Investigation on the mechanism of Qiangxinhuoli prescription in the treatment of chronic heart failure based on p38-MAPK signaling pathway

Background:The aim of this study is to investigate the mechanism of action underlying the therapeutic effects of the national patent Chinese medicine compound“Qiangxinhuoli prescription(QXHLF)”on chronic heart failure(CHF).Methods:In vitro,the H_(9)C_(2) cell model was induced by ANGII,and cell proliferation and related protein expression were detected by Cell Counting Kit-8 and Western blot.In vivo,A rat model of CHF was prepared by ligation of the left anterior descending coronary artery.The effects of QXHLF on cardiac function in CHF rats were evaluated by cardiac index,hemodynamic changes,enzyme-linked immunosorbent assay,hematoxylin-eosin staining,immunohistochemistry,Western blot and RT-PCR.The expression of pro-apoptotic factors and anti-apoptotic factors,as well as TGFβ1,p-p38,TAK 1 mRNA,and protein,were detected.Results:In vitro,QXHLF has a significant inhibitory effect on the proliferation of H_(9)C_(2) cells.QXHLF can reduce the expression levels of TAK 1,TGFβ1,p-p38,Caspase3 and BAX proteins in H_(9)C_(2) cells,and increase the expression level of BCL_(2) protein.In vivo,QXHLF has the potential to increase left ventricular systolic pressure,m aximum rate of change in left ventricular pressure while decreasing left ventricular end diastolic pressure,and inhibiting the serum levels of brain natriuretic peptide.Moreover,QXHLF exhibits significant improvements in the pathological alterations of myocardial cells and fibers in CHF rats,leading to enhanced myocardial tissue morphology and notable advantages in combating myocardial fibrosis.QXHLF can reduce the levels of BAX and Caspase3 and up-regulate the expression of BCL_(2),thereby inhibiting cardiomyocyte apoptosis.Furthermore,QXHLF demonstrates inhibitory effects on the mRNA and protein expression levels of TGFβ_(1),TAK_(1),and p-p38 in the heart tissue of the CHF rat model.Conclusion:These findings indicate that QXHLF has a therapeutic effect on CHF by inhibiting the p38-MAPK signaling pathway,reducing myocardial fibrosis,preventing apoptosis,inhibiting cell proliferation,and restoring myocardial injury.

The Antidepressant Mechanism of JiaWeiWenDan Decoction Regulating p38MAPK-ERK5 Signal Transduction Pathway

Objective: To investigate the anti-depression mechanism of JiaWeiWenDan Decoction in regulating p38MAPK-ERK5 signal transduction pathway. Methods: Depression model rats were randomly divided into Blank Control Group, Model Control Group, Chinese Medicine Treatment Group, and Western Medicine Treatment Group (hereinafter referred to as Blank Group, Model Group, Chinese Medicine Group, and Western Medicine Group), with 48 rats in each group. The mice were treated with p38MAPK-ERK5 on the 7th day, 14th day and 21st day, respectively, and the mice were treated for 28 days. The key targets and cytokines in p38MAPK-ERK5 signal transduction pathway were detected. Results: Compared with the Blank Group, the expression of p38MAPKmRNA in the hippocampus of the Model Group was increased. The Chinese Medicine Group and Western Medicine Group could reduce the expression of p38MAPK mRNA (P P P P Conclusion: The anti-inflammatory effect of JiaWeiWenDan Decoction may be related to the regulation of p38MAPK-ERK5 signaling pathway. With the advance of the treatment week, the best effect was obtained when the treatment was started on the 7th day of modeling.

p38 MAPK is a Component of the Signal Transduction Pathway Triggering Cold Stress Response in the MED Cryptic Species of Bemisia tabaci

Cold stress responses help insects to survive under low temperatures that would be lethal otherwise.This phenomenon might contribute to the invasion of some Bemisia tabaci cryptic species from subtropical areas to temperate regions.However,the molecular mechanisms regulating cold stress responses in whitefly are yet unclear.Mitogen-activated protein kinases（MAPKs）which including p38,ERK,and JNK,are well known for their roles in regulating metabolic responses to cold stress in many insects.In this study,we explored the possible roles of the MAPKs in response to low temperature stresses in the Mediterranean cryptic species（the Q-biotype）of the B.tabaci species complex.First,we cloned the p38 and ERK genes from the whitefly cDNA library.Next,we analyzed the activation of MAPKs during cold stress in the Mediterranean cryptic species by immuno-blotting.After cold stress,the level of phospho-p38 increased but no significant change was observed in the phosphorylation of ERK and JNK,thus suggesting that the p38 might be responsible for the defense response to low temperature stress.Furthermore,we demonstrated that：i）3 min chilling at 0°C was sufficient for the activation of p38 MAPK pathway in this whitefly;and ii）the amount of phosphorylated p38 increased significantly in the first 20 min of chilling,reversed by 60 min,and then returned to the original level by 120 min.Taken together,our results suggest that the p38 pathway is important during response to low temperature stress in the Mediterranean cryptic species of the B.tabaci species complex.

