-2 family members are involved in Triol-induced apoptosis.     

Expression of Smoothened and its role in endothelial cells in synovial tissues of rheumatoid arthritis

AIM: To investigate the expression of Sonic Hedgehog signaling pathway-associated factor Smoothened (Smo) and its role in endothelial cells in synovial tissue of active rheumatoid arthritis (RA). METHODS: Smo expression in synovial tissue from 4 RA patients and 4 patients with trauma or meniscal injury (without arthritis, used for control) was detected by the method of immunohistochemistry. Human umbilical vein endothelial cell line EA.hy926 was used as the model of synovial vascular endothelial cells. The expression of Smo was detected by Western blotting after TNF-α treatment. The small interfering RNA (siRNA) specifically targeting Smo gene was synthesized and transfected into EA.hy926 cells. The interference efficiency of the siRNA on the production of Smo protein was determined by Western blotting. The cells were treated with TNF-α and actinomycin D (ActD) 24 h after siRNA transfection. The cell survival rate was determined by CCK-8 assay and the apoptotic rate was examined by flow cytometry. RESULTS: Smo was highly expressed in synovial tissue from active RA patients, especially in endothelial cells as compared with control group. TNF-α significantly increased the protein expression of Smo in EA.hy926 cells. EA.hy926 cells transfected with Smo-siRNA showed a significant decrease in the cell viability with the cell survival rate of (24.30±0.45)% and the apoptotic rate of (48.00±1.96)%, as compared with those in negative control group ［(36.86±0.62）% and （31.70±0.82）%, respectively］. CONCLUSION: Smo may play a role in the regulation of apoptosis in endothelial cells in RA synovium.     

Combination of dihydromyricetin and cisplatin significantly induces overexpression of Noxa and Bim in PC3 cells

AIM:To investigate the adjuvant effect of dihydromyricetin on cisplatin-based chemotherapy in prostate cancer and its mechanisms. METHODS:The viability of LNCaP and PC3 cells treated with different concentrations of dihydromyricetin and cisplatin was measured by MTT assay. The expression of FOXO1, Noxa and Bim, release of cytochrome C from mitochondria, and activation of caspase-9 and caspase-3 in PC3 cells treated with dihydromyricetin and cisplatin were determined by Western blot. Co-immunoprecipitation was performed to detect the interaction of apoptotic protease-activating factor-1 (Apaf-1) and caspase-9 in the PC3 cells. The apoptotic rate of PC3 cells was analyzed by flow cytometry. RESULTS:Adjuvant therapy of dihydromyricetin significantly enhanced the anti-tumor effect of cisplatin against prostate cancer in vitro. Dihydromyricetin significantly promoted the expression of FOXO1 in the PC3 cells. However, transfection with FOXO1 small interfering RNA (siRNA) obviously suppressed the adjuvant effect of dihydromyricetin. Combination of cisplatin and dihydromyricetin significantly induced the overexpression of Noxa and Bim, the release of cytochrome C, the interaction of Apaf-1 and caspase-9, the activation of caspase-9 and caspase-3, and the apoptosis in the PC3 cells. On the other hand, transfection with FOXO1 siRNA obviously suppressed the apoptotic pathway of PC3 cells treated with dihydromyricetin and cisplatin. CONCLUSION:Dihydromyricetin enhances the cytotoxicity of cisplatin against prostate cancer through the FOXO1-Bim/Noxa pathway in vitro.     

Effect of HIPK2 gene on viability,apoptosis and JAK2/STAT3 signaling pathway in NRK-52E renal tubular epithelial cells induced by hypoxia and reoxygenation

