Effects and mechanism of pterostilbene on wound healing in diabetic skin ulcer model rats / 中国药房

OBJECTIVE To explore the effects of pterostilbene （PTE） on wound healing in diabetic skin ulcer model rats and its mechanism. METHODS Ten SD rats were grouped into control group； after diabetic skin ulcer model of other rats was induced by giving high-fat and high-sugar diet+intraperitoneal injection of streptozotocin+cutting off the skin and subcutaneous tissue in the marked area of the back， model rats were randomly divided into model group， PTE low-dose group （40 mg/kg）， PTE high-dose group （80 mg/kg）， PTE high-dose+PP2 group （80 mg/kg PTE+2 mg/kg SRC inhibitor PP2）， with 10 rats in each group. On the second day after modeling， the rats in each drug group were intraperitoneally injected with corresponding drug solutions， while the rats in control group and model group were intraperitoneally injected with normal saline， once a day， for 14 consecutive days. The wound healing rate of rats in each group was measured on the 7th and 14th day of administration； the contents of interleukin-1β （IL-1β）， IL-6， tumor necrosis factor-α （TNF-α） and vascular endothelial growth factor （VEGF） in the serum of rats were detected； the pathological changes of wound granulation tissue were observed， and the expressions of SRC/mitogen-activated protein kinase kinase （MEK）/extracellular signal-regulated kinase （ERK） signaling pathway-related proteins in wound granulation tissue were detected. RESULTS Compared with control group， the wound healing rate， serum content of VEGF， the phosphorylation levels of SRC， MEK1/2 and ERK1/2 were decreased significantly （P＜0.05）， while serum contents of IL- 1β， IL-6 and TNF-α were increased significantly （P＜0.05）； there was obvious infiltration of inflammatory cells in the wound granulation tissue， and the number of new blood vessels decreased. Compared with model group， above indexes of PTE low-dose and high-dose groups were improved significantly （P＜0.05）， and the pathological injury of granulation tissue in wound was improved. PP2 significantly reversed the improvement effects of PTE on the above indexes （P＜0.05）. CONCLUSIONS PTE can promote the wound healing of diabetic skin ulcer model rats， the mechanism of which may be related to activating SRC/MEK/ERK signaling pathway.

Research progress on targeted drugs for vascular malformations / 口腔疾病防治

@#Vascular malformations, which mainly occur in the head and neck region, are a group of congenital disorders that cannot involute and dilate gradually as patients grow. Traditional therapeutic strategies for vascular malformations include laser therapy, sclerotherapy, interventional embolization, surgical resection, etc. However, for some cases with a relatively larger range of lesions, traditional therapeutic strategies might fall short of the goals. With the development of molecular genetics, gene mutations are currently recognized as the root cause of the occurrence of vascular malformations. The progression of vascular malformation lesions is further promoted by the activation of related pathways. Low-flow vascular malformations mainly involve activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway, whereas high-flow vascular malformations mainly involve activation of the rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase kinase (MAPKK)/extracellular-signal regulated protein kinase (ERK) pathway. Targeted drugs against relevant gene mutations and signaling pathways have also been applied in the treatment of vascular malformations, and previous studies have shown that the mTOR inhibitor rapamycin is effective and now widely used in the treatment of low-flow vascular malformations. The PI3K inhibitor alpelisib is also promising in the treatment of venous malformations, and the MAPKK inhibitor trametinib has shown good results in the treatment of arteriovenous malformations. Therefore, traditional therapies supplemented by targeted drugs may bring new breakthroughs to the treatment of vascular malformations.

Inhibitory Effect of Liuwei Dihuangwan on Triple-negative Breast Cancer Through MAPKKK1 and KLF4 / 中国实验方剂学杂志