Effect of the Tiaobufeishen decoction on Caveolin-1-p38 MAPK signaling pathway and mechanism of improving the tracheobronchomalacia in chronic obstructive pulmonary disease

Objective: Reliving the rela ti onship of the Caveolin-1-p38 MAPK signaling pathway and COPD tr acheob ronchomalacia, and resea rch the mechanism of Tiaobufeishen decoc tion imp rove the regression of the weasand cartilage cells. Methods: Flow cytometry was used to analyze the apoptosis rate to determine the optimal concentration of Tiaobufeishen decoction and CSE, CCK8 assay was used to dete rmine the op ti mal concent ration of P38-MAPK specific inhibitor. The COPD cell model was created by tracheal chondrocyte which dispose by optimal concent ration CSE, then add the IL-1P set up the chond rocyte degene ration model, use the method of toluidine blue staining and immunohistochemical authenticate degeneration of cartilage. This research included control group, model group, model-Tiaobufeishen group, model-blocker group. When the model was set up succeed, add the Tiaobufeishen decoction and P38-MAPK blocke r in the model-Tiaobufeishen and model-blocke r gr oups, r espectively. Weste rn Blot was used to detect the exp ression of caveolin-1 and p-p38 in the chond rocyte. RT-PCR was used to detect the expression of MMP3 and caveolin-1 in the matrix. Results: The cell activity was not influence by the concentration of Tiaobufeishen decoction and blocker, the concentration of the CSE model was moderation. Compared with control group, the level of caveolin-1, p38MAPK, MMP3 in the model group was significant increase, moreover, the result of toluidine blue staining and immunohistochemical methods show that the chond rocyte has obvious reg ression. The exp ression of caveolin-1, p38MAPK, and MMP3 have significant decrease than the control group, and the reduction of chondrocyte degeneration. Conclusion: The caveolin-1-p38MAPK signaling pathway play an important role in the morbidity of the tracheobronchomalacia. Tiaobufeishen decoction could decrease the exp ression of the caveolin-1, p-p38, MMP3, inhibit the activa tion of the caveolin-1-p38MAPK signaling pathway, therefore, it can improve the tracheobronchomalacia.

To investigate the effects of butylphthalide on reducing neuronal apoptosis in rats with cerebral infarction by inhibiting the JNK/P38 MAPK signaling pathway

Objective:To investigate the effects of butylphthalide on reducing neuronal apoptosis in rats with cerebral infarction by inhibiting the JNK/P38 MAPK signaling pathway.Methods:Forty-eight SD male rats were divided into DZ group(control group),CI group(model group)and NBP group(butylphthalide group).Rats in CI group and NBP group were used to establish cerebral infarction models.NBP group used NBP.The solution(80 mg/(kg?d))was administered orally,and the remaining two groups were administered with the same volume of peanut oil.After 14 consecutive days of treatment,the Zea Longa score was used to evaluate the neurological function of DZ,CI and NBP rats.Scoring,TTC staining was used to observe the cerebral infarction volume of rats in DZ group,CI group and NBP group,HE staining was used to observe the pathological morphology of brain tissue in DZ group,CI group and NBP group.Neuronal apoptosis,Western blot was used to detect the expression of p-JNK and p-p38MAPK in brain tissues of DZ group,CI group and NBP group.Results:The neurological function of the rats in the CI group was higher than that in the DZ group,and the difference was statistically significant(P<0.05).The neurological function score of the rats in the NBP group was reduced compared with the CI group,and the difference was statistically significant(P<0.05).The cerebral infarction volume in the group was 35.56%higher than that in the DZ group,and the difference was statistically significant(P<0.05).The minor infarct volume in the NBP group was 21.59%,which was less than that in the CI group,and the difference was statistically significant(P<0.05).Nerve cells are neatly sorted,with a large number.The gap between blood vessels and interstitial tissue in the CI group is enlarged,the cells are severely contracted,and the neuron structure is incomplete.Compared with the CI group,the NBP group has reduced neuron contraction and increased number;The dead nerve cells were brown.The apoptosis rate of nerve cells in the CI group was 79.65%higher than that in the DZ group was 5.82%.The difference was statistically significant(P<0.05).The nerve cell apoptosis rate in the NBP group was 30.23%.Compared with CI group,the difference was statistically significant(P<0.05);Western blot results showed that p-JNK and p-p38MAPK protein expression in CI group was higher than that in DZ group,and the difference was statistically significant(P<0.05).The levels of p-JNK and p-p38MAPK proteins in the NBP group were lower than those in the CI group.There was statistically significant(P<0.05).Conclusion:Butylphthalide can improve neurological damage,reduce apoptotic nerve cells,and reduce infarct volume in rats with cerebral infarction,which is related to the inhibition of JNK/P38 MAPK pathway expression.

