Effect of Hedgehog signaling pathway inhibitor on biological behavior of intrahepatic cholangiocarcinoma cell line RBE

AIM: To investigate the effect of cyclopamine, a Hedgehog(Hh) signaling pathway inhibitor, on the biological behavior of intrahepatic cholangiocarcinoma cell line RBE. METHODS: The proliferation of RBE cells was detected by cell counting with Typan blue staining and MTT assay, and the apoptosis was analyzed by the flow cytometry. The Transwell invasive cabin assay was used to detect the invasion ability, and Western blot was used to determine the protein expression of Gli1 and MMP-9 in the RBE cells before and after cyclopamine treatment. RESULTS: Cyclopamine inhibited the growth of RBE cells in a time-and dose-dependent manner. After cyclopamine treatment for 24 h, 48 h and 72 h, the apoptotic rates were significantly higher than those in control group. In control group, the number of cells invading through the Matrigel of invasion chamber was 154.52±13.61, while in experimental group it was 62.00±12.17, indicating that the invasion ability of the cells declined significantly. Furthermore, Western blot showed that the protein levels of Glil and MMP-9 in the RBE cells were decreased after treatment with cyclopamine for 24 h and 48 h. CONCLUSION: Blockage of the Hh signaling pathway with cyclopamine suppresses the proliferation, promotes the apoptosis and inhibits the invasion ability of RBE cells.     

Effects of Qiancaofang fumigation on expression of YAP/Smad3 in rats with knee osteoarthritis

AIM: To observe the effects of Qiancaofang fumigation on the expression of Yes-associated protein (YAP) and Smad3 in the cartilage of the rats with knee osteoarthritis (KOA), and to investigate the protective effects and mechanisms of Qiancaofang fumigation on the cartilage of the rats with KOA. METHODS: SD rats (n=32) were randomly divided into 4 groups:control group, model group, Chinese medicine group and hyaluronic acid (HA) group. The improved Hulth method was used to construct the rat model of KOA in model group, Chinese medicine group and HA group. The rats in Chinese medicine group were treated with Qiancaofang fumigation. The rats in HA group were treated with intra-articular injection of HA. After treatments, the cartilage tissues and serum of the rats in each group were taken. Hematoxylin-eosin (HE) staining and Mankin's scores were measured. The expression levels of cartilage oligomefic matrix protein (COMP) and C-telopeptide of type Ⅱ collagen (CTX-Ⅱ) in the serum were detected. The method of TdT-mediated dUTP nick-end labeling (TUNEL) was used to observe the apoptosis of the cartilage cells. Immunohistochemistry was performed to detect the expression of YAP and high mobility group protein B2 (HMGB2). Western blot was used to detect the expression of YAP, Smad3, bone morphogenetic protein 2 (BMP2), Bax and Bcl-2. RESULTS: The results of HE staining showed that the cartilage cells in model group were disordered and the structure was not clear. Compared with model group, the cartilage surface in Chinese medicine group was more smooth, the structure was clearer, and the staining of the cartilage matrix was more uniform. Compared with model group, the Mankin's scores in Chinese medicine group and HA group were significantly decreased (P<0.05), the serum concentration of CTX-Ⅱ was significantly increased, the serum concentration of COMP was significantly decreased, and the apoptosis rate of the cells was decreased (P<0.05). The results of immunohistochemistry and Western blot showed that the expression levels of YAP, HMGB2, Smad3 and Bcl-2 were increased in Chinese medicine group and HA group, and the expression levels of BMP2 and Bax were decreased as compared with model group (P<0.05). CONCLUSION: Qiancaofang fumigation relieves KOA partially through the YAP/Smad3 signaling pathway.     

Preventive effect of viartrils on the cartilage degenration of osteoarthritis in rabbits

AIM: To determine whether the viartrils could provide a beneficial effect on the prevention of early/middle stage osteoarthritis(OA) and affect the proliferation of chondrocytes. METHODS: An OA model was produced with severing the anterior, posterior cruciate ligaments of the knee in 24 adult New Zealand rabbits. The animals were then randomly divided into viartrils group and control group. After surgical operation, viartrils (mainly contains glucosamine sulphate) 2 pills per day were administered to the animals in viartrils group. The animals were sacrificed and specimens were taken from the weight-bearing portion of the femoral condylar seven weeks after operation. Each case was evaluated according to a modified histological-histochemical grading system(HHGS) using HE and safranin O/fast green staining slides, and immunohistochemical method was used to detect the proliferation of chondrocytes in articular cartilage. RESULTS: The method of severing the anerior, posterior cruciate ligaments of the knee could successfully induce the early/middle stage model of OA. The pathological remark in control group was significantly higher than that in the viartrils group (P<0.05). The proliferative index of viartrils group chondrocytes was also higher than that in control (P<0.05). CONCLUSION: These data indicate that viartrils could increase the proliferation of chondrocytes and prevent the development of OA pathologic process.     

