Mannitol inhibits the proliferation of neural stem cell by a p38 mitogen-activated protein kinase-dependent signaling pathway / 中华创伤杂志(英文版)

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. The in 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-Whitney U 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.

Impact of Folic Acid on the Resistance of Non-small Cell Lung Cancer Cells to Osimertinib by Regulating Methylation of DUSP1 / 中国肺癌杂志

BACKGROUND@#Drug resistance is the main cause of high mortality of lung cancer. This study was conducted to investigate the effect of folic acid (FA) on the resistance of non-small cell lung cancer (NSCLC) cells to Osimertinib (OSM) by regulating the methylation of dual specificity phosphatase 1 (DUSP1).@*METHODS@#The OSM resistant NSCLC cell line PC9R was establishd by gradually escalation of OSM concentration in PC9 cells. PC9R cells were randomly grouped into Control group, OSM group (5 μmol/L OSM), FA group (600 nmol/L FA), methylation inhibitor decitabine (DAC) group (10 μmol/L DAC), FA+OSM group (600 nmol/L FA+5 μmol/L OSM), and FA+OSM+DAC group (600 nmol/L FA+5 μmol/L OSM+10 μmol/L DAC). CCK-8 method was applied to detect cell proliferation ability. Scratch test was applied to test the ability of cell migration. Transwell assay was applied to detect cell invasion ability. Flow cytometry was applied to measure and analyze the apoptosis rate of cells in each group. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) method was applied to detect the expression level of DUSP1 mRNA in cells. Methylation specific PCR (MSP) was applied to detect the methylation status of the DUSP1 promoter region in each group. Western blot was applied to analyze the expression levels of DUSP1 protein and key proteins in the DUSP1 downstream mitogen-activated protein kinase (MAPK) signaling pathway in each group.@*RESULTS@#Compared with the Control group, the cell OD450 values (48 h, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the OSM group were obviously decreased (P<0.05); the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of extracellular regulated protein kinases (ERK) were obviously increased (P<0.05); the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the DAC group were obviously increased (P<0.05); the apoptosis rate, the expression of p38 MAPK protein, the phosphorylation level of ERK, and the methylation level of DUSP1 were obviously reduced (P<0.05). Compared with the OSM group, the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the FA+OSM group were obviously decreased (P<0.05); the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of ERK were obviously increased (P<0.05). Compared with the FA+OSM group, the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the FA+OSM+DAC group were obviously increased; the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of ERK were obviously reduced (P<0.05).@*CONCLUSIONS@#FA may inhibit DUSP1 expression by enhancing DUSP1 methylation, regulate downstream MAPK signal pathway, then promote apoptosis, inhibit cell invasion and metastasis, and ultimately reduce OSM resistance in NSCLC cells.

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 / 中华医学杂志(英文版)

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.

Effect and mechanism of Bovis Calculus on ulcerative colitis by inhibiting IL-17/IL-17RA/Act1 signaling pathway / 中国中药杂志

This study aimed to elucidate the effect and underlying mechanism of Bovis Calculus in the treatment of ulcerative colitis(UC) through network pharmacological prediction and animal experimental verification. Databases such as BATMAN-TCM were used to mine the potential targets of Bovis Calculus against UC, and the pathway enrichment analysis was conducted. Seventy healthy C57BL/6J mice were randomly divided into a blank group, a model group, a solvent model(2% polysorbate 80) group, a salazosulfapyridine(SASP, 0.40 g·kg~(-1)) group, and high-, medium-, and low-dose Bovis Calculus Sativus(BCS, 0.20, 0.10, and 0.05 g·kg~(-1)) groups according to the body weight. The UC model was established in mice by drinking 3% dextran sulfate sodium(DSS) solution for 7 days. The mice in the groups with drug intervention received corresponding drugs for 3 days before modeling by gavage, and continued to take drugs for 7 days while modeling(continuous administration for 10 days). During the experiment, the body weight of mice was observed, and the disease activity index(DAI) score was recorded. After 7 days of modeling, the colon length was mea-sured, and the pathological changes in colon tissues were observed by hematoxylin-eosin(HE) staining. The levels of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interleukin-6(IL-6), and interleukin-17(IL-17) in colon tissues of mice were detected by enzyme-linked immunosorbent assay(ELISA). The mRNA expression of IL-17, IL-17RA, Act1, TRAF2, TRAF5, TNF-α, IL-6, IL-1β, CXCL1, CXCL2, and CXCL10 was evaluated by real-time polymerase chain reaction(RT-PCR). The protein expression of IL-17, IL-17RA, Act1, p-p38 MAPK, and p-ERK1/2 was investigated by Western blot. The results of network pharmacological prediction showed that Bovis Calculus might play a therapeutic role through the IL-17 signaling pathway and the TNF signaling pathway. As revealed by the results of animal experiments, on the 10th day of drug administration, compared with the solvent model group, all the BCS groups showed significantly increased body weight, decreased DAI score, increased colon length, improved pathological damage of colon mucosa, and significantly inhibited expression of TNF-α,IL-6,IL-1β, and IL-17 in colon tissues. The high-dose BCS(0.20 g·kg~(-1)) could significantly reduce the mRNA expression levels of IL-17, Act1, TRAF2, TRAF5, TNF-α, IL-6, IL-1β, CXCL1, and CXCL2 in colon tissues of UC model mice, tend to down-regulate mRNA expression levels of IL-17RA and CXCL10, significantly inhibit the protein expression of IL-17RA,Act1,and p-ERK1/2, and tend to decrease the protein expression of IL-17 and p-p38 MAPK. This study, for the first time from the whole-organ-tissue-molecular level, reveals that BCS may reduce the expression of pro-inflammatory cytokines and chemokines by inhibiting the IL-17/IL-17RA/Act1 signaling pathway, thereby improving the inflammatory injury of colon tissues in DSS-induced UC mice and exerting the effect of clearing heat and removing toxins.