AIM: To investigate the effect of homeodomain-interacting protein kinase 2 (HIPK2) on the viabi-lity, apoptosis and JAK2/STAT3 signaling pathway in NRK-52E renal tubular epithelial cells induced by hypoxia and reoxygenation (H/R). METHODS: HIPK2 small interfering RNA (siRNA) was transfected into NRK-52E cells by Lipofectamine^TM 2000, and normal control group (control group) and negative control group (HIPK2-NC group) were set up. After H/R, the cell viability was measured by CCK-8 assay, the apoptotic rate and Ca²⁺ fluorescence intensity were analyzed by flow cytometry, and the protein levels of Ki67, cleaved caspase-3, caspase-12, Bcl-2, Bax, p-JAK2 and p-STAT3 were determined by Western blot. RESULTS: Compared with control group, the protein expression of HIPK2 in the NRK-52E cells was significantly decreased after transfection with HIPK2 siRNA (P<0.05). Compared with control group, the cell viability and the protein expression of Ki67 and Bcl-2 in H/R group were also significantly decreased, and the apoptotic rate, the Ca²⁺ fluorescence intensity and the protein levels of cleaved caspase-3, caspase-12, Bax, p-JAK2 and p-STAT3 were significantly increased (P<0.05). Compared with H/R group, the cell viability and the protein expression of Ki67 and Bcl-2 in HIPK2-siRNA+H/R group were significantly increased, while the apoptotic rate, the Ca²⁺ fluorescence intensity and the protein levels of cleaved caspase-3, caspase-12, Bax, p-JAK2 and p-STAT3 were significantly decreased (P<0.05). CONCLUSION: Inhibition of HIPK2 gene expression promotes H/R-induced growth of NRK-52E renal tubular epithelial cells, and reduces the apoptosis. The mechanism is related to down-regulating the JAK2/STAT3 signaling pathway.     

Role of retinoic acid X receptor in regulation of autophagy in rat alveolar type Ⅱ epithelial cells induced by hypoxia/reoxygenation

AIM To investigate the regulatory effect of retinoic acid X receptor （RXR） on autophagy induced by hypoxia/reoxygenation （H/R） in rat alveolar type Ⅱ epithelial cells （AEC Ⅱ） and its molecular mechanism. METHODS AEC Ⅱ were cultured in normoxia. The cells growing to logarithmic growth phase were randomly divided into 5 groups： （1） control （Con） group： cells were cultured for 30 h under normal operation； （2） H/R group： cells were cultured in hypoxia condition for 6 h and then in reoxygenation condition for 24 h； （3） DMSO group： cells were pretreated 1.5 h with medium containing less than 0.1% DMSO before modeling， and the rest were treated the same as the H/R group； （4） 9-cis-retinoic acid （9-RA） group： cells were pretreated for 1 h with 9-RA （100 nmol/L） before hypoxia； （5） HX531 group： cells were treated with 9-RA （100 nmol/L） for 0.5 h， then treatment with HX531 （2.5 μmol/L） for 1 h. CCK-8 assay was used to detect the cell viability. Immunofluorescence staining was used to observe the expression of RXRα. Transmission electron microscope was used to observe the changes of intracellular ultrastructure， and the mRNA expression of adenosine AMP-activated protein kinase （AMPK）， beclin 1， LC3， mammalian target of rapamycin （mTOR） and P62 was detected by RT-PCR. Western blot was used to detected the protein levels of p-AMPK， beclin 1， LC3-Ⅱ， p-mTOR and P62. RESULTS Compared with Con group， the cell viability in H/R， DMSO， 9-RA and HX531 groups were significantly decreased. The mRNA expression of AMPK， beclin 1 and LC3 was significantly increased， and the protein levels of p-AMPK， beclin 1 and LC3-Ⅱ were also increased. The mRNA expression of mTOR and P62 was decreased， and the protein levels of p-mTOR and P62 were also decreased （P<0.05）. The cell injury in 9-RA group was alleviated and autophagy level was significantly lower than that in H/R， DMSO and HX531 groups （P<0.05）， and no significant difference among H/R， DMSO and HX531 groups was observed （P>0.05）. CONCLUSION H/R induces autophagy of AEC Ⅱ. Activating RXR reduce the damage of AEC Ⅱ cells induced by H/R， and its mechanism may be related to the inhibition of autophagy.     

Acetyl-L-carnitine attenuates H2O2-induced oxidative damage of PC12 cells

AIM: To investigate the effect of acetyl-L-carnitine (ALC) on H₂O₂-induced oxidative damage in PC12 cells and its possible mechanism. METHODS: A moderate oxidative damage PC12 cell model was induced by exposure of the PC12 cells to H₂O₂. ALC at different concentrations (100, 200 and 400 μmol/L) was applied to the PC12 cells cultured in vitro, and CCK8 assay was used to detect the cell viability. The cells were divided into control group, H₂O₂ group, and low-ALC, medium-ALC and high-ALC groups. The apoptosis of the cells was analyzed by flow cytometry. The protein levels of Nrf2 and cleaved caspase-3 were determined by Western blot. The nuclear translocation of Nrf2 was observed by immunofluorescence staining. RESULTS: ALC at different concentrations (100, 200 and 400 μmol/L) significantly inhibited H₂O₂-induced PC12 cell apoptosis, and the medium concentration group had the best effect. Compared with H₂O₂ group, low, medium and high concentrations of ALC significantly increased the viability of the PC12 cells induced by H₂O₂, inhibit cell apoptosis (P<0.05), significantly down-regulated the protein level of cleaved caspase-3 (P<0.05), up-regulated the protein level of Nrf2 (P<0.05), and promoted the translocation of Nrf2 from the cytoplasm to the nucleus. CONCLUSION: Acetyl-L-carnitine attenuates H₂O₂-induced oxidative damage of PC12 cells, inhibits the apoptosis and increases the viability, which is related to the activation of Nrf2 signaling pathway.     