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 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.     

Effects of Youguiwan on microstructure and Hsp90 of chondrocytes in rats with knee osteoarthritis

AIM: To observe the effect of Youguiwan (YGW) on the expression of heat shock protein 90α (Hsp90α) in articular cartilage tissues of knee osteoarthritis (KOA) rats, and to further reveal its mechanism. METHODS: SD rats (n=60) were randomly divided into 6 groups:sham control (SC) group, model (M) group, glucosamine sulfate (GS) group, and high-, middle-and low-dose YGW (YGW-H, YGW-M and YGW-L) groups. The modified Hulth method was used to make KOA models for 6 weeks. The rats were gavaged with corresponding drugs for 8 weeks. HE staining was used and Makin score was evaluated. The expression of Hsp90α, fibronectin (Fn) and collagen type Ⅱ (COL-II) was determined by immunohistochemistry. The expression of interleukin-1β (IL-1β), matrix metalloproteinase (MMP)-3 and MMP-13 was analyzed by RT-qPCR. The protein expression of Hsp90α and COL-II was determined by Western blot. RESULTS: As compared with SC group, the Makin score was obviously raised in M group, the expression levels of Hsp90α, IL-1β, MMP-3 and MMP-13 were evidently increased, the expression levels of COL-II and Fn were evidently decreased (P < 0.01), articular cartilage was seriously damaged, and the chondrocytes were disarranged. As compared with M group, the Makin score and the expression of Hsp90α were obviously decreased in YGW-H group, the protein expression of Fn was evidently increased in YGW-H group, the protein expression of COL-II was evidently increased in YGW-H and YGW-M groups, the mRNA expressions of IL-1β, MMP-3 and MMP-13 were evidently decreased in YGW-H, YGW-M and YGW-L groups (P < 0.05 or P < 0.01), cartilage structure tended to be normal, the chondrocytes distribution was uneven, and articular cartilage surface was not smooth. CONCLUSION: Youguiwan is effective for treatment of KOA. Youguiwan protects against articular cartilage degeneration by down-regulating the expression of Hsp90α and up-regulating the expression of Fn, and reducing the secretion of inflammatory factors and the degradation of extracellular matrix.     

Protective effect of curcumin on MRL/lpr mice with lupus nephritis and its mechanism

AIM To observe the effect of curcumin （Cur） on lupus nephritis （LN） and its possible mechanism. METHODS Thirty 10-week-old MRL/lpr lupus mice were randomly divided into MRL/lpr group, Cur-L and Cur-H group with 10 mice in each group, and C57BL/6 mice （n=10） served as normal control （NC） group. The mice in Cur-L group and Cur-H group were given intragastric administration of Cur at 100 and 200 mg·kg^-1·d^-1 for 12 weeks, respectively, and the same volume of normal saline was given to the mice in NC group and MRL/lpr group. The urine protein was detected, and the morphological changes of the renal tissue were observed by HE staining after treatment. The levels of serum creatinine （SCr）, blood urea nitrogen （BUN） and serum anti-double-stranded DNA （dsDNA）, and interleukin-1β （IL-1β）, IL-6 and tumor necrosis factor-α （TNF-α） levels in serum and renal tissues were detected. The protein levels of p-IκB, NF-κB, NLRP3 and caspase-1 in the renal tissues were determined by Western blot. RESULTS Compared with MRL/lpr group, the content of urine protein in Cur groups was significantly reduced, and the renal injury was relieved. The SCr, BUN, serum anti-dsDNA, and the serum and renal levels of IL-1, IL-6 and TNF-α were all significantly reduced, and the protein levels of p-IκB, NF-κB, NLRP and caspase-1 in the renal tissue were significantly decreased （P<0.05）. CONCLUSION Cur has a certain protective effect on the kidney of MRL/lpr mice, and its mechanism may be related to the inhibition of NF-κB and NLRP3 signaling pathways.     

