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Objective @#To study the effect of light-emitting diode (LED) red light on the osteogenic/odontogenic differentiation of human dental pulp stem cells (hDPSCs) and its mechanism were discussed. @*Methods@#This study has been reviewed and approved by the Ethics Committee. hDPSCs were cultured by tissue block enzyme digestion. The proliferative capacity of hDPSCs was detected by the CCK-8 at days 1, 3, 5 and 7 under stimulation with 0, 1, 5 and 10 μg/mL lipopolysaccharide (LPS), and the LPS stimulatory concentration was screened. The CG group (mineralization induction), LPS+CG group, and LPS+CG+ (2, 4, 6, 8, and 10 J/cm2) LED red light groups were set. On day 7, alkaline phosphatase (ALP) staining and ALP activity were determined. Relative expression levels of the ALP, osterix (OSX), dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP) genes were measured by qRT-PCR. On day 21, alizarin red staining and calcium nodule quantitative determination were performed to screen the best light energy. The LPS+CG group and LPS+CG+LED group (optimal energy) were set up, and the secretion and expression levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were detected by ELISAs on days 1, 3, 5 and 7. The relative expression levels of the extracellular regulated protein kinases 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and extracellular regulated protein kinases 5 (ERK5) proteins and their phosphorylated proteins in the MAPK signaling pathway were detected by Western blots. After the pathway was blocked, the relative expression levels of the ALP, OSX, DMP-1, and DSPP proteins after LED red light irradiation on day 7 were detected by Western blots.@*Results@# CCK-8 assays showed that the proliferation of hDPSCs induced by 10 μg/mL LPS was lower than that of the 0, 1, and 5 μg/mL groups on the 5th and 7th days (P<0.05), and 10 μg/mL was selected as the LPS stimulatory concentration in the follow-up experiment. ALP staining, ALP activity, gene expression levels of ALP, OSX, DMP-1 and DSPP and calcium nodule quantification in the LPS+CG+4 J/cm2 group were higher than those in the other treatment groups (P<0.05). 4 J/cm2 LED red light had the strongest ability to promote osteogenic/odontogenic differentiation and was used as the LED light energy density in subsequent experiments. ELISA showed that the secretion and expression levels of TNF-α and IL-1β in the LPS+CG+LED group were lower than those in the LPS+CG group on the 5th and 7th days (P<0.05). Western blot analysis showed that 4 J/cm2 LED red light promoted the expression levels of the p-ERK1/2, p-p38, p-JNK and p-ERK5 proteins. After the MAPK pathway was blocked, the expression levels of the ALP, OSX, DMP-1, and DSPP proteins in the LPS+CG+LED+U0126 (ERK1/2 inhibitor), SP600125 (JNK inhibitor), and BIX02189 (ERK5 inhibitor) groups were lower than those in the LPS+CG+LED group (P<0.001). The protein expression levels of ALP, OSX and DMP-1 in the LPS+CG+LED+SB203580 (p38 inhibitor) group were not significantly different from those in the LPS+CG+LED group (P>0.05).@*Conclusion@#In inflammatory conditions, LED red light promotes osteogenic/odontogenic differentiation of hDPSCs. This effect may be attributed to enhancement of the ERK1/2, JNK, and ERK5 signaling pathways, which reduces the production of the inflammatory cytokines TNF-α and IL-1β.