LASS2/TMSG1 overexpression inhibits proliferation and promotes apoptosis of human lung cancer A549 cells possibly by upregulating ceramide and p38 MAPK to activate a signaling cascade / 南方医科大学学报

OBJECTIVE@#To investigate the effects of LASS2/TMSG1 gene overexpression on proliferation and apoptosis of human lung cancer A549 cells and explore the possible mechanism.@*METHODS@#We examined LASS2/TMSG1 expression level in a previously constructed A549 cell line overexpressing LASS2/TMSG1 using Western blotting. The proliferation and apoptosis of the cells were detected using colony-forming assay, CCK-8 assay, Hoechst/PI double staining and flow cytometry. Fourteen nude mice were randomized into 2 groups (n=7) to receive subcutaneous injection of A549 cells with or without LASS2/TMSG1 overexpression on the back of the neck, and the cell proliferation in vivo was observed. The expression levels of p38 MAPK protein and p-p38 MAPK protein in the xenografts were detected with Western blotting. ELISA was used to detect the levels of ceramide and p38 MAPK protein in cultured A549 cell supernatants and the xenografts in nude mice.@*RESULTS@#Compared with the negative control cells, A549 cells with LASS2/TMSG1 overexpression had significantly lowered proliferation ability in vitro with increased early apoptosis rate (P < 0.05), and showed obvious growth inhibition after inoculation in nude mice(P < 0.05). Western blotting showed that in both cultured A549 cells and the xenografts in nude mice, LASS2/TMSG1 gene overexpression significantly increased the expression levels of p38 MAPK protein and p-p38 MAPK protein (P < 0.05); the results of ELISA also revealed significantly increased levels of ceramide and p38 MAPK protein in the cell supernatant andxenografts as well (P < 0.05).@*CONCLUSION@#Overexpression of LASS2/TMSG1 gene can significantly inhibit the proliferation and promote early apoptosis of human lung cancer A549 cells both in vitro and in vivo possibly by upregulating the expressions of ceramide and p38 MAPK protein to activate a signal transduction cascade.

Analysis of the effect of midazolam on pain in a rat model of lumbar disc herniation based on the p38 MAPK signaling pathway / 中国骨伤

OBJECTIVE@#To investigate the effect of midazolam on pain in lumbar disc herniation model rats based on p38 MAPK signaling pathway.@*METHODS@#Fifty SPF-grade Sprague-Dawley healthy rats, half male and half female, were selected and randomly divided into normal group, model group, and low-dose, medium-dose, high-dose groups. Model group and low-dose, medium-dose, high-dose groups were initially modeled for lumbar disc herniation. Intraperitoneal injection of saline was performed in rats of normal and model groups; and in the low-dose, medium-dose, and high-dose groups, intraperitoneal injection of midazolam was performed with doses of 30, 60, and 90 mg/kg, respectively. Interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), 5-hydroxytryptamine (5-HT), β-endorphin (β-EP), substance P (SP), neuropeptide Y (NPY) were detected in the serum of rats by enzyme-linked immunoassay. The expression of p38 MAPK and matrix metalloproteinase-3(MMP-3) protein were detected by Western blot in the tissues of rats of each group.@*RESULTS@#The levels of TNF-α, IL-1β and β-EP were higher and the level of 5-HT was lower in the model group than in the normal group(P<0.05);the levels of TNF-α, IL-1β and β-EP were lower and the level of 5-HT was higher in the low-dose, medium-dose and high-dose groups than in the model group(P<0.05). The levels of SP and NPY increased in the model group compared with the normal group (P<0.05) and the levels of SP and NPY decreased in the low-dose, medium-dose and high-dose groups compared with the model group (P<0.05). The expression of p38 MAPK and MMP-3 increased in the model group compared with the normal group (P<0.05); the expression of p38 MAPK and MMP-3 decreased in the low-dose, medium-dose and high-dose compared with the model group(P<0.05).@*CONCLUSION@#Midazolam may ameliorate the immune inflammatory response in rats with a model of lumbar disc herniation, possibly regulated through the p38MAPK signaling pathway.