Protective effect of chrysin on mice with insulin resistance

AIM: To explore the effects of chrysin on insulin resistance (IRe) in a mouse model. METHODS: Male C57 mice were randomly divided into control group, IRe group, low-dose chrysin group (IRe+chrysin-low) and high-dose chrysin group (IRe+chrysin-high). After 24 weeks, the body weight, liver index and fat mass in all mice were detected. The blood glucose, insulin level and HOMA-IR were measured to determine the changes of the insulin resistance in the animals. The oxidative stress (SOD, GSH-Px and MDA) was also measured. The mRNA expression of insulin signaling pathway molecules (IR, IRS1, IRS2, Glut2 and Glut4) and inflammatory factors (TNF-α, IL-1β, IL-6 and NF-κB) was analyzed by real-time PCR. The protein levels of IRS1 and p65, and their phosphorylation were detected by Western blot. RESULTS: After 24-week intervention, the indicators in IRe group were higher than those in control group, including body fat deposition, serum glucose, serum insulin, HOMA-IR and liver oxidative stress (P<0.01), indicating that the model of insulin resistance was successfully established. Low dose and high dose of chrysin decreased the body weight, serum glucose, serum insulin and HOMA-IR in the IRe mice (P<0.05). The liver oxidative stress was also reduced in both groups (P<0.05). However, no statistical difference of the indexes between IRe+chrysin-low group and IRe+chrysin-high group was observed. Chrysin upregulated the mRNA expression of IR, IRS1, IRS2, Glut2 and Glut4 (P<0.05), and down-regulated the mRNA expression of various inflammatory factors. The inhibitory effect of chrysin on the mRNA expression of NF-κB was observed (P<0.05), especially in high dose group (P<0.05). It was confirmed that the effect of chrysin on liver IRe was related with the increase in the p-IRS1 levels and decrease in the p-p65 levels by Western blot. CONCLUSION: Chrysin inhibits obesity, hyperglycemia and hyperinsulinemia, and relieves insulin resistance and oxidative stress, which might be closely related to the regulation of insulin signaling pathway and the inhibition of inflammatory factor expression.     

Protective effects of DAPT on rat model with atherosclerotic ischemic brain stroke by blocking Notch pathway

AIM: To investigate the protective effects of DAPT on rat model with atherosclerotic (AS) ischemic brain stroke by blocking Notch pathway. METHODS: SD rats (n=24) were randomly divided into control group and model group, and the rats in model group were fed high-fat diet for 6 weeks to establish the AS model. The AS rats were randomly divided into 3 groups (n=6 in each group):AS-sham group, AS rats with ischemia (AS-ishemia) group, and DAPT administration (AS-ishemia-DAPT) group. The histopathological changes of carotid aorta were observed by HE staining. The serum levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were measured by automatic biochemical analyzer. The levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were detected by ELISA. The protein levels of Notch1 and Hes1 in rat artery, and nuclear factor-κB (NF-κB) and Toll-like receptor 4 (TLR4) in rat brain were determined by Western blot. RESULTS: Notch signaling pathway inhibitor DAPT significantly reduced intimal thickening, vascular stenosis and the formation of AS plaque. Compared with AS-ischemia group, the serum levels of lipids and inflammatory factors were decreased significantly in AS-ischemia-DAPT group, and the protein levels of Notch1 and Hes1 in the rat carotid artery and NF-κB and TLR4 protein expression in rat brain were also decreased significantly (P<0.05). CONCLUSION: Blocking Notch pathway by DAPT not only improves the blood lipid levels, but also inhibits the serum inflammatory cytokine release and NF-κB/TLR4 pathway activation.     

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.     

Changes of BMP signaling pathway in mammary glands of mice with mammary hyperplasia

AIM: To determine the expression patterns of bone morphogenetic protein (BMP) signaling components in the mammary glands with hyperplasia (MGH) and the activation of this signaling pathway. METHODS: The female C57BL/C mice (8-weeks-old) were randomly divided into control group and hormone group with 15 mice in each group. The mice in control group were treated with PBS, while the mice in hormone group were intragastric administration with estradiol valerate (2.5 mg/kg) for 25 d, followed by progesterone (4 mg/kg) peritoneal injection for 5 d. At the end of treatments, the mammary glands were collected to determine the morphological changes, the mRNA expression of BMP signaling components and the phosphorylation level of Smad1/5/9. RESULTS: In MGH, a part of BMP signaling pathway molecules were differentially expressed (P<0.05), including BMP ligands BMP2, 4, 5, 6, 7, 9, 13 and 14, BMP receptor BMPR1A, and antagonists Chrdl1 and Twsg, whereas the expression levels of some molecules, such as BMP3, BMP12, BMPR1B, BMPR2, Chrd, Chrdl2 and Noggin, were not altered. The phosphorylation levels of Smad1/5/9 was up-regulated in MGH (P<0.05). CONCLUSION: BMP signaling pathway is activated in MGH through the abnormal expression of its components. BMP signaling pathway may be a new target for MGH therapy.     