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Objective: Taxifolin is a natural flavonoid compound that can be isolated from onions, grapes, oranges and grapefruit. It also acts as a medicine food homology with extraordinary antioxidant and anti-inflammatory activity. This study aims to explain the protective effects and potential mechanisms of taxifolin against inflammatory reaction. Methods: Levels of interleukin (IL)-6, IL-1β and intracellular reactive oxygen species (ROS) were assessed in different time after the treatment of taxifolin in RAW264.7 cells induced by lipopolysaccharide (LPS). Subsequently, the mRNA and protein levels of inducible nitric oxide synthase (iNOS), vascular endothelial growth factor (VEGF), cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α and the phosphorylation expression levels of the MAPK signal pathway were also evaluated. A silico analysis was used to explain the binding situation for the investigation of taxifolin and MAPK signal pathway. And then MAPK inhibitors were used to reveal the expression level of iNOS, VEGF, COX-2 and TNF-α in RAW264.7 cells. Results: It was demonstrated that cell inflammatory damage induced by LPS was significantly alleviated after the treatment of taxifolin. Then, the mRNA and protein levels of iNOS, VEGF, COX-2 and TNF-α were reduced and the phosphorylation expression levels of the MAPK signal pathway were down-regulated remarkably as well. In silico analysis, taxifolin could form a relatively stable combination with MAPK signal pathway. MAPK inhibitors showed increasing or decreasing effect in the mRNA levels of iNOS, VEGF, COX-2 and TNF-α, which suggesting that taxifolin down-regulated iNOS, VEGF, COX-2 and TNF-α expressions were not entirely through the MAPK pathway. Conclusion: This finding demonstrated that taxifolin improved the inflammatory responses that partly involved in the phosphorylation expression level of MAPK signal pathway in RAW264.7 cells exposed to acute stress.
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Objective: To investigate the effect of hesperidin on apoptosis of gastric cancer AGS cells and its related molecular mechanisms. Methods: MTT assay was used for the killing effect of hesperidin on human gastric cancer AGS cells; Annexin V-FITC/PI double staining and flow cytometry was used to detect the apoptosis induced by hesperidin on AGS cells, the level of reactive oxygen species, and the addition of NAC Post-apoptosis changes; Western blotting was used to detect the expression of apoptosis-related proteins and signaling pathway-related proteins. Results: MTT assay showed that hesperidin had a good inhibiting effect on AGS cells. After treated with hesperidin, AGS cells showed apoptosis such as nuclear condensation and cell shrinkage. Annexin V-FITC/PI double staining and flow cytometry showed that hesperidin can induce mitochondrial dependent apoptosis of AGS cells and increase the level of intracellular reactive oxygen species. After pretreatment of NAC, hesperidin induced apoptosis inhibition. The results of Western blotting showed that the expression of p-JNK, p-p38, Bad, cleaved Caspase-3, and cleaved PARP increased, and the expression of anti-apoptotic proteins p-ERK and Bcl-2 decreased, which indicated that hesperidin activated the MAPK signaling pathway and mitochondria-dependent apoptosis in AGS cells. Conclusion: Hesperidin has a good killing effect on human gastric cancer AGS cells, and induces mitochondria-dependent apoptosis in AGS cells by increasing the level of reactive oxygen species in AGS cells and regulating MAPK signaling pathway.
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Objective To explore the mechanism of gastric carcinoma cell SGC-7901 apoptosis induced by chlorogenic acid-like compounds extracted from Broussonetia papyrifera (CALCBP) bark. Methods The SGC-7901 cells were used to evaluate the anti-tumour activity of the extract in vivo, and the proliferation of cells was examined by MTT assay. The cell morphological changes of cells were observed by DAPI staining; The cell apoptosis and the cell cycle were detected respectively by flow cytometry after PI and Annexin V/PI staining; The intracellular ROS were determined under the fluorescence microscope using DCHF-DA probe, the changes of mitochondrial membrane potential were observed by JC-1 staining. The protein expression of p53, Bcl-2, Bax, and Cytochrome C, p-p38, p-JNK, JNK, p-ERK, ERK were analyzed by Western blotting. Results The proliferation of SGC-7901 cells was inhibited significantly by CALCBP in dose-dependent and time-dependent manner, the condensed chromosome and apoptotic body can be observed in the treated cells and the cell cycle was arrested in G2/M phase, the mitochondrial membrane potential was significantly decreased, whereas the cellular ROS levels of the treated cells were significantly increased. Moreover, the protein expression of p53, Bax, Cytochrome C, and p-p38 were significantly up-regulated and p-ERK and Bcl-2 expression were significantly down-regulated. Conclusion The apoptosis of gastric cancer cell SGC-7901 induced by CALCBP was probably related to oxidative stress of the cell mitochondrial via p38-MAPK and ERK-MAPK signal pathways.