EGCG antagonizes hypoxia-induced autophagy and apoptosis in PC12 cells by mTOR regulation

AIM:Hypoxia (evoked by CoCl2)-induced apoptosis and autophagy are emerging as crucial events in the etiopathology of many neurodegenerative diseases. Epigallocatechin gallate (EGCG) is the active ingredient in tea polyphenols with abilities of anti-apoptosis and anti-autophagy, but the mechanism has not been fully elucidated. In recent years, studies have reported that the mammalian target of rapamycin (mTOR) involved in the regulation of a variety of neurological like differentiation and maturation of nerve cells, anti-oxidative stress, etc. Therefore, we investigate that whether EGCG protects PC12 from hypoxia-induced apoptosis and autophagy by enhancing mTOR expression. METHODS:The expression of mTOR and beclin-1 were detected by Western blotting. The expression of caspase-3 was measured by ELISA. The cell viability was detected by CCK-8 assay. The LC-3 expression in nucelus was observed by immunofluorescence. RESULTS:Hypoxia induced apoptosis and autophagy in PC12 cells. EGCG antagonized hypoxia-induced apoptosis and autophagy by enhancing mTOR expression. Blocking the pathway of mTOR reversed the protective effect of EGCG on PC12 cells. CONCLUSION: EGCG antagonizes hypoxia-induced autophagy and apoptosis in PC12 cells by controlling mTOR regulation.     

Effect of CHOP expression knock-down on apoptosis of renal tubular epithelial cells

AIM:To study the effect of C/EBP homologous protein (CHOP) on the apoptosis of renal tubular epithelial HK2 cells. METHODS:The serum mRNA levels of CHOP in the patients with acute kidney injury and healthy controls were detected by qPCR. In vitro, renal tubular epithelial HK2 cells were divided into control group, negative group (transfected with negative control siRNA), si-CHOP group (transfected with CHOP siRNA), and induced by transforming growth factor-β1 (TGF-β1). The viability of the cells was measured by MTT assay, and the apoptotic rate was analyzed by flow cytometry. The protein levels of nuclear antigen Ki-67, proliferating cell nuclear antigen (PCNA), caspase-3 and cleaved caspase-3 were determined by Western blot. RESULTS:Compared with the healthy controls, the serum mRNA levels of CHOP in the patients with acute kidney injury were increased significantly (P<0.05). Transfection with CHOP siRNA significantly decreased the expression of CHOP in the renal tubular epithelial HK2 cells (P<0.05). Knock-down of CHOP expression by siRNA significantly increased the viability of renal tubular epithelial HK2 cells (P<0.05), decreased the apoptotic rate (P<0.05), increased the expression of Ki-67 and PCNA (P<0.05), and down-regulated the protein level of cleaved caspase-3 (P<0.05). CONCLUSION:The serum mRNA levels of CHOP were increased in the patients with acute kidney injury. Knock-down of CHOP expression inhibits the apoptosis of renal tubular epithelial cells by regulating the expression of proliferation-and apoptosis-related proteins.     

Expression of histone chaperone ASF1B in prostate cancer cells and its effect on cell viability in vitro