α1-antitrypsin combined with bone marrow mesenchymal stem cells regulates retinopathy in diabetic rats via p38 MAPK/NF-κB signaling pathway

Objective:To investigate the effect ofα1-antitrypsin combined with bone marrow mesenchymal stem cells on retinopathy in diabetic rats and its mechanism.Methods:A model of diabetic retinopathy was established by intraperitoneal injection of streptozotocin.The 30 Wistar rats successfully modeled were randomly divided into a model group,a bone marrow mesenchymal stem cell group and a combined group(α1-antitrypsin combined with bone marrow Mesenchymal stem cells),the blood glucose and serum insulin levels of diabetic rats were measured 4 weeks after treatment.Enzyme-linked immunosorbent assay(ELISA)for measuring serum inflammatory factors IL-1β,IL-6 and TNF-α in rats.Observing the pathological morphology of rat retina under hematoxylin-eosin staining(HE).TUNEL staining to observe the apoptosis of rat retinal nerve cells.Immunohistochemical method to detect the expression level of CD45 in retinal tissue.Real-time fluorescence quantitative PCR was used to detect the expression of retinal vascular endothelial growth factor(VEGF),hypoxiainducible factor-1α(HIF-1α),and angiotensinⅡ(ANGⅡ)mRNA.Western blot was used to detect the expression of p38 MAPK/NF-κB signaling pathway-related proteins in the retinal tissue of each group of rats.Results:Compared with the control group,the rats in the model group had increased blood glucose,decreased insulin levels,increased serum IL-1β,IL-6,and TNF-α levels,and had obvious lesions in the retina.CD45 showed high expression in retinal tissue,VEGF,HIF-1α,ANGⅡ mRNA expression increased,p-p38,p-p65,p-IκBα protein expression increased(P<0.05).Compared with the model group,the bone marrow mesenchymal stem cell group and the combined group have decreased blood glucose,increased insulin levels,and decreased serum IL-1β,IL-6 and TNF-α levels.Retinopathy is improved,apoptosis of retinal nerve cells is reduced,CD45 expression in retinal tissue is reduced,VEGF,HIF-1α,ANGⅡ mRNA expression is decreased,and p-p38,p-p65,p-IκBα protein expression is decreased.Compared with the bone marrow mesenchymal stem cell group,the effect of the combined group was more obvious(P<0.05).Conclusion:α1-antitrypsin combined with bone marrow mesenchymal stem cell transplantation can improve the degree of retinopathy in diabetic rats.The mechanism may be related to the inhibition of p38 MAPK/NF-κB signaling pathway.

Betulinic acid protects against ovarian impairment by decreasing F-2 toxin-induced oxidative stress and inflammation associated with the downregulation of p38 expression in mice

F-2 toxin is an estrogenic mycotoxin that causes reproductive disorders in animals.Betulinic acid(BA)is a natural pentacyclic lupane-structure triterpenoid that has diverse pharmacological activities.In this study,the antioxidative and anti-inflammatory effects of BA and its underlying mechanism are explored in F-2 toxin-triggered mouse ovarian damage.We found that BA alleviated the F-2 toxin-induced ovarian impairment by stimulating follicle growth,reducing inflammatory cell infiltration,repairing damaged mitochondria and endoplasmic reticulum.Simultaneously,BA not only reversed F-2 toxin-induced reduction of follicle stimulating hormone(FSH)and luteinizing hormone(LH)levels in the serum,but also restrained the protein expression of the estrogen receptors a(ERa)and ERβ.Moreover,BA restored the balance of F-2 toxin-induced ovarian redox system disorders.Subsequently,we found that 0.25 mg/kg BA played an anti-inflammatory role in the F-2 toxin-induced ovarian impairment by decreasing interleukin-1β(IL-1β).IL-6,and tumor necrosis factor-α(TNF-α)mRNA expression,as well as inhibiting p38 protein expression.These data demonstrated that BA exerts its protective effect on F-2 toxin-induced ovarian oxidative impairment and inflammation by inhibiting p38 expression,which implies a natural product-based medicine to ameliorate F-2 toxin-caused female reproductive toxicity and provides a detoxifying method for food contaminated by mycotoxin.