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OBJECTIVE: To investigate the effects of Fritillariae cirrhosae bulbus on airway inflammation and ERK/MAPK signal pathway of asthma model mice, and to explore its possible mechanism of the treatment of asthma. METHODS: The asthma model was induced by egg albumin. A total of 40 model mice were randomly divided into model group (0. 5％ carboxymethyl cellulose, intragastric administration), positive control group (0. 5 mg/kg dexamethasone, intraperitoneal injection), Fritillariae cirrhosae bulbus low-dose and high-dose groups (9. 0, 18. 0 mg/kg, intragastric administration), with 10 mice in each group. Other 10 normal mice were included in normal group (0. 5％ carboxymethyl cellulose, intragastric administration). They were given medicine once a day for consecutive 28 d. After medication, the number of total cells and differential cells (neutrophils, macrophages, lymphocytes and eosinophils) in bronchoalveolar lavage fluid (BALF) of mice were counted. The pathological morphology of bronchial smooth muscle in mice was observed under light microscope, and the inflammatory score was scored; the activities of ERK, phosphorylated ERK (p-ERK), p38 MAPK and phosphorylated p38 MAPK (p-p38 MAPK) were measured by ELISA. The protein expression of ERK, p-ERK, p38 MAPK and p-p38 MAPK in lung tissue were determined by Western blot assay. mRNA expression of ERK and p38 MAPK were determined by real time fluorescent quantitative PCR. RESULTS: Compared with normal group, the number of total cells and differential cells in BALF of mice, inflammation score, the activities of p-ERK and p-p38 MAPK in lung tissues were increased significantly of mice in model group (P<0. 01); the protein expression of p-ERK and p-p38 MAPK, mRNA expression of ERK and p38 MAPK were increased significantly in lung tissue of mice in model group (P<0. 01). Compared with model group, above indexes of treatment groups were all improved significantly (P<0. 05 or P<0. 01). CONCLUSIONS: Fritillariae Cirrhosae Bulbus can improve airway inflammation in asthma model mice, the mechanisms of which may be related to inhibiting the activation of ERK/MAPK signal pathway.
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Objective To explore the effect of FGF21 on MSG-induced nonalcoholic fatty liver diseases of inflammation and TLR4/p38MAPK signal pathway in rats.Methods Newborn SD rats were randomly divided into control group,MSG group and FGF21 group(n =10).Rats of MSG group and FGF21 group were adiministered with solution of MSG 4g/(kg · d) at 2nd,4th,6th,8th and 10th postnataldays.After being fed for thirteen weeks,rats of FGF21 group was intraperitoneal injected with FGF21 1 mg/(kg · d) for a continuous 32 days.Liver pathology of the rats was analyzed by HE staining.The liver weight,aminotrasferases were observed.The expression of IL-6,TNF-α,TLR4,p38MAPK in liver tissue were determined by fluorescence quantitative PCR and Western blot.Results Compared with the control group,liver tissues of the MSG group changed to bullous steatosis and had inflammatory cell infiltration,the liver weighted,serum activities of ALT,AST,ALP were increased (P ＜ 0.01,P ＜ 0.01,P ＜ 0.01,P ＜ 0.01).The expression of IL-6,TNF-α,TLR4,p38MAPK mRNA and protein expression of TLR4,p38MPAK,p-p38MAPK in rats hepatocyte were increased than those in control group (P ＜ 0.01).After reatment with FGF21,the bullous steatosis and inflammatory cell,liver weighted,serum activities of ALT,AST,ALP were signficantly decreased (P ＜ 0.05,P ＜ 0.01,P ＜ 0.01,P ＜ 0.05),and the levels of IL-6,TNF-a,TLR4,p38 MAPK mRNA and protein expression of TLR4,p38-MPAK,p-p38MAPK were reduced than those of MSG group(P ＜0.01).Conculsion The FGF21 may regulate MSG-induced NAFLD of inflammation through TLR4/p38MAPK signaling pathway in rats.
