Effect of cyclopamine on aristolochic acid-induced phenotypic transformation and Hedgehog pathway in renal epithelial cells

AIM: To investigate the effect of cyclopamine on Hedgehog (HH) signaling, phenotypic transformation and matrix accumulation induced by aristolochic acid (AA) in renal tubular epithelial cell NRK-52E. METHODS: NRK-52E cells were randomly divided into control group (treated with solvent only), AA group (treated with AA at concentrations of 1, 5, 10 mg/L) and cyclopamine group (treated with AA at concentration of 10 mg/L plus cyclopamine at concentrations of 1, 5, 10 μmol/L). After cultured for 24 h, the mRNA expression of Ptch1, Smo, α-SMA, E-cadherin, ZO-1, BMP-7, type I collagen and type III collagen was quantified by real-time PCR. The protein levels of Shh and TGF-β1 were detected by ELISA. Immunofluorescence staining was used to evaluate the expression of Ptch1, Smo, α-SMA, E-cadherin and type III collagen in the NRK-52E cells. RESULTS: AA increased the expression of TGF-β1, α-SMA and type III collagen, decreased the expression of E-cadherin and ZO-1 protein, and down-regulated the expression of Ptch1, Shh and Smo mRNA in the NRK-52E cells, indicating that AA activated HH signaling, and phenotypic transformation and matrix accumulation occurred in AA-treated NRK-52E cells. Treatment with cyclopamine inhibited HH signaling by decreasing Smo expression and increasing Ptch1 expression. Moreover, cyclopamine also down-regulated the expression of TGF-β1, α-SMA, type I collagen and III collagen, and up-regulated the expression of BMP-7, ZO-1 and E-cadherin. CONCLUSION: AA induces phenotypic transformation and matrix accumulation in renal tubular epithelial cells, which can be inhibited by cyclopamine treatment. The possible mechanism is that cyclopamine suppresses the activation of HH signaling, resulting in the reduction of epithelial-to-mesenchymal transition and matrix deposition.     

Expression of Hedgehog signaling molecules in rat livers after partial hepatectomy

AIM: To detect the expression of Hedgehog signaling molecules in rat livers after partial hepatectomy. METHODS: The model of rat partial hepatectomy was established by resecting the middle and left lobes of the liver. Eighteen male Sprague-Dawley rats were divided into 3 groups: sham operation group (group A), partial hepatectomy group 1 (group B) and partial hepatectomy group 2 (group C). Hepatic tissues were collected 24 h after the operation in group A and group B, and 48 h after the operation in group C. The expression of Ki-67,Sonic Hedgehog(Shh),Indian Hedgehog(Ihh) and Glioblastoma-2(Gli-2) in the hepatic tissues were detected by immunohistochemical staining and Western blotting. RESULTS: The edema and spotty necrosis in the hepatic tissues were observed in group B and group C by HE staining. The cells of different dividing stages were found in the hepatic tissues of group C. Compared with group A, the expression of Ki-67, Shh, Ihh and Gli-2 in group B (P<0.01) and group C (P<0.01) was significantly elevated, and the expression levels in group C were higher than those in group B (P<0.01). CONCLUSION: Hedgehog signaling in rat livers may be activated after partial hepatectomy and stimulate liver regeneration.     

Preliminary study of Sonic Hedgehog signaling pathway in rheumatoid arthritis

AIM: To investigate the expression of Sonic Hedgehog (Shh) signaling pathway-associated factors in peripheral blood mononuclear cells (PBMCs) and synovial tissues of rheumatoid arthritis (RA). METHODS: The mRNA expression levels of Shh, Ptch1 and Gli1 in PBMCs of 35 RA patients, and 35 age-and sex-matched healthy controls were analyzed by real-time PCR. The expression of Shh, Ptch1 and Gli1 in synovial tissues was detected by immunohistochemisty assay in 10 RA patients and 5 patients with traumatic or meniscal injury (no arthritis) as control group. All patients accorded with the American College of Rheumatology (ACR) 1987 revised classification criteria for determining RA, and the score of DAS28 was ≥3.2. RESULTS: The results of real-time PCR showed that the expression of Shh and Gli1 mRNA in RA patients was higher than that in the controls (Shh and Gli1 in RA were 1.36±1.48 and 1.15±0.68, while Shh and Gli1 in control group were 0.47±0.25 and 0.49±0.05, respectively). The mRNA expression of Ptch1 between the 2 groups had no significant difference. Similarly, the results of immunohistochemistry assay showed that the positive staining rates of Shh and Gli1 in RA group were higher than those in control group. However, no difference of Ptch1 positive staining rate between the 2 groups was observed (P>0.05). CONCLUSION: The positive expression of Shh and Gli1 indicates the activation of Shh signaling pathway in the RA patients.     