MiR-204-3p overexpression inhibits gastric carcinoma cell proliferation by inhibiting the MAPK pathway and RIP1/MLK1 necroptosis pathway to promote apoptosis

BACKGROUND Gastric carcinoma(GC)is the third most frequent cause of cancer-related death,highlighting the pressing need for novel clinical treatment options.In this regard,microRNAs(miRNAs)have emerged as a promising therapeutic strategy.Studies have shown that miRNAs can regulate related signaling pathways,acting as tumor suppressors or tumor promoters.AIM To explore the effect of miR-204-3p on GC cells.METHODS We measured the expression levels of miR-204-3p in GC cells using quantitative real-time polymerase chain reaction,followed by the delivery of miR-204-3p overexpression and miR-204-3p knockdown vectors into GC cells.CCK-8 was used to detect the effect of miR-204-3p on the proliferation of GC cells,and the colony formation ability of GC cells was detected by the clonal formation assay.The effects of miR-204-3p on GC cell cycle and apoptosis were detected by flow cytometry.The BABL/c nude mouse subcutaneous tumor model using MKN-45 cells was constructed to verify the effect of miR-204-3p on the tumorigenicity of GC cells.Furthermore,the study investigated the effects of miR-204-3p on various proteins related to the MAPK signaling pathway,necroptosis signaling pathway and apoptosis signaling pathway on GC cells using Western blot techniques.RESULTS Firstly,we found that the expression of miR-204-3p in GC was low.When treated with the lentivirus overexpression vector,miR-204-3p expression significantly increased,but the lentivirus knockout vector had no significant effect on miR-204-3p.In vitro experiments confirmed that miR-204-3p overexpression inhibited GC cell viability,promoted cell apoptosis,blocked the cell cycle,and inhibited colony formation ability.In vivo animal experiments confirmed that miR-204-3p overexpression inhibited subcutaneous tumorigenesis ability in BABL/c nude mice.Simultaneously,our results verified that miR-204-3p overexpression can inhibit GC cell proliferation by inhibiting protein expression levels of KRAS and p-ERK1/2 in the MAPK pathway,as well as inhibiting protein expression levels of p-RIP1 and p-MLK1 in the necroptosis pathway to promote the BCL-2/BAX/Caspase-3 apoptosis pathway.CONCLUSION MiR-204-3p overexpression inhibited GC cell proliferation by inhibiting the MAPK pathway and necroptosis pathway to promote apoptosis of GC cells.Thus,miR-204-3p may represent a new potential therapeutic target for GC.

Activation and signaling of the p38 MAP kinase pathway

The family members of the mitogen-activated protein (MAP) kinases mediate a wide variety of cellular behaviors in response to extracellular stimuli. One of the four main sub-groups, the p38 group of MAP kinases, serve as a nexus for signal transduction and play a vital role in numerous biological processes. In this review, we highlight the known characteristics and components of the p38 pathway along with the mechanism and consequences of p38 activation. We focus on the role of p38 as a signal transduction mediator and examine the evidence linking p38 to inflammation, cell cycle, cell death, development, cell differentiation, senescence and tumorigenesis in specific cell types. Upstream and downstream components of p38 are described and questions remaining to be answered are posed. Finally, we propose several directions for future research on p38.

Promotion effect of FOXCUT as a microRNA sponge for miR-24-3p on progression in triple-negative breast cancer through the p38 MAPK signaling pathway