AIM To investigate the expression of histone chaperone anti-silencing function 1B （ASF1B） in prostate cancer cells and its effect on cell viability in vitro. METHODS Human prostate cancer PC-3 cells were used， and knockdown of ASF1B was conducted by small interfering RNA （siRNA） transfection into the cells. The cells were divided into control group， siRNA negative control vector （mock） group and siRNA-ASF1B group. The viability of the PC-3 cells treated with ASF1B-siRNA for 12， 24 and 48 h was measured by MTT assay. Flow cytometry was used to analyze cell apoptosis and cell cycle distribution. The mRNA expression of apoptosis-related molecules was detected by RT-qPCR， and the expression levels of MAPK/JNK/ERK signaling pathway-related proteins were determined by Western blot. RESULTS The protein level of ASF1B in the normal cells （benign prostatic hyperplasia） was significantly lower than that in the PC-3 cells （P<0.01）. Compared with control group and mock group， the protein expression level of ASF1B in the PC-3 cells transfected with siRNA-ASF1B plasmid and the viability of the PC-3 cells were significantly decreased （P<0.01）. The apoptotic rate of the PC-3 cells transfected with siRNA-ASF1B plasmid was significantly higher than that in control group （P<0.01）， and the cell cycle was arrested at G₁ phase. The mRNA levels of p53， caspase-3， Bax and PARP-1 in the PC-3 cells transfected with siRNA-ASF1B plasmid were up-regulated compared with those in control group and Mack group （P<0.01）. In addition， the protein levels of MAP2K4 and p-JNK in the PC-3 cells in siRNA-ASF1B group were significantly higher than those in mock group （P<0.01）， while the protein level of p-ERK was significantly lower than that in mock group （P<0.01）. CONCLUSION ASF1B silencing induces G₁ arrest and promotes apoptosis of PC-3 cells. Activating MAPK/JNK/ERK signaling pathway may be a possible contributor to the anti-prostate cancer effect of siRNA-ASF1B.     

Ferulic acid protects against apoptosis of PC12 cells induced by kainic acid

AIM：To investigate the effect of ferulic acid (FA) on the apoptosis of PC12 cells induced by kainic acid (KA) in vitro. METHODS：In order to establish an Alzheimer disease neuronal cell model, the rat pheochromocytoma cell line PC12 was treated with KA at a concentration of 50 μmol/L. These model neurons were divided into KA model group and 3 groups treated with FA at doses of 25, 50 and 100 μmol/L, respectively. At the same time, normal group was established without KA pretreatment. The viability of the PC12 cells was detected by MTT assay. The expression of Bcl-2, Bax and cytochrome C (Cyt C) was determined by immunocytochemical method. Apoptotic rate of the PC12 cells was measured by flow cytometry with annexin V/PI double staining. The protein levels of Bcl-2, Bax and Cyt C were analyzed by Western blotting. RESULTS：The cell survival rate, the expression of Bcl-2 and the ratio of Bcl-2 to Bax in KA model group were significantly decreased (P<0.01),while the expression of Bax and Cyt C was obviously increased compared with normal control group (P<0.01). The apoptotic rate in KA model group was obviously increased compared with normal control group (P<0.01) After the intervention of FA, the cell survival rates were increased and the apoptotic rates were decreased. Furthermore, the positive rate and expression of Bcl-2, and the ratio of Bcl-2 to Bax in each dose of FA treatment group were significantly increased, while the expression of Bax and Cyt C in each dose group was significantly reduced as compared with KA model group (P<0.05 or P<0.01). CONCLUSION：KA obviously induces apoptosis of PC12 cells. FA had obvious protective effect on PC12 cells against the toxicity of KA. FA blocks endogenous apoptic pathway through inhibiting the expression of Bax and Cyt C and increasing the expression of Bcl-2 and the ratio of Bcl-2/Bax, thus improving the survival rate of PC12 cells.     

ROCK promotes hypoxia-induced cardiomyocyte apoptosis by inhibiting PI3K/Akt pathway

AIM: To investigate the expression of Rho-associated coiled-coil protein kinase (ROCK) at different hypoxic phases and to explore its role in myocardial cell apoptosis. METHODS: The rat cardiomyocytes were primarily cultured and identified by an antibody targeting α-actin of striated muscle. The myocardial cell hypoxic model was established by exposing the cells in hypoxic liquid for 1 h, 3 h, 6 h and 9 h. The cell apoptotic rate was assessed by flow cytometry. The cell survival rate was determined by MTT assay. The protein levels of ROCK-1, ROCK-2, caspase-3 activation fragment, PI3K and p-PI3K at different hypoxia phases were determined by Western blotting.RESULTS: After exposed to hypoxic liquid for 1 h, 3 h, 6 h and 9 h, the apoptotic rates of the cardiomyocytes were （8.76±1.51）%, （15.36±2.34）%, （26.50±3.43）% and （41.96±4.22）%, respectively, significantly higher than those in control group ［(2.60±0.34）%, P<0.01］. The survival rates were （93.20±4.12）%, （86.14±3.10）%, （75.53±7.25）%and （60.21±6.75）%, respectively, signficantly lower than those in control group ［（97.60±1.12）%, P<0.05］. After 1 h of hypoxic exposure, the levels of ROCK-1 and ROCK-2 began to rise, reached its peak at 3～6 h, and began to decrease after 9 h, which were significantly higher than those in control group (P<0.05). After 1 h of hypoxic exposure, the caspase-3 activation fragment began to rise, which was sustained in a high level at following observed time points as compared with control group (P<0.01). No difference of the PI3K expression in the course of hypoxia was observed. However, after 1 h of hypoxic exposure, the p-PI3K level began to rise, reached its peak at 3 h, began to decrease at 6 h, and was almost undetectable at 9 h. CONCLUSION: Hypoxia stimulates the cardiomyocytes to increase the expression of ROCK-1 and ROCK-2, and is in parallel with the cardiomyocyte apoptosis. ROCKs may play an important role in the process of hypoxia-induced cardiomyocyte apoptosis by inhibiting the p-PI3K pathways.     