Protective effect of paeoniflorin on nerve cells in APP/PS1 mice and its mechanism

AIM: To investigate the therapeutic and preventive effects of paeoniflorin (PF) on APP/PS1 mice, and to explore the possible mechanism. METHODS: Fifteen male 5-month-old APP/PS1 non-dominant mice were chosen as normal control group, 15 male 5-month-old APP/PS1 double transgenic mice were used as model group, and 15 male 5-month-old APP/PS1 double transgenic mice treated with 5 mg/kg PF by intraperitoneal injection were allocated in administation group. The learning and memory ability of the mice in each group was detected by Morris water maze. The apoptosis was assessed by TUNEL fluorescence staining. The protein expression of PI3K, Akt, p-PI3K, p-Akt, caspase-3, caspase-9, Bcl-2 and Bax in cerebral cortex and hippocampus was detected by Western Blot. The protein expression levels and distribution of caspase-3 and caspase-9 were detected by immunohistochemistry. RESULTS: (1) Compared with normal control group, the learning and memory ability declined in APP/PS1 model group. Compared with APP/PS1 model group, PF obviously improve the ability of learning and memory in mice. (2) Compared with normal control group, the apoptosis of nerve cells in APP/PS1 model group significantly increased and distributed in wider areas, while that in PF group was reduced (P<0.05). (3) Compared with APP/PS1 model group, PF could significantly lower pro-apoptotic factors, caspase-3, caspase-9 and Bax (P<0.05), and increase the expression of anti-apoptotic factors, p-PI3K, p-Akt and Bcl-2 (P<0.05). CONCLUSION: PF can up-regulate the expression of Bcl-2 and down-regulate the expression levels of caspase-9, caspase-3 and Bax via the activation of PI3K/Akt pathway, thereby inhibiting the nerve cell apoptosis and protecting the nerve cells, so as to treat neurodegenerative diseases.     

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.     

Role of EndoMT in HHPH based on TGF-β/Smads signaling pathway and regulated by Yiqi-Wenyang-Huoxue-Huatan formula

AIM: To investigate the relationship between transforming growth factor-β (TGF-β)/Smads signaling pathway and pulmonary arterial endothelial-mesenchymal transition (EndoMT) in hypoxia-hypercapnia pulmonary hypertension (HHPH) process and the regulatory effect of Yiqi-Wenyang-Huoxue-Huatan formula (YWHHF). METHODS: Healthy male SD rats were randomly divided into 5 groups:normal control (N) group, hypoxia-hypercapnia (HH) group, high-dose YWHHF (YH) group, middle-dose YWHHF (YM) group and low-dose YWHHF (YL) group. The rats in N group was housed in normoxic environment, and the rats in the other 4 groups were housed in hypoxia-hypercapnia environment (9%~11% O₂ and 5%~6% CO₂) for 4 weeks, 8 h/d, 6 d/week. The excess water vapor was absorbed by anhydrous CaCl₂, and CO₂ was absorbed by sodium hydroxide. The rats in YWHHF groups were put into the oxygen chamber before the same volume of YWHHF at different concentrations were given (200 g/L for YH group, 100 g/L for YM group and 50 g/L for YL group). The average pulmonary artery pressure and the average carotid artery pressure were measured during the operation. After operation, the right ventricular free wall and left ventricle plus interventricular septum were collected for determining the right ventricular hypertrophy index. Moreover, the morphological changes of the lung tissues were observed under light microscope. The mRNA and protein levels of α-smooth muscle actin (α-SMA), CD31, TGF-β1 and Smad2/3, and the protein level of p-Smad2/3 were detected by RT-PCR and Western blot. RESULTS: Compared with N group, the pulmonary artery mean pressure, the mRNA and protein expression of α-SMA, TGF-β1 and Smad2/3, and the protein level of p-Smad2/3 were increased, the levels of CD31 were decreased (P<0.05), and the lung tissue damage was observed in the other 4 groups. Compared with HH group, the pulmonary artery mean pressure, the mRNA and protein expression of α-SMA, TGF-β1 and Smad2/3, and the protein level of p-Smad2/3 were decreased, while the mRNA and protein levels of CD31 were increased. Moreover, the lung tissue damage was reduced in YH, YM and YL groups. CONCLUSION: TGF-β/Smads pathway may be involved in the process of EndoMT under hypoxia and hypercapnia condition, and YWHHF may reduce EndoMT by inhibiting the expression of TGF-β/Smads pathway-related molecules.     