Cangxi Tongbi Capsules promote chondrocyte autophagy by regulating circRNA＿0008365/miR-1271/p38 MAPK pathway to inhibit development of knee osteoarthritis / 中国中药杂志

To investigate the mechanism by which Cangxi Tongbi Capsules promote chondrocyte autophagy to inhibit knee osteoarthritis(KOA) progression by regulating the circRNA＿0008365/miR-1271/p38 mitogen-activated protein kinase(MAPK) pathway. The cell and animal models of KOA were established and intervened with Cangxi Tongbi Capsules, si-circRNA＿0008365, si-NC, and Cangxi Tongbi Capsules combined with si-circRNA＿0008365. Flow cytometry and transmission electron microscopy were employed to determine the level of apoptosis and observe autophagosomes, respectively. Western blot was employed to reveal the changes in the protein levels of microtubule-associated protein light chain 3(LC3)Ⅱ/Ⅰ, Beclin-1, selective autophagy junction protein p62/sequestosome 1, collagen Ⅱ, a disintegrin and metalloproteinase with thrombospondin motifs 5(ADAMTS-5), and p38 MAPK. The mRNA levels of circRNA＿0008365, miR-1271, collagen Ⅱ, and ADAMTS-5 were determined by qRT-PCR. Hematoxylin-eosin staining was employed to reveal the pathological changes of the cartilage tissue of the knee, and enzyme-linked immunosorbent assay to measure the levels of interleukin-1β(IL-1β) and tumor necrosis factor-alpha(TNF-α). The chondrocytes treated with IL-1β showed down-regulated expression of circRNA＿0008365, up-regulated expression of miR-1271 and p38 MAPK, lowered autophagy level, increased apoptosis rate, and accelerated catabolism of extracellular matrix. The intervention with Cangxi Tongbi Capsules up-regulated the expression of circRNA＿0008365, down-regulated the expression of miR-1271 and p38 MAPK, increased the autophagy level, decreased the apoptosis rate, and weakened the catabolism of extracellular matrix. However, the effect of Cangxi Tongbi Capsules was suppressed after interfering with circRNA＿0008365. The in vivo experiments showed that Cangxi Tongbi Capsules dose-dependently inhibited the p38 MAPK pathway, enhanced chondrocyte autophagy, and mitigated articular cartilage damage and inflammatory response, thereby inhibiting the progression of KOA in rats. This study indicated that Cangxi Tongbi Capsules promoted chondrocyte autophagy by regulating the circRNA＿0008365/miR-1271/p38 MAPK pathway to inhibit the development of KOA.

Effect of Jingfang Granules on carrageenan-induced tail thrombosis in mice based on ERK/p38 MAPK signaling pathway / 中国中药杂志

The present study explored the anti-inflammatory and anti-thrombotic mechanism of Jingfang Granules on tail thrombosis induced by carrageenan in mice. Thirty-two male ICR mice were randomly divided into a control group, a model group, a Jingfang Granules group, and a positive drug(aspirin) group, with eight mice in each group. The thrombosis model was induced by intraperitoneal injection of carrageenan(45 mg·kg~(-1)) combined with low-temperature stimulation, and the mice were treated with drugs for 7 days before modeling. Twenty-four hours after modeling, blood was detected for four blood coagulation indices in each group. The enzyme-linked immunosorbent assay(ELISA) was used to detect the activity of plasma interleukin-6(IL-6), interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), and other inflammatory factors. The tails of mice in each group were cut off to observe tail lesions and measure the length of the thrombus. The protein expression and phosphorylation level of extracellular signal-regulated kinase 1/2(ERK1/2) and p38 mitogen-activated protein kinase(p38 MAPK) in spleen tissues were detected by Western blot. The results showed that dark red thrombus appeared in the tails of mice in each group. The length of the black part accounted for about 40% of the total tail in the model group. Additionally, the model group showed prolonged prothrombin time(PT), increased fibrinogen(FIB) content, and shortened activated partial thromboplastin time(APTT). Compared with the model group, the groups with drug intervention displayed shortened black parts in the tail and improved four blood coagulation indices(P<0.05). As revealed by ELISA, the expression levels of TNF-α, IL-1β, and IL-6 in the mouse plasma were significantly up-regulated in the model group, and those in the groups with drug intervention were reduced as compared with the model group(P<0.05). As demonstrated by Western blot, the protein expression and phosphorylation levels of ERK1/2 and p38 MAPK in the spleen tissues were significantly elevated in the model group, while those in the Jingfang Granules group were down-regulated as compared with the model group with a significant difference. Jingfang Granules can inhibit tail thrombosis of mice caused by carrageenan presumedly by inhibiting the activation of ERK1/2 and p38 MAPK signaling pathways.