Effect of lncRNA FEZF1-AS1 on viability and apoptosis of LPS-induced vascular endothelial cells by regulating miR-363-3p expression

AIM To investigate the mechanism of long noncoding RNA （lncRNA） FEZF1-AS1 regulating microRNA-363-3p （miR-363-3p） on the viability and apoptosis of lipopolysaocharide （LPS）-induced vascular endothelial cells. METHODS Human umbilical vein endothelial cells （HUVECs） were cultured in vitro. pcDNA-NC， pcDNA-FEZF1-AS1， anti-miR-NC， anti-miR-363-3p， miR-NC and miR-363-3p mimics were transfected into the HUVECs and LPS stimulation was applied for 24 h. RT-qPCR was used to detect the expression of FEZF1-AS1 and miR-363-3p. The cell viability was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The dual-luciferase reporter experiment was used to verify the targeted regulation of FEZF1-AS1 and miR-363-3p. Western blot was used to determined the expression of cyclin D1， Ki67 and cleaved caspase-3. RESULTS Compared with control group， the expression level of FEZF1-AS1 in LPS group was significantly reduced （P<0.05）， and the expression level of miR-363-3p was significantly increased （P<0.05）. Compared with pcDNA-NC+LPS group， the cell viability in pcDNA-FEZF1-AS1+LPS group was significantly increased （P<0.05）， the apoptotic rate was significantly reduced （P<0.05）， the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）， and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Compared with anti-miR-NC+LPS group， the cell viability in anti-miR-363-3p+LPS group was significantly increased （P<0.05）， the apoptotic rate was significantly reduced （P<0.05）， the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）， and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Dual-luciferase reporter experiment confirmed that FEZF1-AS1 targeted miR-363-3p. Compared with miR-NC+pcDNA-FEZF1-AS1+LPS group， the cell viability in miR-363-3p+pcDNA-FEZF1-AS1+LPS group was significantly reduced （P<0.05）， the apoptotic rate was significantly increased （P<0.05）， the protein levels of cyclin D1 and Ki67 were significantly reduced （P<0.05）， and the protein level of cleaved caspase-3 was significantly increased （P<0.05）. CONCLUSION Over-expression of FEZF1-AS1 promotes the viability and inhibits apoptosis of LPS induced vascular endothelial cells by inhibiting the expression of miR-363-3p.     

Mangiferin attenuates hypoxia/reoxygenation-induced injury of human myocardial cells

AIM To investigate the effect of mangiferin on hypoxia/reoxygenation (H/R)-induced injury of human myocardial cells and its mechanism. METHODS Human myocardial AC16 cells were divided into normal group, H/R group and H/R + mangiferin (50, 100 and 200 μmol/L) treatment groups. The mRNA and protein expression levels of Kelch-like epichlorohydrin-associated protein-1 (Keap-1), Bax, Bcl-2, caspase-3, caspase-9 and superoxide dismutase 2 (SOD2) were detected by RT-qPCR and Western blot, respectively. The protein expression of nuclear factor E2-related factor 2 (Nrf-2) in nucleus was determined by Western blot. The expression of microRNA-432-3p (miR-432-3p) was detected by RT-qPCR. The generation of reactive oxygen speciess (ROS) in the cells was measured by DCFH-DA probing. The cell viability was measured by CCK-8 assay. Apoptosis was analyzed by flow cytometry. RESULTS No significant difference in the expression of miR-432-3p and Keap-1 between normal group and H/R group was observed. Compared with normal group, the nuclear translocation of Nrf-2, the ROS level, and the mRNA and protein expression of Bax, caspase-3 and caspase-9 were significantly increased in H/R group （P<0.05）. The mRNA and protein expression of SOD2 and Bcl-2, and the cell viability significantly decreased in H/R group compared with normal group, while the apoptosis was increased significantly （P<0.05）. Treatment with mangiferin resulted in an increase in the miR-432-3p expression and a decrease in the ROS level, and the expression of Keap-1, Bax, caspase-3 and caspase-9 was also inhibited as compared with H/R group （P<0.05）. The Nrf-2 nuclear translocation, and the protein levels of SOD2 and Bcl-2 in mangiferin treatment groups were significantly increased as compared with H/R group （P<0.05）. The cell viability was increased significantly, and the apoptosis was decreased significantly in mangiferin treatment groups as compared with H/R group （P<0.05）. The effects of mangiferin in middle- and high-dose groups were better than those in low-dose group, and no significant difference between middle- and high-dose groups was found. CONCLUSION Mangiferin inhibits the decrease in myocardial cell viability and the apoptosis induced by H/R injury. The mechanism may be related to the up-regulation of Nrf-2 antioxidant stress effect via enhancing the expression of miR-432-3p.     