Background:Triple-negative breast cancer(TNBC)is a type of highly invasive breast cancer with a poor prognosis.According to new research,long noncoding RNAs(lncRNAs)play a significant role in the progression of cancer.Although the role of lncRNAs in breast cancer has been well reported,few studies have focused on TNBC.This study aimed to explore the biological function and clinical significance of forkhead box C1 promoter upstream transcript(FOXCUT)in triple-negative breast cancer.Methods:Based on a bioinformatic analysis of the cancer genome atlas(TCGA)database,we detected that the lncRNA FOXCUT was overexpressed in TNBC tissues,which was further validated in an external cohort of tissues from the General Surgery Department of the First Affiliated Hospital of Nanjing Medical University.The functions of FOXCUT in proliferation,migration,and invasion were detected in vitro or in vivo.Luciferase assays and RNA immunoprecipitation(RIP)were performed to reveal that FOXCUT acted as a competitive endogenous RNA(ceRNA)for the microRNA miR-24-3p and consequently inhibited the degradation of p38.Results:lncRNA FOXCUT was markedly highly expressed in breast cancer,which was associated with poor prognosis in some cases.Knockdown of FOXCUT significantly inhibited cancer growth and metastasis in vitro or in vivo.Mechanistically,FOXCUT competitively bounded to miR-24-3p to prevent the degradation of p38,which might act as an oncogene in breast cancer.Conclusion:Collectively,this research revealed a novel FOXCUT/miR-24-3p/p38 axis that affected breast cancer progression and suggested that the lncRNA FOXCUT could be a diagnostic marker and therapeutic target for breast cancer.

Coronary microembolization induced myocardial contractile dysfunction through a p38 mapk pathway

Background Cathepsin S and its endogenous inhibitor cystatin C are implicated in the pathogenesis of atherosclerosis,especially in the plaque destabilization and rupture leading to acute coronary syndrome.However, whether circulating cathepsin S and cystatin C also change in association with coronary plaque morphology is unknown yet. Methods Sprague-Dawley rats were randomly divided into three groups;Sham group,CME group and SB203580 group (n=10 per group).CME rats were produced by injection of 42μm microspheres into the left ventricle with occlusion of the ascending aorta.SB203580,a p38 MAPK inhibitor,was injected into femoral vein after finishing the injection of microspheres in SB203580 group.Left ventricular Ejection Fraction was determined by echocardiography.The level of phosphorylated and total P38 MAPK in myocardium was assessed by Western Blot.Results Left ventricular(LV) Ejection Fraction was depressed at 3 hours and until up to 12 hours in CME group.The increased p38 MAPK activation was observed in CME group.The administration of SB203580 partly inhibited the p38 MAPK activity and preserved cardiac contractile function.Conclusions p38 MAPK is significantly activated by CME and the inhibition of p38 MAPK can partly preserve cardiac contractile function.

Realgar transforming solution is involved of JNK, P38 MAPK pathway in down-regulating Ras/MAPK pathway in Caenorhabditis elegans

OBJECTIVE To investigate the effect and the mechanisms of realgar transforming solution(RTS)on down-regulating over activated ras.METHODS we used the optimizing technical processes to obtain the RTS,and eval⁃uate the mechanisms of RTS on down-regulating overactivated ras in Caenorhabditis elegans.RESULTS We found that the mRNA level of let60 and lin45 significantly decreased following exposure to RTS,but mRNA levels of mpk1 were not statistically significant in let60/ras(gf)mutant.RTS together with sorafenib(RAF inhibitors)significantly enhanced the activity of RTS on down-regulating overactivated ras more than RTS only,but 50μmol·L^-1 PD98059,a specific ERK inhibitor did not.Lin45 gene RNAi decreased the ability of RTS on down-regulating overactivated ras significantly,but mpk1 gene RNAi did not,indicating that the activity of RTS on down-regulating overactivated ras mainly through targeting to lin45/raf.In addition,RTS significantly increased mRNA level of pmk1/p38 and jnk1/jnk in let-60/ras(gf)mutant,50μmol·L^-1 SB203580(p38 inhibitor)and SP600125(JNK inhibitor)significantly attenuated the effects of RTS on down-regulating overactivated ras in some degree,and pmk1,jnk1 gene RNAi displayed the similar results,suggesting that P38 and JNK/MAPK pathways in some degree were involved in the effects of RTS on down-regulating overactivated ras in C.ele⁃gans.CONCLUSION Realgar transforming solution down-regulate the Ras/MAPK pathway through JNK and P38 MAPK pathways.

ANTI-OXIDATIVE MECHANISMS OF PRAVASTATIN PREVENTING AORTIC ATHEROSCLEROSIS IN apoE KNOCKOUT MICE:ROLE OF p38 MAPK PATHWAY