Effect of up-regulation of Shh-Gli1 signaling pathway on radioresistance and cell cycle in human esophageal cancer cell line Eca109

AIM:To investigate the relationship between Sonic Hedgehog (Shh) signaling pathway and cell cycle and radioresistance of esophageal cancer by up-regulating Gli1, a key factor in Shh signaling pathway. METHODS:The human esophageal cancer cell line Eca109 was transfected with plasmid to induce Gli1 over-expression, which served as Eca109-ox-Gli1 group. In addition, Eca109 cells transfected with empty plasmid served as negative control group and the untreated Eca109 cells were used as normal control group. The expression of Gli1 was confirmed by real-time PCR and Western blot. The radiosensitivity of the cells in the 3 groups was determined by colony formation assay. The effect of irradiation on the cell cycle was analyzed by flow cytometry. RESULTS:The expression of Gli1 in Eca109-ox-Gli1 group was higher than that in the other 2 groups (P<0.05). The survival fraction at dose of 2 Gy in Eca109-ox-Gli1 group was higher than that in normal control group, indicating that the radioresistance of the Eca109 cells transfected with Gli1 plasmid was increased. The cells in Eca109-ox-Gli1 group showed higher S phase proportion than that in normal control group and negative control group (P<0.01). After irradiation at dose of 6 Gy, all cells in the 3 groups found that the cell cycle was arrested at G₂/M phase, while the cells in normal control group showed higher G₂/M phase proportion than that in Eca109-ox-Gli1 group (P<0.01). CONCLUSION:The up-regulation of Gli1 may enhance the radioresistance of esophageal cancer by regulating the cell cycle.     

Effect of Sonic Hedgehog signaling blockade on growth of hepatocarcinoma cells

AIM: To investigate the effect of Sonic Hedgehog (Shh) signaling blockade on the growth of hematocarcinoma cells and underlying mechanisms. METHODS: The expression of Shh signaling molecules in hematocarcinoma cell lines BEL-7402, Huh7 and HepG2 was detected by RT-PCR. The cell viability was detected by MTT assay. The cell cycle and apoptosis were analyzed by flow cytometry. The expression of apoptosis-related proteins was determined by Western blot. RESULTS: Shh signaling molecules were all expressed in BEL-7402, Huh7 and HepG2 cells. The mRNA expression of Patched (Ptch), Gli1 and Gli2 was down-regulated by anti-Shh antibody. Blockade of Shh signaling pathway inhibited the proliferation of hepatocarcinoma cells with increasing cells in G₀/G₁ phase and induced the apoptosis of hepatocarcinoma cells. Treatment with anti-Shh antibody down-regulated the protein expression of pro-caspase-3, pro-caspase-8 and pro-caspase-9, while up-regulated the protein levels of cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9 in BEL-7402 cells. CONCLUSION: Blockade of Shh signaling pathway inhibits the growth of hepatocarcinoma at different levels by cell cycle arrest and inducing apoptosis of hematocarcinoma cells.     

Effect of protease inhibitor bortezomib on treatment of rheumatoid arthritis by affecting IL-33/ST2 signaling pathway