ObjectiveTo study the underlying mechanism of Liuwei Dihuangwan in inhibiting triple-negative breast cancer through mitogen-activated protein kinase kinase kinase 1 （MAPKKK1） and Krüppel-like factor 4 （KLF4）. MethodFour hundreds SPF female Kunming mice aged 11.5 months were palpated once every 3 days. The model mice of spontaneous tumors were randomly divided into a model group （normal saline）， a paclitaxel group （0.025 g·kg-1·d-1， ip， 21 days）， and high-， medium- and low-dose Liuwei Dihuangwan groups （7.2， 3.6， 1.8 g·kg-1·d-1， ig）. Tumor tissues were separated until the moribund stage. The tumor volume and weight were measured， and the tumor inhibition rate and the survival time of the tumor mice were calculated （after 6 months， tumor-free mice were assigned into the normal group）. SPF SD rats were selected to prepare serum samples containing Liuwei Dihuangwan of different concentrations for cell culture， and MAPKKK1 in MDA-MB-231 cells was silenced. The protein expression of MAPKKK1 and KLF4 was detected by immunofluorescence and Western blot. ResultThe in vivo experimental results showed that compared with the conditions of the normal group， the protein expression of MAPKKK1 and KLF4 in tumor tissues of the model group dropped （P<0.01）. Compared with the model group， all medication groups showed reduced tumor volume and weight （P<0.05， P<0.01）， increased tumor inhibition rate， prolonged survival time of tumor mice （P<0.05）， and increased protein expression of MAPKKK1 （P<0.01）. Additionally， the paclitaxel group and the high-dose Liuwei Dihuangwan group exhibited increased protein expression of KLF4 （P<0.01）. The in vitro experiments showed that compared with the conditions of the normal group， the fluorescence intensities of MAPKKK1 and KLF4 in MDA-MB-231 cells in all medication groups were potentiated， and the protein expression of MAPKKK1 in the paclitaxel group and the high- and medium-dose Liuwei Dihuangwan groups， and the protein expression of KLF4 in the paclitaxel group and high-dose Liuwei Dihuangwan group increased （P<0.01）. After silencing of MAPKKK1， compared with the conditions of the negative plasmid group （unsilenced MAPKKK1）， the fluorescence intensities of MAPKKK1 and KLF4 and the protein expression decreased in the RNAi-27 positive plasmid group （silenced MAPKKK1） （P<0.05， P<0.01）. Compared with the RNAi-27 positive plasmid group， all medication groups had enhanced fluorescence intensities of MAPKKK1 and KLF4 and protein expression to different degrees （P<0.05， P<0.01）. ConclusionLiuwei Dihuangwan can inhibit the growth of triple-negative breast cancer， and the underlying molecular mechanism is related to the up-regulation of MAPKKK1 and activation of KLF4 expression.

Expression of mitogen-activated protein kinase kinase 1 in gastric cancer and its clinical significance / 解剖学报

Objective To investigate the expression of mitogen-activated protein kinase kinase 1 (MAP2K1) in gastric cancer and its clinical significance. Methods Immunohistochemistry and Western blotting were used to detect the protein expression of MAP2K1 in gastric cancertissues and cells. The morphology and the expression position of MAP2K1 were observed by immunofluorescence. MAP2K1 mRNA expression in gastric cancer tissues was analyzed by data mining of Starbase database and Oneomine database. The correlation between MAP2K1 mRNA expression and clinicopathological features was analyzed by UALCAN database. Survival analysis was performed using Kaplan Meier-Plotter online analysis tools. GEPIA2 database mining the relationship between MAP2K1 and gastric cancer stem cell related factors and drug resistance related factors. Results Immunohistochemistry, immunofluorescence and Western blotting showed that MAP2K1 protein was highly expressed in gastric cancer tissues and cells, and MAP2K1 was expressed in the cytoplasm of gastric cancer. According to the analysis of various databases, the expression of MAP2K1 mRNA in gastric cancer tissue was higher than that in normal gastric tissue, and the expression of MAP2K1 mRNA was closely related to gastric cancer stage, grade, lymph node metastasis and patient gender, and the overall survival rate of gastric cancer patients in the group with high MAP2K1 mRNA expression was significantly lower than that in the group with low MAP2K1 mRNA expression, which may be related to the characteristics of gastric cancer stem cells and drug resistance. Conclusion MAP2K1 is highly expressed in gastric cancer, and its expression level may affect the poor prognosis of patients by regulating stem cell related factors and drug resistance related factors. MAP2K1 may be a new diagnostic marker to determine the prognosis of gastric cancer patients.