Objective To determine whether pravastatin exerts anti-oxidative effects on preventing aortic＂ atherosclerosis via modulating p38 MAPK pathway. Methods Male 8-week-old apoE^-/- mice fed a diet containing 1.25% cholesterol （wt/wt） were divided into pravastatin group administered with pravastatin （80 mg. kg ^-1· d^-1 ） and atherosclerosis group administered with PBS; and male 8-week-old C57BL/6J mice fed a normal diet were as control group （ n = 12 ）. In thoracoabdominal aortas of mice, levels of Malondialdehyde （ MDA ） and activities of superoxide dismutase （ SOD ） were measured and expression of phosphorylated p38 MAPK （ p-p38 MAPK） and phosphorylated signal transducer and activator of transcr（ption 1 （pSTAT1） were examined by Western blotting. Results After eight weeks, atherosclerosis in aortic root was significantly prevented by pravastatin. In aortic atherosclerosis lesion, the level of MDA was significantly reduced; adversely the activity, of SOD was increased. Expressions of p-p38 MAPK and pSTAT1 were significantly decreased in aortic atherosclerosis lesion. Conclusion Our results suggests that anti-oxidative mechanisms of pravastatin preventing aortic atherosclerosis may partially depend on modulating p38 MAPK signal pathway.

Safflower polysaccharide inhibits hepatocellular carcinoma cells through P38MAPK activation

Background:Safflower is a traditional medicine that is widely used to treat various diseases.Safflower polysaccharide is one of the effective constituents of safflower.This study explored the anti-tumor effect of safflower polysaccharide and its possible mechanism.Methods:The MTT assay was used to detect the proliferation of hepatocellular carcinoma cells(SMMC-7721 cells).We observed cell apoptosis with a fluorescence microscope.And we used qRT-PCR and western blot to detect the expression of cell cycle related factors.Results:Safflower polysaccharide could inhibit the growth of SMMC-7721 cells in a dose-dependent manner and induce its apoptosis.Compared with the blank group,the cell proliferation-related protein of CDC25B and Survivin were down-regulated in SMMC-7721 cells treated with safflower polysaccharide,while the cell apoptosis-related protein levels of P53 and P38MAPK were up-regulated(P<0.05).In addition,safflower polysaccharide inhibited the protein levels of CDC2(P<0.05).Conclusion:Safflower polysaccharide can inhibit the growth of hepatocellular carcinoma cells by inhibiting proliferation and promoting apoptosis,and the P38MAPK pathway may play an important role in this process.

Mannitol inhibits the proliferation of neural stem cell by a p38 mitogen-activated protein kinase-dependent signaling pathway

Purpose:Mannitol is one of the first-line drugs for reducing cerebral edema through increasing the extracellular osmotic pressure.However,long-term administration of mannitol in the treatment of cerebral edema triggers damage to neurons and astrocytes.Given that neural stem cell(NSC)is a subpopulation of main regenerative cells in the central nervous system after injury,the effect of mannitol on NSC is still elusive.The present study aims to elucidate the role of mannitol in NSC proliferation.Methods:C57 mice were derived from the animal house of Zunyi Medical University.A total of 15 pregnant mice were employed for the purpose of isolating NSCs in this investigation.Initially,mouse primary NSCs were isolated from the embryonic cortex of mice and subsequently identified through immunofluorescence staining.In order to investigate the impact of mannitol on NSC proliferation,both cell counting kit-8 assays and neurospheres formation assays were conducted.Thein vitro effects of mannitol were examined at various doses and time points.In order to elucidate the role of Aquaporin 4(AQP4)in the suppressive effect of mannitol on NSC proliferation,various assays including reverse transcription polymerase chain reaction,western blotting,and immunocytochemistry were conducted on control and mannitol-treated groups.Additionally,the phosphorylated p38(p-p38)was examined to explore the potential mechanism underlying the inhibitory effect of mannitol on NSC proliferation.Finally,to further confirm the involvement of the p38 mitogen-activated protein kinase-dependent(MAPK)signaling pathway in the observed inhibition of NSC proliferation by mannitol,SB203580 was employed.All data were analyzed using SPSS 20.0 software(SPSS,Inc.,Chicago,IL).The statistical analysis among multiple comparisons was performed using one-way analysis of variance(ANOVA),followed by Turkey’’s post hoc test in case of the data following a normal distribution using a Shapiro-Wilk normality test.Comparisons between 2 groups were determined using Student’s t-test,if the data exhibited a normal distribution using a Shapiro-Wilk normality test.Meanwhile,data were shown as median and interquartile range and analyzed using the Mann-WhitneyU test,if the data failed the normality test.A p<0.05 was considered as significant difference.Results:Primary NSC were isolated from the mice,and the characteristics were identified using immunostaining analysis.Thereafter,the results indicated that mannitol held the capability of inhibiting NSC proliferation in a dose-dependent and time-dependent manner using cell counting kit-8,neurospheres formation,and immunostaining of Nestin and Ki67 assays.During the process of mannitol suppressing NSC proliferation,the expression of AQP4 mRNA and protein was downregulated,while the gene expression of p-p38 was elevated by reverse transcription polymerase chain reaction,immunostaining,and western blotting assays.Subsequently,the administration of SB203580,one of the p38 MAPK signaling pathway inhibitors,partially abrogated this inhibitory effect resulting from mannitol,supporting the fact that the p38 MAPK signaling pathway participated in curbing NSC proliferation induced by mannitol.Conclusions:Mannitol inhibits NSC proliferation through downregulating AQP4,while upregulating the expression of p-p38 MAPK.