AIM To investigate the effect of bortezomib, a protease inhibitor, on the treatment of rheumatoid arthritis （RA） and it mechanism, based on interleukin-33 （IL-33）/suppression of tumorigenicity 2 （ST2） signaling pathway. METHODS A total of 40 Wistar rats were randomly divided into 4 groups： control group, model group, and low- and high-dose bortezomib groups, with 10 rats in each group. In addition to control group, the rats in other groups were used to construct RA model. Bortezomib was given intraperitoneally at 0.2 mg/kg and 0.5 mg/kg in low- and high-dose bortezomib groups, respectively, while the rats in control group and model group were injected with the same amount of saline, once a day for 21 d. The general situation of the rats in each group was observed, the swelling degree of the foot was calculated, and the inflammation score was evaluated. HE staining was used to observe the pathological changes of ankle joint. The automatic biochemical analyzer was used to detect blood hemoglobin content, the total number of platelets （PLT）, serum creatinine （SCr） level and blood urea nitrogen （BUN） level. The serum levels of IL-6, tumor necrosis factor-α （TNF-α）, IL-33 and ST2 were measured by ELISA. The protein expression of IL-33 and ST2 in ankle tissues of each group was determined by Western blot. RESULTS On the 7th, 14th and 21th days after modeling, compared with control group, the degree of paw swelling in model group was significantly increased （P<0.05）. Compared with model group, the swelling degree of paw in low- and high-dose groups was decreased （P<0.05）. At the end of administration, compared with control group, the synovial cells in model group were increased and in disorder, with a lot of inflammatory exudates in the articular cavity, and the inflammatory score, the levels of PLT, SCr and BUN, the serum levels of IL-6, TNF-α, IL-33 and ST2, and the protein expression of IL-33 and ST2 in ankle tissues were significantly increased （P<0.05）. Compared with model group, the inflammatory exudates in the articular cavity of the rats in low- and high-dose bortezomib groups were decreased, and the inflammatory score, the levels of PLT, SCr and BUN, the serum levels of IL-6, TNF-α, IL-33 and ST2, and the protein expression of IL-33 and ST2 in ankle tissues were decreased （P<0.05）. CONCLUSION Bortezomib may reduce the inflammation and swelling of the joints in RA rats by regulating the IL-33/ST2 signaling pathway.     

Effect of IQGAP1 siRNA on biological characteristics of glioma cells by regulating STAT3 signaling pathway

AIM: To investigate the effect of IQGAP1 gene expression knock-down on invasion, migration and immunosuppression of glioma cells and its mechanism. METHODS: Human glioma U251 cells were randomly divided into blank group, negative control group and si-IQGAP1 group. AG490, an inhibitor of STAT3 signaling pathway, was used to treat the cells for 48 h. The cell viability was measured by MTT assay. The protein levels of IQGAP1, vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), STAT3 and p-STAT3 were determined by Western blot. The cell invasion and migration abilities were detected by Transwell assays. RESULTS: The protein expression of IQGAP1 in si-IQGAP1-1 group and si-IQGAP1-2 group was significantly lower than that in blank group (P<0.05). Compared with blank group, the viability, the invasion ability and the migration ability of the cells in si-IQGAP1 group and AG490 group were significantly decreased, while the protein levels of VEGF, TGF-β1 and p-STAT3 were significantly decreased (P<0.05). Compared with AG490 group, the cell viability, invasion ability and migration ability in AG490+si-IQGAP1 group were significantly decreased, and the protein levels of VEGF and TGF-β1 were significantly decreased (P<0.05). CONCLUSION: Silencing of IQGAP1 gene expression reduces the invasion and migration abilities of glioma cells and decreases the protein expression of cellular immunosuppression molecules VEGF and TGF-β1, which is related to down-regulation of STAT3 signaling pathway.     

Quercitrin promotes apoptosis of gastric cancer cell line SGC7901 by inhibiting PI3K/AKT signaling pathway

AIM: To investigate whether quercitrin induces apoptosis of gastric cancer cell line SGC7901 by inhibition of PI3K/AKT signaling pathway. METHODS: The human gastric cancer SGC7901 cells were selected as the research object. The cytotoxicity of quercitrin was detected by MTT assay, and IC₅₀ value of quercitrin was calculated. The SGC7901 cells were divided into control group, quercitrin group (incubated with 200 μmol/L quercitrin), insulin-like growth factor-1 (IGF-1) group (incubated with 100 μg/L IGF-1) and quercitrin+IGF-1 group (incubated with 200 μmol/L quercitrin and 100 μg/L IGF-1). After 48 h, the apoptosis of SGC7901 cells was analyzed by flow cytometry, and the protein levels of cleaved caspase-3, p-AKT (Ser473), AKT, p-PI3K (Tyr508) and PI3K were determined by Western blot. RESULTS: The viability of SGC7901 cells was significantly decreased as the concentration of quercitrin increased, starting at 100 μmol/L (P<0.05). The IC₅₀ value of quercitrin for 48 h was 275.40 μmol/L. After treatment with 200 μmol/L quercitrin for 48 h, the apoptosis rate and the protein level of cleaved caspase-3 in quercitrin group were significantly increased (P<0.05), and the phosphorylated levels of AKT and PI3K were significantly decreased compared with control group (P<0.05). Treatment with quercitrin and IGF-1 inhibited the effect of quercitrin on SGC7901 cells compared with quercitrin group. CONCLUSION: Quercitrin may induce apoptosis of gastric cancer cell line SGC7901 by inhibiting the activation of PI3K/AKT signaling pathway.     