Effect of Panax Notoginseng Saponins on Breast Cancer Cell Line 4T1 in Tumor-bearing Mice Through MEKK1/SEK1/JNK1/AP-1 Pathway / 中国实验方剂学杂志

Objective:To explore the potential mechanisms of Panax Notoginseng Saponins （PNS） on growth inhibition of breast cancer cell line 4T1 in tumor-bearing mice by investigating the mitogen-activated protein kinase kinase kinase 1 （MEKK1）/stress activated protein kinase （SAPK）/extracellular regulated protein kinases （Erk） Kinase （SEK1）/c-Jun N-terminal kinase 1 （JNK1）/activator protein-1 （AP-1） signaling pathways. Method:The 4T1 breast cancer mice model was established. Forty-eight mice with successful modeled and randomly divided into the low， medium and high-dose PNS groups （10， 20， 40 mg·kg-1） and the model control group （12 mice in each group）. The PNS groups received intraperitoneal injection with dosage of 10 mL·kg-1， while the controlled group was given the same dosage of saline. After administration with PNS for 28 days， tumor tissues were isolated， weighed， sliced and homogenized. Tumor cell apoptosis was detected by TdT mediated-dUTP nick end labeling （TUNEL） staining. The mRNA expressions of MEKK1， SEK1， JNK1 and AP-1 in tumor tissue were detected by Real-time polymerase chain reaction（Real-time PCR）. The protein expressions of MEKK1， SEK1， JNK1 and AP-1 in tumor tissue were detected by immunofluorescence staining and Western blot. Result:Compared with model group， the tumor weights of medium-dose and high-dose PNS groups were decreased significantly （P<0.05）. TUNEL staining showed that the number of apoptotic tumor cells increased with the rise of dosage of PNS （P<0.05）. The medium-dose and high-dose PNS groups showed a significant increase in the mRNA expressions of MEKK1， SEK1， JNK1 and AP-1 as well as the protein expressions of MEKK1， SEK1， JNK1 and AP-1 in tumor tissues （P<0.05）， with statistically significant differences （P<0.05）. Conclusion:PNS could inhibit the tumor growth of breast cancer cell line 4T1 in tumor-bearing mice， which may be related to the activation of MEKK1/SEK1/JNK1/AP-1 signaling pathways.

Mechanism of Duanteng Yimu Decoction on Expression of MEKK2 in Rheumatoid Arthritis Synovial Fibroblasts and Arthritis in CIA Mice / 中国实验方剂学杂志

Objective::Duanteng Yimu decoction(DTYMD)is effective in treatment of rheumatoid arthritis (RA) by relieving joint inflammation and down-regulating some inflammatory factors in a short period of time, but the mechanism is still unclear. We aimed to investigate upstream kinase of mitogen activated protein kinases(MAPK) and define the anti-inflammatory mechanism of DTYMD. Method::Fibroblasts-like synovial cells(FLSs) were divided into blank group, model group (IL-1β), high-dose DTYMD group (1 000 mg·L-1), medium-dose DTYMD group (800 mg·L-1), low-dose DTYMD group (600 mg·L-1) and armour ammonia butterfly(MTX) group (20 μmol·L-1). The protein and mRNA expressions of mitogen-activated protein kinase kinase kinase 2 (MEKK2) were analyzed by real-time fluorescence quantitative PCR(Real-time PCR). Totally 42 male DBA/1J mice were randomly divided into 6 groups, with 7 mice in each group, namely normal group, model group and MTX group (2 mg·kg-1), low-dose DTYMD group (6.25 mg·kg-1), medium-dose DTYMD group (12.5 mg·kg-1), and high-dose DTYMD group (25 mg·kg-1). Except for the normal group, the other five groups were included in collagen-induced arthritis(CIA) model by secondary immunoassay. After administration, the posterior limbs and ankle joints were stained with htoxylin-eosin(HE), and the pathological scores of the joints were evaluated. Result::Compared with the model group, DTYMD inhibited the activity of FLSs in a concentration-dependent manner (P<0.01). Compared with the blank control group, the cell proliferation rate of the model group increased (P<0.01). Compared with the model group, high and middle-dose DTYMD groups could inhibit protein and mRNA expressions of MEKK2 (P<0.01), but there was no significant difference in low-dose group. However, the expression of DTYMD protein in high/medium/low-dose groups was significantly higher than that in blank group (P<0.01), but there was no significant difference in MTX group. Compared with the model group, the expressions of matrix metalloprotease-1 (MMP-1), tumor necrosis factor-α(TNF-α) and interleukin(IL)-6 were negatively regulated in different DTYMD groups(P<0.01), and the expressions of MMP-1, IL-6, TNF-α in the model group were significantly higher than those in the blank group (P<0.05, P<0.01). In the animal experiment, compared with the model group, high/middle-dose DTYMD groups could decrease the degree of joint swelling in CIA mice (P<0.01), but there was no significant difference in the low dose group, and the joint swelling in the model group was significantly higher than that in the blank group (P<0.05). In HE staining of ankle joint of CIA mice, the pathological scores of high/small-dose DTYMD groups were significantly lower those of model group (P<0.05, P<0.01), and the pathological score of model group was higher than that of blank group (P<0.01). Conclusion::DTYMD might down-regulate MEKK2 to negatively regulate inflammatory cytokines IL-6, TNF-α and MMP-1, thereby alleviating the inflammatory response in rheumatoid arthritis.