Tetrahydropalmatine alleviated diabetic neuropathic pain by inhibiting activation of microglia via p38 MAPK signaling pathway / 中国中药杂志

Neuropathic pain is one of the common complications of diabetes. Tetrahydropalmatine(THP) is a main active component of Corydalis Rhizoma with excellent anti-inflammatory and pain-alleviating properties. This study aims to investigate the therapeutic effect of THP on diabetic neuropathic pain(DNP) and the underlying mechanism. High-fat and high-sugar diet(4 weeks) and streptozotocin(STZ, 35 mg·kg~(-1), single intraperitoneal injection) were employed to induce type-2 DNP in rats. Moreover, lipopolysaccharide(LPS) was used to induce the activation of BV2 microglia in vitro to establish an inflammatory cellular model. Fasting blood glucose(FBG) was measured by a blood glucose meter. Mechanical withdrawal threshold(MWT) was assessed with von Frey filaments, and thermal withdrawal latency(TWL) with hot plate apparatus. The protein expression levels of OX42, inducible nitric oxide synthase(iNOS), CD206, p38, and p-p38 were determined by Western blot, the fluorescence expression levels of OX42 and p-p38 in the dorsal horn of the rat spinal cord by immunofluorescence, the mRNA content of p38 and OX42 in rat spinal cord tissue by qRT-PCR, and levels of nitric oxide(NO), interleukin-1β(IL-1β), interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), interleukin-10(IL-10), and serum fasting insulin(FINS) by enzyme-linked immunosorbent assay(ELISA). RESULTS:: showed that the mo-del group demonstrated significant decrease in MWT and TWL, with pain symptoms. THP significantly improved the MWT and TWL of DNP rats, inhibited the activation of microglia and p38 MAPK signaling pathway in rat spinal cord, and ameliorated its inflammatory response. Meanwhile, THP promoted the change of LPS-induced BV2 microglia from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, suppressed the activation of the p38 MAPK signaling pathway, decreased the expression levels of inflammatory factors NO, IL-1β, IL-6, and TNF-α, and increased the expression level of anti-inflammatory factor IL-10. The findings suggested that THP can significantly ameliorate the pain symptoms of DNP rats possibly by inhibiting the inflammatory response caused by M1 polarization of microglia via the p38 MAPK pathway.

Mechanism of paeoniflorin inhibiting apoptosis of hippocampal neurons of rats induced by lead acetate / 中华劳动卫生职业病杂志

Objective: To investigate the effect and underlying mechanism of paeoniflorin on hippocampal neuron apoptosis induced by lead acetate. Methods: In September 2020, primary hippocampal neuronal cells were isolated and cultured from fetal rats, and identified using cellular immunofluorescent. MTT assay was used to measure the cell viability to determine the concentration and time of lead acetate-induced hippocampal neuron apoptosis. MTT was also used to evaluate the effect of paeoniflorin concentration on the apoptosis of hippocampal neurons induced by lead acetate. According to the results, different concentrations of paeoniflorin were selected to intervene hippocampal neuron cells, after 24 h, lead acetate was added to the cells, meanwhile, blank and model groups were set up, the content of reactive oxygen species (ROS) , superoxide dismutase (SOD) , lactate dehydrogenase (LDH) , malondialdehyde (MDA) and Caspase-3 were measured. Extracellular signal regulated kinase (ERK) , phosphorylated ERK (p-ERK) , p38 mitogen -activated protein kinases (p38MAPK) , phosphorylated p38MAPK (p-p38MAPK) , c-Jun N-terminal kinase (JNK) and phosphorylated JNK (p-JNK) protein expression in hippocampal neuronal cells were determined by Western blotting. Results: The isolated and cultured hippocampal neurons were identified by immunofluorescence chemical staining and then treated with lead acetate, MTT results showed that lead acetate had the best toxicity effect when treated for 24 h at a concentration of 25 μmol/L. Paeoniflorin showed no cytotoxic effect on hippocampal neuronal cells when the concentrations below 80 μmol/L. Compared with the model group, the activity of hippocampal neuronal cells was significantly increased after treating with 20, 40 or 80 μmol/L paeoniflorin (P<0.05) . Compared with the blank group, the ROS activity, LDH release level, MDA content and caspase-3 content were significantly increased (P<0.01) , and the SOD activity was significantly decreased (P< 0.01) in the hippocampal neuronal cells of the model group. Compared with the model group, the ROS activity, LDH release level, MDA content and caspase-3 content were obviously decreased (P<0.05) , SOD activity was significantly increased (P <0.01) after hippocampal neuronal cells were treated with 40 or 80 μmol/L paeoniflorin. Relative to the model group, the ratio of p-ERK/ERK were significantly up-regulated (P<0.01) , while the ratios of p-p38MAPK/p38MAPK and p-JNK/JNK were significantly down-regulated after hippocampal neuronal cells were treated with 40 or 80 μmol/L paeoniflorin (P<0.05) . Conclusion: Paeoniflorin may down-regulate the expression of p-p38MAPK and p-JNK protein, up-regulate the expression of p-ERK protein, and inhibit the apoptosis of hippocampal neurons induced by lead acetate through the MAPK signaling pathway.