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Objective To investigate the protective effect and possible mechanism of Gossypol on isolated myocardial ischemia/reperfusion injury in rats.Methods 40 male Sprague-Dawley rats were randomly divided into 4 groups:Blank group,heart ischemia reperfusion group (MI/R group),high and low-dose Gossypol group (40,20 mg/L).The model of the myocardial ischemia/reperfusion injury were established using the Langendorff method.The hemodynamicsindexes,cardiac enzymes AST and LDH,inflammatory cytokines (NF-κB,ICAM-1,TNF-α and IL-6) were measured.The effect and mechanism of Gossypol on early-stage MI/R of the oxidative stress response and the JNK/p38 MAPK signal pathway were investigated.Results Experimental results showed that Gossypol could significantly improve the functional capacity of the heart,reduce the contents of AST,LDH and inflammatory cytokines in reperfused heart tissue,and increase superoxide dismutase levels to protect the heart.The mechanism of this substance may involve anti-lipid peroxidation and inhibition of p38 kinase phosphorylation and JNK,and reduction of oxidative stress injury and apoptosis damage induced by MI/R.Conclusion This study confirm that Gossypol exerts extensive anti-MI/R effects.Its mechanism may be related to the interfering with the oxidative stress response and suppressing the JNK/p38 MAPK signal pathway.
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To explore the inhibitory effect of timosaponin AⅢ on the proliferation of human glioblastoma cell line U87MG and investigate its related mechanism. As compared with the model group, the tumor weight was significantly reduced in timosaponin AⅢ-treated group. Timosaponin AⅢinhibited the proliferation of U87MG cell line in a dose-dependent manner. It up-regulated the gene and protein expression levels of p21, meanwhile inhibited the protein expression levels of β-Catenin, Cyclin D1 and Bcl-2. It also inhibited the translocation of β-Catenin into nucleus, suppressed the phosphorylation expression of ERK, but increased the phosphorylation expression of p38 and JNK. Combined use of JNK inhibitor SP600125 and p38 inhibitor SB203580 could decrease p21 and increase β-Catenin protein expressions. Timosaponin AⅢ inhibited the proliferation of human glioblastoma cell line U87MG partly by intervening MAPK and Wnt/β-Catenin signal pathways.
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OBJECTIVE To explore the estrogen- like neuroprotective effects and the related mechanism of quercetin by using PC12 cells induced with Aβ25-35, provided thought and strategy for the drug therapy of AD. METHODS Cells were cultured with Aβ25- 35 for 24 h, 17β-estradiol (0.1 μmol·L- 1), genistein(50 μmol·L-1) and three different concentrations of quercetin (200 μmol·L-1, 300 μmol·L-1 and 400 μmol·L-1) were respectively added after 24h. The effects of quercetin on activity of AD model were tested by MTT. Immunohistochemical stain and Western blot were used to detect the expression of estrogen receptors alpha and beta, p-ERK1/2 and apoptosis related proteins.The mechanism of quercetin estrogen-like neuroprotective effects was detected using estrogen receptor antagonist ICI182,780 and MAPKK inhibitor U0126. RESULTS The results revealed thatthe toxic effects showed in a dose-dependent increase of Aβ25- 35 on PC12 cells.Comparing with the control group,cells injury was observed after cultured with 10 μmol·L-1 Aβ25-35 for 24 h(P<0.01). The MTT results showed that 17β-estradiol, genistein and three different concentrations of quercetin could significantly enhance the cell survival rate compared with the model group (P<0.05). Compared with model group,Immunofluorescence and Western blot results show that quercetin could improve the estrogen receptor alpha and p- ERK1/2 protein expression (P<0.05), and the expression of estrogen receptor beta protein is increased without significant difference. And in the Western blot experiments, the ratio of Bcl- 2 and Bax was increased and the expression of Caspase 3 was decreased( P<0.05).When estrogen receptor inhibition ICI182,780 were reduced,the expression of p- ERK1/2 was decreased (P<0.05) and the ratio of Bcl- 2 and Bax was decreased, Caspase 3 protein expression was increased (P<0.05). In addition,pretreatment of cells with U0126 would reduce Bcl-2/Bax ratio and increase Caspase 3 protein expression increased (P<0.05). CONCLUSION Quercetin protected PC12 cells, which suffered from Aβ25- 35-induced cytotoxicity and exert neuroprotective effects. The estrogen-like neuroprotective effect can reduce the apoptosis in the classic estrogen receptor pathway and MAPK pathway. And quercetin can also active MAPK signaling pathways by the mediation of estrogen receptor alpha.