Effect of Shaofu-Zhuyu decoction on MAPK/ERK signaling pathway in rats with endometriosis

AIM: To observe the effects of Shaofu-Zhuyu decoction (SFZY) on mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway in the rats with endometriosis (EM), and to explore the mechanism of SFZY for treatment of EM.METHODS: Healthy female SD rats were used to establish the EM model. The rats were randomly divided into blank control group, model group, positive control group, and low dose, middle dose and high dose of SFZY groups. The pathological changes of the endometriotic tissue were observed by HE staining. The levels of tumor necrosis factor-α (TNF-α), interleukin-6(IL-6) and IL-8 in the uterine tissue were detected by ELISA. The mRNA expression of ERK, vascular endothelial growh factor (VEGF) and matrix metalloprotein-9 (MMP-9) was detected by RT-qPCR. The protein expression of nuclear factor-κB (NF-κB), MAPK and MAPK-ERK kinase (MEK) was determined by Western blot.RESULTS: Compared with model group, the levels of TNF-α, IL-6 and IL-8 in the uterine tissue of the rats in middle dose and high dose of SFZY groups were significantly decreased (P<0.05), the mRNA expression of ERK, VEGF and MMP-9 was significantly reduced, and the protein expression of NF-κB, MEK and MAPK was decreased significantly in the rat endometriotic tissues (P<0.05).CONCLUSION: SFZY may play a key role in the treatment of EM by regulating MAPK/ERK signaling pathway.     

Effects of pterostilbene on cervical cancer cell autophagy and SIRT1-FoxO signaling pathway

AIM To study the effect of pterostilbene (PTE) on autophagy and SIRT1-FoxO signaling pathway in cervical cancer cells. METHODS Human cervical cancer HeLa cells were used as the study objects. CCK-8 assay and flow cytometry were used to determine the effects of PTE at different concentrations on the viability and apoptotic rate of HeLa cells. The mRNA expression of SIRT1 and FoxO in the cells was detected by qPCR. The number of autophagosomes in the cells was observed under transmission electron microscope.The protein levels of SIRT1, FoxO, LC3-Ⅰ,LC3-Ⅱ, p62, Bax and Bcl-2 in the cells were determined by Western blot. RESULTS After treatment with PTE for 24 and 48 h, the viability inhibitory rate of the HeLa cells was increased with increasing PTE concentration. Compared with 0 μmol/L PTE, after 15, 30 and 60 μmol/L PTE treatment, the apoptotic rate of the HeLa cells, the number of autophagosomes in the cells, and the protein levels of Bax, LC3-Ⅱ/LC3-Ⅰ, SIRT1 and FoxO were increased (P<0.05), while the protein levels of Bcl-2 and p62 were decreased (P<0.05) in a concentration-dependent manner. CONCLUSION PTE may induce autophagy and apoptosis of HeLa cells by activating SIRT1-FoxO signaling pathway, and inhibit HeLa cell viability.     