High concentration of extracellular ATP causes autophagy and apoptosis of SH-SY5Y cells

AIM：To examine the effects of high concentration of extracellular ATP on human neuroblastoma SH-SY5Y cell injury. METHODS：Cultured SH-SY5Y cells were grouped according to the concentrations of ATP and treatment time. The cell viability was detected by CCK-8 assay. The variation of autophagic vacuoles was observed with monodansylcadaverine staining. The cell apoptosis was analyzed by Hoechst 33258 staining. Meanwhile, apoptotic rate was detected by flow cytometry. The levels of caspase-3 and microtubule-associated protein 1 light chain 3-Ⅱ (LC3-Ⅱ) were determined by Western blotting. RESULTS：Compared with control group, the survival rate of SH-SY5Y cells was significantly reduced by ATP at different concentrations (3, 6, 9, 12 and 15 mmol/L for 3 h) and different treatment time (1, 2, 3 and 6 h with 6 mmol/L ATP, peaking at 3 h). The autophagic vacuoles of SH-SY5Y cells were significantly increased at 1 h with ATP treatment, trended to decrease over time and returned to control level at 6 h. The protein expression of LC3-Ⅱ was significantly increased at 1 h with ATP treatment, which was consistent with the time points of increasing autophagic vacuoles. LC3-Ⅱ expression level gradually decreased at 2~3 h with ATP treatment, and returned to control level at 6 h. Compared with control group, the apoptotic rate and the expression level of caspase-3 were enhanced synchronously. The peak of apoptotic rate occurred at 3 h, and kept until 6 h.The level of cleaved caspase-3 expression peaked at 6 h. CONCLUSION：High concentration of extracellular ATP induces the autophagy and apoptosis of SH-SY5Y cells. The increased autophagy shows up, followed by the climax of apoptosis until 6 h. With the prolonged duration of ATP, apoptosis is the main process in the cells.     

Effect of transcription factor BACH2 over-expression on viability and apoptosis of human acute lymphoblastic leukemia T lymphocytes

AIM To investigate the effect of over-expression of BTB and CNC homology 2 （BACH2） on the viability and apoptosis of human acute lymphoblastic leukemia T lymphocytes CCRF-CEM. METHODS CCRF-CEM cells were divided into 3 groups： control group， empty vector group， and BACH2 over-expression group. The BACH2 over-expression vector was transfected into CCRF-CEM cells of BACH2 over-expression group by liposome transfection method. The difference in mRNA expression of BACH2 between CCRF-CEM cells and peripheral blood mononuclear cell （PBMC） was detected by qPCR. CCK8 assay was performed to evaluate the viability of CCRF-CEM cells. Flow cytometry was used to analyzed the apoptosis of CCRF-CEM cells. The protein expression of BACH2 and cyclin D3 in the CCRF-CEM cells was observed by immunofluorescence staining. The protein expression of cyclin D3， Bcl-2 and caspase-3 was determined by Western blot. RESULTS The mRNA expression of BACH2 in CCRF-CEM cells was significantly lower than that in PBMC （P<0.05）. Compared with control group， BACH2 over-expression significantly suppressed the viability，increased the apoptotic rate and caspase-3 expression， and decreased the expression of cyclin D3 and Bcl-2 in CCRF-CEM cells （P<0.05）. CONCLUSION BACH2 expression is decreased in T lymphocytes of human acute lymphoblastic leukemia. Over-expression of BACH2 inhibited the viability of human acute lymphoblastic leukemia T lymphocyte and induced apoptosis.