Lotus leaf flavonoids induce apoptosis of human lung cancer A549 cells through the ROS/p38 MAPK pathway

BACKGROUND: Leaves of the natural plant lotus are used in traditional Chinese medicine and tea production. They are rich in flavonoids. METHODS: In this study, lotus leaf flavonoids (LLF) were applied to human lung cancer A549 cells and human small cell lung cancer cells H446 in vitro to verify the effect of LLF on apoptosis in these cells through the ROS/p38 MAPK pathway. RESULTS: LLF had no toxic effect on normal cells at concentrations up to 500 µg/mL, but could significantly inhibit the proliferation of A549 cells and H446 cells. Flow cytometry showed that LLF could induce growth in A549 cells. We also found that LLF could increase ROS and MDA levels, and decrease SOD activity in A549 cells. Furthermore, qRT-PCR and western blot analyses showed that LLF could upregulate the expression of p38 MAPK (p-p38 MAPK), caspase-3, caspase-9, cleaved caspase-3, cleaved caspase-9 and Bax and downregulate the expression of Cu/Zn SOD, CAT, Nrf2, NQO1, HO-1, and Bcl-2 in A549 cells. Results of HPLC showed that LLF mainly contain five active substances: kaemp-feritrin, hyperoside, astragalin, phloridzin, and quercetin. The apoptosis-inducing effect of LLF on A549 cells came from these naturally active compounds. CONCLUSIONS: We have shown in this study that LLF is a bioactive substance that can induce apoptosis in A549 cells in vitro, and merits further research and development.

Jiawei Baitouweng Decoction affects intestinal mucosal tight junction proteins in rats with ulcerative colitis through p38 MAPK-MLCK signaling pathway / 中国中药杂志

The aim of this paper was to explore the effect and mechanism of Jiawei Baitouweng Decoction(JWBTW) against ulcerative colitis(UC) from the perspective of intestinal mucosal tight junction proteins. From 60 SPF-grade male SD rats, 10 were randomly selected as the blank control, and the remaining 50 were treated with 3% dextran sodium sulfate(DSS) solution to induce UC and then randomized into the model group, mesalazine group, and low-, medium-, and high-dose JWBTW( L-JWBTW, M-JWBTW and H-JWBTW) groups, with 10 rats in each group. After successive medication for 14 days, the rat general conditions like body weight and stool were observed and the disease activity index(DAI) was calculated. The pathological changes in colon tissue was observed under a microscope for injury severity scoring and histopathological scoring. The serum endotoxin content was determined by limulus assay, followed by the measurement of protein expression levels of ZO-1, occludin, claudin-1, p38 MAPK, MLCK, MLC2 and p-MLC in colon tissue by Western blot. The results showed that compared with the blank group, the model group exhibited significantly reduced body weight, elevated DAI, injury severity and histopathological scores and serum endotoxin content, up-regulated protein expression levels of p38 MAPK, MLCK, MLC2 and p-MLC, and down-regulated ZO-1, occludin and claudin-1. Compared with the model group,mesalazine and JWBTW at each dose obviously increased the body weight, lowered the DAI, injury severity and histopathological scores and serum endotoxin content, down-regulated the protein expression levels of p38 MAPK, MLCK, MLC2 and p-MLC, and up-regulated the ZO-1, occludin and claudin-1, with the most obvious changes noticed in the H-JWBTW group. All these have indicated that JWBTW exerts the therapeutic effect against UC by inhibiting the activation of p38 MAPK/MLCK pathway, reversing the protein expression levels of occludin, claudin-1 and ZO-1, decreasing the serum endotoxin content, promoting the repair of intestinal mucosal mechanical barrier, maintaining the integrity of tight junctions, and reducing the permeability of intestinal mucosa.