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[Objective] To investigate the effects of platycodin D(PD) on the proliferation and apoptosis of human stomach cancer SGC7901 and the related mechanism.[Methods] SGC7901 was cultured in virto and was treated with 5～20μm·L-1 concentrations of PD.Cell proliferation was examined by MTT assay.Cell apoptosis was detected by Annexin V FITC/PI double staining.The change of mitochondrial trans-membrane potential was measured by JC-1 staining.The potein expression of cleaved caspase-3,cleaved caspase-9,cleaved PARP,bcl-2,bax,p-ERK,ERK,p-JNK,JNK,p-p38 and p38 detected by Western blot.[Results] MTT results showed that PD inhibited the growth of SGC7901 cells in a dose-dependent manner at 24h and 48h.SGC7901 cells treated with PD for 24h showed significantly enhanced apoptosis and weakened mitochondrial membrane potential compared with the control cells.Western blot results showed that PD could up-regulate expression of cleaved PARP,cleaved caspase-3,cleaved caspase-9,bax,p-JNK,p-p38 protein,decreased bcl-2,p-ERK protein,the expression of ERK,JNK,p38 protein did not change significantly.[Conclusion] PD may inhibit the proliferation and induce the apoptosis of SGC7901 cells.These findings indicated that PD inhibited cell proliferation by inhibiting the ERK signaling.PD effect on bax and bcl-2 by activation of JNK and p38 signaling pathway resulted in the decrease of mitochondrial membrane potential and activation of caspase,which induced the apoptosis of cancer cells.
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This paper was aimed to study the effects of icariin (ICA) on the proliferation of vascular smooth muscle cell (VSMC) induced by oxidized low density lipoprotein (ox-LDL), and the molecular mechanism of the expression of proliferating cell nuclear antigen (PCNA) and MAPK signaling pathway. In this study, VSMC was induced by ox-LDL (50 mg•L⁻¹),the effect of ICA on the proliferation of VSMC was detected by MTT assay, Western blot and Real-time PCR. The results showed that after stimulation of ox-LDL, the proliferation activity of VSMC was increased, S phase, G₂/M phase cells were increased, G₀/G₁ phase cells were decreased, PCNA protein expression was enhanced; ICA (40, 20, 10 μmol•L⁻¹) could effectively inhibit ox-LDL-induced VSMC proliferation, S phase and G₂/M phase cells were decreased, the percentage of cells in G₀/G₁ phase were increased, PCNA expression was decreased, p38MAPK and ERK1/2 activation were inhibited. These results indicate that ICA can inhibit the proliferation of VSMC by reducing the expression of PCNA and blocking the p38MAPK and ERK1/2 signaling pathway.
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Objective To investigate the apoptosis induction of Bullatacin on A549 cell line of pulmonary adenocarcinoma. Methods The MTT assay was used to detect the growth inhibition rates of A549 cells cultured with Bullatacin in different concentrations (6.25, 12.5, 25, 50, 100μg/mL). 25μg/mL Bullatacin was used to culture A549 cells for 0, 12, 24, 48 h. The cell cycle distribution and apoptosis were measured by flow cytemetry. The protein expressions of ERK, JNK, and p38 were studied by Western blot. Results Dosage dependence was obviously showed after the different concentrations of Bullatacin were used to A549, and 25 μg/mL;Bullatacin blocked A549 cell in G0/G1 periods and induced its apoptosis. Compared with the blank group, protein expressions of P-ERK, P-JNK, and P-p38 were all increased by different degrees. Conclusion Bullatacin significantly inhibits the proliferation and induces the apoptosis of A549 cell. Its mechanism is related to activity of MAPK pathway thought the phosphorylation of the three protein kinases by Bullatacin.