Effect of HDAC1 silencing on apoptosis of squamous cell carcinoma of skin via regulating STAT3 signaling pathway

AIM: To investigate the effect of histone deacetylase 1 (HDAC1) silencing on apoptosis of squamous cell carcinoma of skin. METHODS: Skin squamous cell carcinoma A431 cells were transfected with HDAC1 small interfering RNA (HDAC1 siRNA) or small interfering RNA negative control (siRNA NC). The expression levels of HDAC1 in transfected cells were detected by RT-PCR and Western blot. The cell viability was measured by MTT assay, and the apoptosis was analyzed by flow cytometry. The protein levels of STAT3, p-STAT3 and cleaved caspase-3 were determined by Western blot. The inhibitor of STAT3 signaling pathway was used to treat the A431 cells transfected with HDAC1 siRNA. The cell viability was detected by MTT assay, the apoptosis was analyzed by flow cytometry, and the protein levels of STAT3, p-STAT3 and cleaved caspase-3 were determined by Western blot. RESULTS: HDAC1 siRNA inhibited the expression of HDAC1 at mRNA and protein levels in the A431 cells. After interfering with the expression of HDAC1, the cell viability and the protein level of p-STAT3 in the cells decreased, while the apoptotic rate and the protein level of cleaved caspase-3 in the cells were increased. After treatment with the inhibitor of STAT3 pathway, the viability of A431 cells transfected with siRNA and the protein level of p-STAT3 decreased, while the apoptotic rate and the protein le-vel of cleaved caspase-3 in the cells were increased. CONCLUSION: Interference with HDAC1 expression may regulate the STAT3 signaling pathway to inhibit the viability of skin squamous cell carcinoma cells, thus promoting the apoptosis of squamous cell carcinoma of skin.     

27-Hydroxycholesterol modulates lung cancer cell proliferation by activation of LXR signaling pathway

AIM:To investigate the effect of cholesterol metabolite 27-hydroxycholesterol (27-OHC) on the proliferation of lung cancer cells. METHODS:Human lung cancer A549 cells were treated with 27-OHC at different concentrations (0, 0.3125, 0.625, 1.25, 2.5, 5 and 10 μmol/L) for 24~48 h. The cell viability, cell cycle, cell prolife-ration, the intracellular cholesterol levels and cholesterol metabolism-related molecule expression were subsequently assessed by CCK-8 assay, flow cytometry, EdU staining, tissue total cholesterol detection kit, real-time PCR and Western blot. RESULTS:27-OHC decreased the viability of the A549 cells in a dose-and time-dependent manner (P<0.01) and inhibited the cell proliferation (P<0.05). The expression of typical liver X receptor (LXR) downstream target proteins including ATP-binding cassette transporter A1 (ABCA1), low-density lipoprotein receptor (LDLR), and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CR) were modulated, which promoted the efflux of intracellular cholesterol, and reduced cholesterol influx and de novo synthesis, resulting in decreased intracellular cholesterol levels and cell viability. Furthermore, the inhibitory effect of 27-OHC on A549 cell viability was significantly attenuated after the LXR pathway was partially blocked by 5 μmol/L GSK2033 treatment (P<0.05). CONCLUSION:27-OHC inhibits A549 cell prolife-ration via activation of LXR signaling pathway.     

Glucosylceramide synthase upregulates apoptosis-related gene bcl-2 expression via MEK/ERK signaling pathway in leukemia multidrug-resis-tant cell line

AIM: To investigate whether glucosylceramide synthase (GCS) regulates apoptosis-related gene bcl-2 expression via MEK/ERK signaling pathway, thus enhancing drug resistance of K562/A02 human leukemia multidrug resistant cell line. METHODS: siRNA targeting GCS was transfected into K562/A02 cells. Bcl-2, p-ERK and total ERK expression at mRNA and protein levels after GCS knockdown were detected by real-time PCR and Western blotting. After exposed to MEK-ERK pathway inhibitor U0126, the expression of Bcl-2 at mRNA and protein levels also was analyzed by real-time PCR and Western blotting, respectively. The viability of the cells was evaluated by CCK-8 assay. RESULTS: The expression of GCS and Bcl-2, as well as MEK/ERK signaling were significantly inhibited in K562/A02 cells by GCS siRNA transfection compared with negative control group. Inactivation of MEK/ERK signaling due to U0126 treatment decreased Bcl-2 mRNA and protein levels in a concentration-dependent manner, and sensitized K562/A02 cells to adriamycin. CONCLUSION: GCS may affect the expression of apoptosis-related gene bcl-2 by MEK/ERK signaling pathway, thus regulating multidrug resistance of human leukemia K562/A02 cells.     