Protective effects of Zhouluotong extract Z-6 on Schwann cells damaged by high-glucose and PI3K/Akt/nNOS pathway

AIM:To explore the role of PI3K/Akt/nNOS in Zhouluotong extract resisting diabetic peripheral neuropathy. METHODS:The Schwann cells were divided into normal group (D-glucose 25 mmol/L), model group (D-glucose 100 mmol/L), Zhouluotong extract Z-6 + high glucose group, Zhouluotong + high glucose group, mecobalamine + high glucose group. The viability, nitric oxide content and the Ca2+-ATPase activity in Schwann cells were determined by Cell Counting Kit-8 , nitric oxide assay kit and Ca2+-ATPase assay kit, respectively. The apoptosis of Schwann cells were analyzed by flow cytometry. The expression of Bcl-2, Bcl-xL, Bax, Bak and caspase-3, and the phosphorylation levels of nNOS and Akt were determined by Western blotting. The signal pathway of PI3K/Akt was explored by dominant negative PI3K and Akt (δp85 and DN-Akt) transient transfection assay. RESULTS:Under high-glucose culture, the cell viability, nitric oxide content in culture supernatant, the expression of Bcl-2 and Bcl-xL, and the phosphorylation levels of Akt and nNOS in the Schwann cells were significantly increased. The cell apoptosis, the expression of Bax, Bak and caspase 3 in the Schwann cells were significantly decreased by Zhouluotong extract Z-6, compared with model group. Increased nitric oxide content and the up-regulation of nNOS were observed. However, the effects of blocking PI3K/Akt, the upstream pathway of nNOS , by transfection with DN-δp85 on Akt phosphorylation in the Schwann cells was still unclear. CONCLUSION: Zhouluotong extract Z-6 changes the phosphorylation of nNOS, and the expression of anti-apoptotic factors, caspase-3 and pro-apoptotic factors in Schwann cells under high-glucose culture, thus reducing apoptosis and elevating viability. The relationship to PI3K/Akt/nNOS pathway needs further investigation.     

Fluorocitrate inhibits regulatory effect of ischemic postconditioning on HIF-1α/iNOS signaling pathway after cerebral ischemia in tree shrews with brain injury

AIM: To investigate the regulatory effect of HIF-1α/iNOS signaling pathway on the neuroprotection of ischemic postconditioning (PC) in tree shrews, and to explore the mechanisms of deteriorated cerebral injury after inhibiting astrocyte (AS) metabolism. METHODS: Thrombotic cerebral ischemia was induced by photochemical reaction in tree shrews. Fluorocitrate (FC) was used to inhibit AS metabolism and the ischemic PC was established at 4 h after ischemia followed by clipped ipsilateral common carotid artery on the ischemia side for 3 times, 5 min/time. A total of 67 male tree shrews were randomly divided into 7 groups:control (n=9), ischemia (4 h and 24 h, n=9 for each group), ischemia with PC (4 h and 24 h, n=9 for each group), and FC pretreatment (4 h and 24 h, n=11 for each group). The cerebral infarction size was detected by TTC staining, and the histological changes of hippocampal neurons were observed under light microscope. The regional cerebral blood flow (rCBF) in ischemic cortex was monitored by laser Doppler brain flowmetry. The protein expression of iNOS in hippocampus was detected both by immunohistochemistry and Western blot. The production of NO detected by spectrophotometer. The level of HIF-1α in hippocampus analyzed by ELISA. RESULTS: The cerebral infarct volume was increased with prolonged duration of ischemia, and the changes of ischemia at 24 h were significant (P<0.05). The cortical rCBF was progressively decreased, and it was decreased at 4 h and 24 h after ischemia (P<0.05). The expression of HIF-1α and iNOS in hippocampus was enhanced, and the production of NO was increased significantly (P<0.05). Ischemic PC restored the cortical rCBF (P<0.05), reduced cerebral infarction volume (P<0.05), down-regulated iNOS expression and reduced NO production in the hippocampus (P<0.05). However, the cortical rCBF in FC pretreatment group was significantly lower than that in ischemic group (P<0.05), the neuronal damage was aggravated, and the infarction volume was increased after pretreatment with FC (P<0.05). CONCLUSION: Ischemic PC may reduce cerebral injury by regulating the expression of HIF-1α and iNOS. Inhibition of AS function may attenuate the protective effect mediated by ischemic PC and aggravate brain injury.     