Mechanism of advanced glycation end products inhibiting the proliferation of peripheral blood mononuclear cells and osteoblasts in rats / 北京大学学报(医学版)

OBJECTIVE@#To explore the mechanism of nuclear factor-kappa B (NF-κB), phosphatidylinositol 3-kinase (PI3K)/protein kinase B(PKB/Akt) and mitogen-activated protein kinase (MAPK) signaling pathways after intervention of advanced glycosylation end products (AGEs) in peripheral blood mononuclear cells (PBMCs) and osteoblasts (OB) in rats, so as to provide certain experimental basis and theoretical basis for further research on the clinical treatment of periodontal tissue inflammation caused by diabetes mellitus.@*METHODS@#AGEs were prepared, PBMCs and OB were isolated and cultured in vitro. CCK-8 was used to detect the cell viability intervened by different concentrations and time of AGEs. Western blot and qRT-PCR were used to detect the expression changes of genes related to NF-κB, PI3K/PKB and MAPK signaling pathways.@*RESULTS@#OB and PBMCs were successfully isolated and cultured in vitro. The activity of PBMCs and OB cells was significantly correlated with the concentration, time and interaction of AGEs. With the increase of AGEs concentration and time, the activity of PBMCs and OB cells significantly decreased (P < 0.001). AGEs stimulation significantly increased the expression of NF-κB in PBMCs and the contents of tumor necrosis factor α(TNF-α), interleukin-1β(IL-1β) (P < 0.01). TNF-α, IL-1β levels were significantly reduced after inhibition of NF-κB pathway (P < 0.01). NF-κB p65, JNK, and p38 phosphorylated and non-phosphorylated proteins increased significantly after AGEs stimulation of OB (P < 0.05). The phosphorylated protein expression of IκB was significantly increased, while the expression of non-phosphorylated protein was decreased (P < 0.01).The expressions of NF-κB p65, JNK, and IκB were significantly increased at the mRNA levels, and the expressions of IκB mRNA were significantly decreased (P < 0.05). There was no difference in the expression of Akt in either phosphorylated or non-phosphorylated proteins or at the mRNA level (P>0.05). With the addition of MAPK signaling pathway inhibitors, the phosphorylation and non-phosphorylated protein expressions of NF-κB p65, p38 and JNK were significantly reduced, and the phosphorylated protein of IκB was significantly decreased and the non-phosphorylated protein was significantly increased compared with the group with AGEs alone (P < 0.05). The results of qRT-PCR showed that the expression of IκB increased significantly after the addition of the JNK pathway blocker (P < 0.05), and the expression of NF-κB p65, p38 and JNK decreased, but the difference was not significant (P>0.05). While NF-κB p65, p38 and JNK were significantly decreased and IκB was significantly increased in the AGEs group after the addition of the p38 pathway blocker (P < 0.05). At this time, there was still no significant change in the expression of Akt at the protein level and mRNA level (P>0.05).@*CONCLUSION@#AGEs inhibit the proliferation of PBMCs and OB, and the NF-κB and MAPK pathways are likely involved in regulating this process, but not the PI3K/PKB pathway.

Acanthus ilicifolius L. Treatment for Oral Candidiasis with Immunosuppressive Conditions Subjected to p38 MAPK Enhancement

ABSTRACT@#Methanolic extract from the leaves of Acanthus ilicifolius L. (A. ilicifolius L.) is a potent inhibitor of Candida albicans (C. albicans) growth and anti-inflammatory. C. albicans causes oral candidiasis in immunosuppressive condition. Mitogen-activated protein kinase (MAPK) signalling via p38 appears to discriminate between yeast and hyphal cells of C. albicans. Activation of p38 MAPK by hyphae results in the upregulation of proinflammatory cytokines. The p38 MAPK activation is known to impair corticosteroid action. The research was conducted to investigate the effect of methanolic extract A. ilicifolius L. treatment of oral candidiasis with the immunosuppressive condition through enhancement of p38 MAPK expression in the epithelial cells. Immunosuppressed conditions were obtained when 16 healthy male Rattus norvergicus (Wistar) was given oral administration of dexamethasone and tetracycline for 14 days and induced with C. albicans (ATCC-10231) 1 McFarland. The subjects were divided into four groups (n = 4/group): immunosuppression (IS), immunosuppression with oral candidiasis without treatment (ISC), immunosuppression with oral candidiasis and nystatin treatment (ISC+N), and immunosuppression with oral candidiasis and A. ilicifolius L. treatment (ISC+AI), and were treated for 14 days. Later, the rats were euthanised, and their tongue were biopsied. The p38 MAPK expression was subjected to immunohistochemical examination, observed under a microscope (400× magnification) and statistically analysed (one-way ANOVA, LSD-test, p < 0.05). The p38 MAPK expression of ISC+AI (36.05 ± 1.54) was higher than IS (26 ± 2.32), ISC (26.4 ± 3.71), IS+N (34.2 ± 0.99). Significant differences existed between ISC+AI and ISC+N to IS and ISC (p < 0.05). No significant differences were present between IS and ISC; ISC+AI and ISC+N (p > 0.05). Therefore, this treatment could enhance p38 MAPK expression in oral candidiasis with the immunosuppressed condition.