S100A6 promotes proliferation and migration of human osteosarcoma cell line 143B through PI3K/Akt signaling pathway

AIM: To investigate the effect of PI3K/Akt signaling pathway on S100A6-induced proliferation and migration of human osteosarcoma cell line 143B. METHODS: Recombinant human S100A6 protein (rhS100A6) was prepared. The 143B cells were treated with rhS100A6 in the presence or absence of PI3K inhibitor (LY294002 or wortmannin) exposure. The final concentrations of rhS100A6, LY294002 and wortmannin were 30 mg/L, 10 μmol/L and 0.5 μmol/L, respectively. The expression levels of total Akt (t-Akt) and phosphorylated Akt (p-Akt) in the 143B cells were analyzed by Western blotting. The cell proliferation and migration were determined by MTT and Transwell assays. RESULTS: rhS100A6 protein was successfully prepared, and significantly increased the proliferation and migration of 143B cells (P<005). rhS100A6 up-regulated the phosphorylation of Akt in 143B cells (P<005). Compared with rhS100A6 group, the level of p-Akt in 143B cells and the proliferation and migration of the cells were decreased in combined treatment group of rhS100A6 with LY294002 or wortmannin (P<005), where the proliferation rate at different time points dropped from 10.3% to 69.7% (P<005), and the migration rate dropped from 34.9% to 47.7% (P<005). CONCLUSION: To some extent, S100A6 promotes proliferation and migration of human ostersarcoma cell line 143B through PI3K/Akt signaling pathway.     

Effect of serum amyloid A on invasion,migration and MAPK signaling pathway in osteosarcoma cells

AIM: To investigate the effect of silencing of serum amyloid A (SAA) on the viability, apoptosis, migration and mitogen-activated protein kinase (MAPK) signaling pathway in osteosarcoma U2OS cells. METHODS: Small interfering RNA (siRNA) targeting SAA was transfected into U2OS cells to silence the expression of SAA gene. The U2OS cells were divided into blank control group, negative control group, and experimental group. The cells in negative control group and experimental group were transfected into negative control siRNA and SAA-siRNA, respectively. The cells in blank control group were without any treatment. The viability of the cells was measured by MTT assay and the apoptotic rate was analyzed by flow cytometry with Annexin V-FITC/PI double staining. The migration and invasion abilities of the cells were detected by Transwell chamber assay. The protein levels of SAA, phosphorylated p38 MAPK (p-p38 MAPK) and phosphorylated c-Jun N-terminal kinase (p-JNK) in the cells were determined by Western blot. RESULTS: The protein expression of SAA in SAA-siRNA group was significantly lower than that in blank control group (P<0.05). Compared with blank control group, the cell viability in SAA-siRNA group was significantly decreased (P<0.05), the apoptotic rate was significantly increased (P<0.05), and the invasion and migration abilities were significantly decreased (P<0.05). The protein levels of p-p38 MAPK and p-JNK in SAA-siRNA group were significantly lower than those in blank control group (P<0.05), and no significant difference of total JNK and p38 protein levels was observed. CONCLUSION: Silencing of SAA expression inhibits the viability of osteosarcoma cells, induces apoptosis and decreases the migration of osteosarcoma cells, which may be related to the activation of MAPK signaling pathway.     

Effect of chelerythrine on TGF-β/Smads signaling pathway in mouse livers with hepatic fibrosis