The p38/MAPK pathway as a therapeutic target to prevent therapeutic escape of breast cancer stem cells

Cancer stem cells(CSCs)play an important role in metastasis development,tumor recurrence,and treatment resistance,and are essential for the eradication of cancer.Currently,therapies fail to eradicate CSCs due to their therapeutic stress-induced cellular escape,which leads to enhanced aggressive behaviors compared with CSCs that have never been treated.However,the underlying mechanisms regulating the therapeutic escape remain unknown.To this end,we established a model to isolate the therapeutic escaped CSCs(TSCSCs)from breast CSCs and performed the transcription profile to reveal the mechanism.Mechanistically,we demonstrated that the behavior of therapeutic escape was regulated through the p38/MAPK signaling pathway,resulting in TSCSCs exhibiting enhanced motility and metastasis.Notably,blocking the p38/MAPK signaling pathway effectively reduced motility and metastasis ability both in vitro and in vivo,which were further supported by downregulated motility-related genes and epithelial-mesenchymal transition(EMT)-related proteins vimentin and N-cadherin.The obtained findings reveal the p38/MAPK pathway as a potential therapeutic target for TSCSCs and would provide profound implications for cancer therapy.

Targeting of p38 Mitogen-activated Protein Kinases to Early Growth Response gene 1 (EGR-1) in the Human Paclitaxel-resistance Ovarian Carcinoma Cells

To investigate the relationship between the expression of early growth response gene 1 （EGR-1） and p38MAPK pathway in the paclitaxel resistance of ovarian carcinoma cells, the effect of p38MAPK inhibitor SB203580 on cell apoptosis was examined by using Hoechst 33258 staining. The intracellular Rh123 （Rhodamine 123） accumulation was detected by the flow cytometry （FCM）. The 50% inhibition concentration （IC50） of paclitaxel for A2780/Taxol cells was determined by MTT method. Electrophoretic motility shift assay （EMSA） was employed to examine the EGR-1DNA binding activity. MDR1 and EGR-1 mRNA were assessed by RT-PCR. The expressed of p-gp, phos- phorylated p53 and p38 were detected by Western blotting. SB203580 could remarkably promote the apoptosis of A2780/Taxol cells, and the cell apoptosis was in a time-dependent manner. Cellular Rh123 accumulation was increased, and the IC50 of paclitaxel for A2780/Taxol cells was decreased significantly. A2780/Taxol cell line after SB203580 treatment was shown to have a significantly higher level of EGR-1 DNA binding activity. SB203580 down-regulated the activity of p38MAPK pathway, but up-regulated EGR-1 expression. SB203580 significantly increased the level of cellular phosphorylated p53 protein, but decreased the p-gp protein level and MDR1 mRNA level in A2780/Taxol cells. There existed a close relationship between p38MAPK pathway and the paclitaxel resistance of ovarian carcinoma cells. The expression of EGR-1 mediated by p38MAPK pathway plays a critical role in paclitaxel resistance of ovarian carcinoma cells.

Guaiane-type Sesquiterpenoid Dimers from Artemisia zhongdianensis and Antihepatoma Carcinoma Activity via the p38MAPK Pathway

17 new guaiane-type sesquiterpenoid dimers(GSDs),artemzhongdianolides B1—B17(1—17),were isolated from Artemisia zhongdianensis under the guidance of bioassay,and elucidated by spectral analyses(HRESIMS,1D and 2D NMR,IR,ECD).The absolute configuration of compounds 1,3,7,9,10,and 13 was determined by single-crystal X-ray diffraction analyses.Structurally,artemzhongdianolides B1(1)and B2(2)were the first example of the GSDs fused via a C-13/C-13'single bond,and artemzhongdianolides B3—B17 were[4+2]Diels–Alder adducts of two monomeric guaianolides.Most of the compounds showed antihepatoma cytotoxicity with IC_(50) values ranging from 9.9 to 170.1μmol/L.Importantly,artemzhongdianolide B9(9)was the most active one against three hepatoma cell lines with IC_(50) values of 13.1μmol/L(HepG2),19.5μmol/L(Huh7),and 19.5μmol/L(SK-Hep-1),and dose-dependently inhibited cell migration and invasion,induced G1 cell cycle arrest and cell apoptosis in HepG2 cells.Compound 9 might suppress HepG2 cells via affecting the p38MAPK signaling pathway suggested by machine learning approach,and significantly upregulated expression of phosphorylated p38 validated by Western blot assay.