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Objective To explore the effect of Jiawei Wendan decoction on whole regulating function of CaMK/MAPK signal pathway in hippocampus of depression model rats.Methods Isolated depression model rats as research object were living unpredictable chronic stress.Based on the examination of mRNA/protein expression which was the key factor of the signal pathway of CaMK/MAPK and the intersection CREB1 mRNA expression,the relationship between CaMK Ⅱ/RSK protein expression and the CREB1 mRNA expression were analyzed with relation analysis method.Results ① Bidirectional correlation analysis:the respective coefficient (r value) of CaMK Ⅱ protein expression and the CREB1mRNA expression in hippocampus of the model group,Chinese medicine treatment group and western medicine treatnent group were-0.502 (P ＜ 0.01),-0.643 (P ＜ 0.01),-0.408 (P＜ 0.05) ;the respective coefficient(r value) of RSK protein expression and the CREB1 mRNA expression in hippocampus of the model group,Chinese medicine treatment group and western medicine treatment group were 0.550 (P ＜ 0.01),0.687 (P ＜ 0.0 l),0.407 (P ＜ 0.01).②Regression analysis:the respective regression coefficient of CaMK Ⅱ protein (positive direction),RSK protein (negative direction) and CREB1 mRNA expression in the model group,Chinese nedicine treatment group and western medicine treatment group were R 2 =0.472,F =12.983(P＜0.01),R2=0.666,F=28.961(P＜0.01),R2=0.356,F=8.004(P＜0.01).Conclusion The anti-depressant effect mechanism of Jiawei Wendan decoction is to regulate the whole function of the CaMK and MAPK signal pathway and then further up-regulate CREB1 mRNA expression.
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ObjectiveTo investigate the activation of MAPK signal pathway in multiple myeloma and the regulation of BLyS expression levels through MAPK signal pathway; preliminarily study the role of MAPK signal pathway in the up-regulation of BLyS expression levels induced by IFN-γ.MethodsActivated MAPK pathway were detected by Western blot,while the expression of BLyS were detected with RT-PCR and Western blot,and Western blot investigated the effect of MAPK pathway on BLyS expression levels induced by IFN-γ.ResultsIn addition to the expression of ERK,JNK,p38,p-JNK was also expressed in MM cell lines,the MAPK pathway inhibitor targeting JNK SP600125 can down-regulate the expression of BLyS,and its activator anisomycin can up-regulate the expression of BLyS.SP600125 restrained the proliferation and survival of MM cells.ConclusionJNK/SAPK pathway was activated in MM cells; The activated degree of JNK/SAPK pathway and the expression level of BLyS was positively correlated.JNK/SAPK pathway play an important role in the up-regulation of BLyS expression levels induced by IFN-γ.
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AIM: To observe the effects of sera containing Kang xian ling (a traditional Chinese medicine) on c-Met and its downstream MAPK signal molecules in HK-2 cells treated with transforming growth factor-β_1(TGF-β_1), so as to further explore the effective mechanism of anti-renal fibrosis of the traditional Chinese medicine. METHODS: Rat sera containing herbs were collected after rats were intragastrically administered with the herbs. HK-2 cells activated by TGF-β_1 were incubated with different heat-inactivated sera. The proliferation and expression of MAPK and the phosphorylation of MAPK in HK-2 cells were detected by Alarm blue and immunoblotting. RESULTS: No significant difference of cell proliferation between any two groups was observed. C-Met and the phosphorylation of its downstream molecules were also observed after incubated with different sera. CONCLUSION: The receptor c-Met of hepatocyte growth factor, and the phosphorylation of its downstream MAPK molecules can be regulated by the sera containing Kang xian ling.
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Objective:To investigate the inhibitory effect of 4'-methylether-scutellarein(4-MS),an extract from Verbena officinalis,on human choriocarcinoma JAR cell line and the possible mechanism.Methods:JAR cells were exposed to 4'-methylether-scutellarein of different concentrations for 48 h.MTT assay was used to examine the anti-proliferative effect of 4'-methylether-scutellarein.Flow cytometry was used to investigate the apoptosis and the changes of cell cycle.AO/EB double staining was applied to discriminate the apoptotic cells from dead ones.The changes of Survivin,p38-MAPK and Caspase 3 mRNA expressions were detected by RT-PCR in JAR cells treated with 4-MS.Furthermore,Western blotting assay was used to determine Survivin protein expression,phosphorylation level of p38 and Caspase 3 in JAR cells before and after 4-MS treatment.Results:4-MS inhibited the proliferation of JAR cells in a dose- and time-dependent manner(P