AIM:To investigate the anti-hepatic fibrosis effect of chelerythrine on mice and the regulation of transforming growth factor-β (TGF-β)/Smads signaling pathway. METHODS:C57BL/6N mice (n=50) were randomly divided into control group, model group and chelerythrine groups (10 mg·kg^-1·d^-1, 20 mg·kg^-1·d^-1 and 40 mg·kg^-1·d^-1, ig). The mouse model of hepatic fibrosis was established by intraperitoneal injection of carbon tetrachloride (CCl₄) in combination with the olive oil for 8 weeks. At the 5th week, different doses of chelerythrine was used to treat hepatic fibrosis in the mice. At the 14th week, hepatic index was detected. Histopathological changes and the degree of hepatic fibrosis were observed by hematoxylin-eosin staining and Van Gieson staining. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hyaluronic acid (HA), and hepatic hydroxyproline (Hyp) content were assayed by spectrophotometry and ELISA. The mRNA expression of TGF-β₁, Smad3, Smad4 and Smad7 in the liver was detected by RT-qPCR, and the protein expression of TGF-β₁, Smad4 and Smad7 was determined by Western blot. RESULTS:The degree of hepatic fibrosis changed markedly in model group compared with control group. The hepatic index, the serum levels of ALT and AST, and the contents of HA and Hyp were significantly increased (P<0.05). The mRNA expression of TGF-β₁, Smad3 and Smad4 was significantly up-regulated, while the mRNA expression of Smad7 was significantly down-regulated (P<0.05). The protein expression of TGF-β₁ and Smad4 was significantly up-regulated, while the protein expression of Smad7 was significantly down-regulated (P<0.05). Compared with model group, the changes of the above indexes in chelerythrine groups were inhibited. CONCLUSION:Chelerythrine protects the mouse liver from CCl₄-induced fibrogenesis injury by regulating TGF-β/Smads signaling pathway.     

Effect of salidroside on activity of endothelial progenitor cells and phosphoinositide 3-kinase/Akt signaling pathway

AIM：To investigate whether salidroside has influence on the activities of endothelial progenitor cells (EPCs) and its mechanism. METHODS：Mononuclear cells from normal human peripheral blood were cultured in fibronectin coated flasks in endothelial progenitor medium. After 7 d, EPCs were characterized as adherent cells with acLDL-DiI uptaking and lectin binding by direct fluorescent staining. The proliferation and migration of EPCs were analyzed by MTT assay and Transwell chamber assay, respectively. The EPCs adhesion assay was performed by re-plating the cells on fibronectin-coated dishes, and then adherent cells were counted. NO and Akt protein were also detected. RESULTS：Salidroside promoted EPCs proliferative, migratory and adhesive capacities in a concentration dependent manner. Salidroside also increased NO secretion, and the level of phosphorylated Akt protein. However, the effects of salidroside on EPCs were inhibited by phosphoinositide 3-kinase inhibitor LY294002. CONCLUSION：Salidroside regulates the activity of EPCs by phosphoinositide 3-kinase/Akt signaling pathway.     

Effect of ixazomib on apoptosis and NF-κB signaling pathway in pancreatic cancer cells

AIM:To investigate the effects of ixazomib on the apoptosis and NF-κB signaling pathway in pancreatic cancer cells. METHODS:Human pancreatic cancer cell lines CFPAC-1 and PANC-1 were cultured, and the cells were treated with ixazomib at 0, 10, 20, 30 and 40 nmol/L for 12, 18, 24 and 48 h. The expression of NF-κB p65, IκB kinase (IKK), Bax and caspase-3 in the cells at mRNA and protein levels was determined by RT-qPCR and Western blot. The cell viability was measured by CCK-8 assay. The apoptosis was analyzed by flow cytometry. RESULTS:Treatment with ixazomib at 10~40 nmol/L inhibited the viability of PANC-1 cells and CFPAC-1 cells, and the inhibitory rate was increased significantly with the increases in the concentration and time (P<0.05). Compared with the control cells, treatment with ixazomib significantly increased the apoptotic rates of PANC-1 cells and CFPAC-1 cells in a dose- dependent manner (P<0.05), and significantly decreased the mRNA expression levels of NF-κB p65 and IKK in the PANC-1 cells and CFPAC-1 cells (P<0.05), while the mRNA expression levels of apoptotic factors Bax and caspase-3 in the PANC-1 cells and CFPAC-1 cells were significantly increased (P<0.05). The results of Western blot showed that treatment with ixazomib significantly decreased the protein levels of NF-κB p65 and IKK in the PANC-1 cells and CFPAC-1 cells (P<0.05), which was consistent with the results of mRNA expression. The protein levels of apoptosis factors Bax and caspase-3 in the CFPAC-1 cells were significantly increased (P<0.05), and the protein level of caspase-3 in the PANC-1 cells was increased significantly (P<0.05). However, Bax protein did not increase significantly in 10 nmol/L ixazomib group. CONCLUSION:Ixazomib, a proteasome inhibitor, inhibits the viability of pancreatic cancer cells and promotes apoptosis by inhibiting the activation of NF-κB signaling pathway in a time- and dose-dependent manner.