Effects of JNK signaling pathway on hnRNP K protein aggregates in NIH3T3 cells treated with sodium arsenite

AIM: To explore the role of JNK signaling pathway in the formation of heterogeneous nuclear ribonucleoprotein (hnRNP) K protein aggregates by observing the expression and localization of hnRNP K in NIH3T3 cells induced by sodium arsenite(NaAsO2).METHODS: The recombinant vector pcDNA3-hnRNP K-HA was constructed and transfected into NIH3T3 cells. The expression and localization of endogenous hnRNP K and distribution of the protein aggregates stimulated with NaAsO2 at different time points were observed under fluorescence microscope. The level of reactive oxygen species (ROS) in the cells was detected by a ROS assay kit. After pretreated with the inhibitors of 5 signaling pathways (JNK, MEK, PI3K/Akt, NF-κB and nuclear transport), the changes of the protein aggregates were observed.RESULTS: The recombinant plasmid was correctly constructed. The hnRNP K was mainly distributed in the nucleus. The intracellular fluorescent aggregates increased with the prolonging stimulation of NaAsO2 in the cells transfected with the recombinant plasmid, and the overexpression of hnRNP K inhibited ROS generation in the cells induced by NaAsO2. SP600125, an inhibitor of JNK signaling pathway, significantly inhibited the formation of protein aggregates. CONCLUSION: The hnRNP K is primarily located in the nucleus of NIH3T3 cells, with a small amount in the cytoplasm. The formation of protein aggregates is significant after NaAsO2 stimulation, which can restrain the level of intracellular ROS, and the process is involved in JNK signaling pathway.     

Inhibitory effect of butylphthalide on apoptosis of hippocampal neurons in Alzheimer disease rats via SIRT1/NF-κB signaling pathway

AIM: To investigate the effect of butylphthalide on apoptosis of hippocampal neurons in Alzheimer disease (AD) rats via SIRT1/NF-κB signaling pathway and its mechanism. METHODS: AD rat model was established by intragastric administration of AlCl₃ and intraperitoneal injection of D-galactose. After treated with butylphthalide at 25 mg/kg (low dose), 50 mg/kg (medium dose) and 100 mg/kg (high dose), the effects of butylphthalide on the morphology of hippocampal neurons, apoptosis rate, and the protein levels of Bcl-2, Bax, cleaved caspase-3 and the SIRT1/NF-κB signaling pathway associated proteins were determined by HE staining, flow cytometry and Western blot, respectively. After treated with SIRT1 agonist SRT1720 and inhibitor sirtinol, the role of SIRT1/NF-κB signaling pathway in hippocampal neuronal apoptosis was observed. On the basis of giving 50 mg/kg butylphthalide, sirtinol was administered, and the effect of butylphthalide on neuronal apoptosis regulated by SIRT1/NF-κB signaling pathway was evaluated. RESULTS: The morphology of hippocampal neurons in the AD rats were improved, the apoptosis rate of hippocampal neurons and the protein levels of Bax and cleaved caspase-3 were inhibited, and the protein levels of Bcl-2 and the activation of SIRT1/NF-κB signaling pathway were promoted by butylphthalide significantly (P<0.05). The protein expression of Bcl-2 and the activation of SIRT1/NF-κB signaling pathway were promoted, and the apoptosis of hippocampal neurons and the protein levels of Bax and cleaved caspase-3 were inhibited by SRT1720 remarkably (P<0.05), whereas the effect of sirtinol was contrary to that of SRT1720. After sirtinol treatment, the inhibitory effect of butylphthalide on apoptosis of hippocampal neurons, the protein levels of Bax and cleaved caspase-3, and the promotion of Bcl-2 protein expression in hippocampal neurons were markedly weakened (P<0.05). CONCLUSION: Butylphthalide inhibits the apoptosis of hippocampal neurons in the AD rats by down-regulating the protein expression of Bax and cleaved caspase-3, and up-regulating the protein expression of Bcl-2 through activating SIRT1/NF-κB 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.