Total flavonoids of Drynariae Rhizoma regulates ER-p38 MAPK signaling pathway to improve scopolamine-induced learning and memory impairments in model mice / 中国中药杂志

This study intended to explore the effect and mechanism of total flavonoids of Drynariae Rhizoma in improving scopola-mine-induced learning and memory impairments in model mice. Ninety four-month-old Kunming(KM) mice were randomly divided into six groups. The ones in the model group and blank group were treated with intragastric administration of normal saline, while those in the medication groups separately received the total flavonoids of Drynariae Rhizoma, Kangnaoshuai Capsules, donepezil, as well as total flavonoids of Rhizoma Drynariae plus estrogen receptor(ER) blocker by gavage. The mouse model of learning and memory impairments was established via intraperitoneal injection of scopolamine. Following the measurement of mouse learning and memory abilities in Morris water maze test, the hippocampal ERβ expression was detected by immunohistochemistry, and the expression levels of ERβ and phosphorylated p38(p-p38) in the hippocampus and B-cell lymphoma 2(Bcl-2), Bcl-2-associated death promoter(Bad), and cysteinyl aspartate-specific protease-3(caspase-3) in the apoptotic system were assayed by Western blot. The contents of malondia-ldehyde(MDA), superoxide dismutase(SOD), and nitric oxide(NO) in the hippocampus were then determined using corresponding kits. Compared with the control group, the model group exhibited significantly prolonged incubation period, reduced frequency of cros-sing the platform, shortened residence time in the target quadrant, lowered ERβ, Bcl-2 and SOD activity in the hippocampus, and increased p-p38/p38, Bad, caspase-3, MDA, and NO. Compared with the model group, the total flavonoids of Rhizoma Drynariae increased the expression of ERβ and SOD in the hippocampus, down-regulated the expression of neuronal pro-apoptotic proteins, up-re-gulated the expression of anti-apoptotic proteins, and reduced p-p38/p38, MDA, and NO. The effects of total flavonoids of Drynariae Rhizoma on the above indexes were reversed by ER blocker. It has been proved that the total flavonoids of Drynariae Rhizoma obviously alleviate scopolamine-induced learning and memory impairments in mice, which may be achieved by regulating the neuronal apoptotic system and oxidative stress via the ER-p38 mitogen-activated protein kinase(ER-p38 MAPK) signaling pathway.

Guanxin Zhitong Capsules attenuate human endothelial cell damage induced by palmitic acid via MAPK signaling pathway / 中国中药杂志

The present study observed the effect of Guanxin Zhitong Capsules(GXZT) on the lipotoxicity of vascular endothelial cells and investigated the mechanism of GXZT in atherosclerosis treatment. The lipotoxicity model in human umbilical vein endothelial cells(HUVECs) was induced by palmitic acid(PA) stimulation. These cells were divided into a normal control group(NC, 15% normal serum), a model group(PA, 0.6 mmol·L~(-1) PA+15% normal serum), a high-dose GXZT group(GXZT-H, 0.6 mmol·L~(-1) PA+15% GXZT-medicated serum), a medium-dose GXZT group(GXZT-M, 0.6 mmol·L~(-1) PA+10% GXZT-medicated serum+5% normal serum) and a low-dose GXZT group(GXZT-L, 0.6 mmol·L~(-1) PA+5% GXZT-medicated serum+10% normal serum). HUVECs were detected for cell viability by cell counting kit-8(CCK-8) assay, apoptosis by flow cytometry, mitochondrial membrane potential(MMP) by JC-1 labeled laser scanning confocal microscopy, and total and phosphorylated proteins of p38, ERK1/2, and JNK1/2 in the mitogen-activated protein kinases(MAPK) signaling pathway by Western blot. The phosphorylated level was calcula-ted. Compared with the NC group, the PA group showed decreased cell viability and MMP(P<0.01, P<0.01), elevated apoptosis(P<0.01), and up-regulated phosphorylated levels of p38, ERK1/2, and JNK1/2(P<0.01, P<0.01, P<0.01). Compared with the PA group, the GXZT-H, GXZT-M, and GXZT-L groups showed increased cell viability and MMP(P<0.01, P<0.01, P<0.01), reduced apoptosis(P<0.01), and down-regulated protein expression and phosphorylated levels of p38, ERK1/2 and JNK1/2 in the MAPK signaling pathway(P<0.01, P<0.01, P<0.01). In conclusion, the results suggest that GXZT functions via blocking MAPK signaling pathway to relieve the damage of HUVECs induced by PA.