Magnesium lithospermate B inhibits lipopolysaccharide-induced endothelial activation through NF-KB pathway

Aim Magnesium lithospermate B （MLB） is the most abundant hydrophilic active component of Salvia rniltiorrhiza Radix, a traditional Chinese herbal medicine mainly used to treat cardiovascular diseases. Studies have shown that endothelial activation contributes to the pathophysiology of cardiovascular diseases such as atherosclero- sis, diabetic vasculopathy, heart failure and hypertension. In the present study, the effects of MLB on endothelial activation were investigated. Lipopolysaccharide （LPS） 1 mg L^-1 was employed to induce endothelial activation, which was determined by relative gene expression and endothelial adhesion assay. Results showed that pretreatment with MLB attenuated LPS-induced ICAM1, VCAM1 and TNF-α upregulation in human dermal microvascular endo- thelial cells （HMEC-1） in dose-dependent manner, which contributed to the reduction of THP-1 adhesion to HMEC-1. Furthermore, it was revealed that 100 μmol · L^-1 MLB significantly decreased the nuclear translocation of NF-KB p65, a critical transcription factor in LPS-indueed inflammatory response, through the inhibition of IKBμ degradation. Besides, the transcriptional activity of NF-KB p65 was also inhibited by the pretreatment of MLB. Mo- reover, MLB pretreatment considerably inhibited LPS-induced p38 phosphorylation, which at least partly contribu- ted to the reduction of ICAM1 expression. In conclusion, these findings suggest that MLB inhibits LPS-induced nu- clear translocation and transcripitional activity of NF-KB, thus attenuates the increased expression of adhesion mole- cules and inflammatory factors, protects endothelial cells from LPS-induced activation.

MAPK/ERK regulation of P53 in human epidermoid carcinoma cell line A431

Objective:To observe the impact of activation and inhibition of mitogen activated protein kinases(MAPK)/extracellular signalregulated protein kinase(ERK)signaling pathway on the proliferation and apoptosis of cutaneous squamous cell carcinoma(SCC).cells and investigate the interaction mechanism between MAPK/ERK signaling pathway and tumor suppressor gene P53 in SCC.Methods:Human A431 cells were cultured and divided into MAPK/ERK inhibition groups with low-,medium-and highconcentration of inhibitors(PD98059+DMSO),MAPK/ERK activation groups with low-,medium-and high-concentration of stimuli(IGF+PBS)and blank control group(DMSO).The cell proliferation in vitro was detected by MTT assay,with the cell apoptosis detected by flow cytometry(FCM)and the protein expression of P-ERK and P53 detected by western blot in each group.Results:The A431 cell proliferation was inhibited by different concentrations of PD98059 with a clear concentration-effect and time-effect relationship(p<.05);and the cell proliferation was promoted by the different concentrations of IGF with a clear concentration-effect and time-effect relationship(p<.05).The FCM results showed a significant increase in the apoptosis rate of A431 cells which were treated with PD98059,with a clear concentration-effect relationship(p<.05);while the apoptosis rate was decreased significantly after A431 cells were treated with IGF,also with a concentration-effect relationship(p<.05).The western blot results showed that the expression of P-ERK protein was decreased but the expression of P53 was increased after A431 cells were treated with PD98059.With the concentration of PD98059 going up,the decrease in P-ERK and the increase in P53 were more significant(p<.05);while the expression of P-ERK protein was increased but the expression of P53 was decreased after A431 cells were treated with IGF.With the concentration of IGF going up,the increase in P-ERK and the decrease in P53 were more significant(p<.05).According to Pearson correlation analysis,the expression of P53 was negatively correlated to that of P-ERK(p<.05).Conclusions:After MAPK/ERK signaling pathway was activated by IGF in A431 cells,the expression of pro-apoptotic factor P53 was decreased with the ability of cell proliferation enhanced and the ability of apoptosis reduced.However,after the inhibition of MAPK/ERK signaling pathway,the expression of pro-apoptotic factor P53 was increased with the ability of cell proliferation reduced and the ability of apoptosis increased.