Effect of IL-33 on pyroptosis of macrophages in mice with sepsis via NF-κB/p38 MAPK signaling pathway

ABSTRACT Purpose To demonstrate the effect of IL-33 on the macrophage pyroptosis in mice with sepsis through the NF-kB/p38 MAPK signal pathway. Methods In total, 24 C57BL/6 mice were divided into the sham operation group (sham) and the cecal ligation and puncture group (CLP). After CLP, 24 IL-33-/- mice were divided into the IL-33-/- group and the IL-33-/- intervention group. The latter group was intraperitoneally injected with IL-33. Mouse mortality was observed after CLP. Macrophage apoptosis in peritoneal lavage fluid was detected by flow cytometry. Serum inflammatory factor level was detected by ELISA. Apoptotic protein expression and NF-κB/p38 MAKP signaling pathway protein expression were detected by qRT-PCR and Western blot. Results Knocking out IL-33 significantly reduced the mortality of CLP mice, as well as the mRNA expression of IL-33 and the levels of serum inflammatory factors, including IL-33, IL-1β, and IL-18. It also reduced the rate of macrophage apoptosis and the expression of the apoptotic protein caspase-1 p10; increased the expression of IκBα; and reduced the protein expression of NF-κB and p38 MAPK. These effects were reversed after exogenous injection of IL-33. Conclusions IL-33 can increase the level of macrophage pyroptosis in mice with sepsis (by activating the NF-kB/p38MAPK signal pathway) and the mortality of these mice.

Effects of propofol on inflammatory response and activation of p38 MAPK signaling pathway in rats with ventilator-induced lung injury

ABSTRACT Purpose: To investigate the effects of propofol on inflammatory response and activation of p38 mitogen-activated protein kinase (MAPK) signaling pathway in rats with ventilator-associated lung injury (VALI). Methods: Thirty-six Sprague Dawley (SD) rats were divided into control, VALI and VALI+propofol groups. The VALI group received the mechanical ventilation for 2 h. The VALI+propofol group received the mechanical ventilation for 2 h, which was accompanied by intravenous injection of propofol with dose of 8 mg·kg-1·h-1. At the end, the mean arterial pressure (MAP) and blood gas indexes were measured, and the lung wet/dry mass ratio (W/D) and biochemical indexes of lung tissue and bronchoalveolar lavage fluid (BALF) were determined. Results: Compared with VALI group, in VALI+propofol group the blood pH, partial pressure of oxygen, partial pressure of carbon dioxide and MAP were increased, the lung W/D, lung tissue myeloperoxidase activity and total protein concentration, white blood cell count, and tumor necrosis factor α, interleukin 1β and interleukin 6 levels in BALF were decreased, and the p-p38 MAPK protein expression level and phosphorylated p38 MAPK (p-p38 MAPK)/p38 MAPK ratio were decreased. Conclusions: Propofol treatment may alleviate the VALI in rats by reducing the inflammatory response and inhibiting the activation of p38 MAPK signaling pathway.

Ca mobilization and signaling pathways induced by rRgpB in human gingival fibroblast / 浙江大学学报·医学版

: To assess the () recombinant gingivalis gingipain R2 (rRgpB)-induced Ca mobilization in human gingival fibroblast (HGF) mediated by protease-activated receptor (PAR) and its downstream signal transduction pathways. : Flow cytometry was used to detect the expression of PAR in HGF. The proliferation of HGF was measured by CCK-8. The dynamic changes of intracellular Ca concentration in HGF induced by rRgpB and the blocking effect of PAR-1 antagonist were observed by laser confocal microscopy. Western blot was performed to determine the phosphorylation levels of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) 1/2, p38 mitogen-activated protein kinase (p38 MAPK) and p65 in HGF. : PAR-1 and PAR-3 were expressed in HGF, and the rRgpB could promote the proliferation of HGF. rRgpB caused a transient increase in [Ca], which could be completely suppressed by vorapaxar, a PAR-1 antagonist. The phosphorylation levels of JNK, ERK1/2 and p65 were significantly up-regulated after the induction of rRgpB for and (all <0.05), which was completely inhibited by vorapaxar. However, the phosphorylation level of p38 MAPK had no significant change after rRgpB stimulation. : rRgpB causes an increase in [Ca] in HGF mediated by PAR-1. JNK, ERK1/2 and nuclear factor-κB may be involved in intracellular signal transduction after PAR-1 activation.