Expression of MAP4K4 in bladder cancer and its biological effect on human bladder cancer T24 cells / 中华泌尿外科杂志

Objective To observe the effect of MAP4K4 targeted shRNA on biological characteristics such as proliferation,invasiveness,and apoptosis in human bladder cancer cell.Methods Differentially expressed genes was screened out through cDNA microarray analysis in 5 pairs of fresh-frozen muscle-invasive bladder cancer(MIBC) and adjacent normal tissue obtained from radical cystectomy.Combining the results of genechip and literature review,MAP4K4 was picked up for further analysis.To verify the result of microarray analysis,16 pairs of fresh muscle-invasive bladder cancer (MIBC) and adjacent tissues were assessed for the expression of MAP4K4 mRNA and protein through RT-PCR,qRT-PCR and Western-blot.T24 cell line was stably trasfected with MAP4K4 targeted shRNA and control shRNA,respectively.The effects of MAP4K4 silencing on proliferation,invasiveness and apoptosis of T24 cells transfected with MAP4K4 targeted shRNA and control shRNA were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),transwell and flowcytometry (FCM) assay.Results MAP4K4 was overexpressed in muscle invasive bladder cancer than in normal tissue.Down regulation of MAP4K4 expression decreased bladder cancer cell proliferation(MAP4K4-targeted versus control,P＜0.001),invasiveness(MAP4K4-targeted versus control,P=0.004)and promoted cell apoptosis(MAP4K4-targeted versus control,P=0.023).Conclusions MAP4K4 is overexpressed in muscle invasive bladder cancer than in normal tissue.Down-regulation of MAP4K4 expression inhibits the invasive ability of bladder cancer.Therefore,MAP4K4 might be a potential therapeutic target for bladder cancer.

lnhibiton of JNK phosphorylation involved in melatonin′s protection of calyculin A-induced tau hyperphosphorylation / 中国药理学与毒理学杂志

OBJECTlVE To explore the mechanisms of tau hyperphosphorylation induced by calyculin A ( CA) in neuroblastoma cells and the effect of melatonin. METHODS N2a cells were treated with CA 5 nmol·L-1 , or CA with melatonin 50 μmol·L-1 , or CA with vitamin E ( Vit E ) 50 μmol·L-1 for 12 h. The level of tau phosphorylation at Ser422 ( recognized by R145d antibody) site and the level of phosphorylated c-Jun N-terminal kinases ( p-JNK ) and phosphorylated mitogen-activated protein kinase kinase 4 ( p-MKK4 ) were detected with immunoblotting, the level of malondialdehyde ( MDA ) was assayed with fluorimetry, and the activity of p38-mitogen activated protein kinase ( p38MAPK ) was assayed by radioimmunobloting. RESULTS CA treatment increased the level of phosphorylated tau at Ser422 site (1.70±0.19, 1.0, P<0.01), and melatonin attenuated the effect of CA (0.98±0.12, 1.70± 0.19, P<0.01). ln addition, CA treatment increased the level of MDA (μmol·g-1 protein:0.241±0.006, 0.141±0.006, P<0.01) and melatonin antagonized the increase of MDA induced by CA (μmol·g-1 protein:0.172±0.004, 0.193±0.005, 0.241±0.006, P<0.01) . CA treatment increased the level of p-JNK (1.91±0.27, 1, P<0.01) and p-MKK4 (1.81±0.09, 1, P<0.01) and melatonin antagonized the effect of CA induced increase of p-JNK (1.11±0.15, 1.91±0.27, P<0.01) and p-MKK4 (1.14±0.06, 1.81±0.09, P<0.01) without changing the level or activity of p38MAPK. Both JNK inhibitor ( SP600125 ) and MKK4/JNK transduction pathway inhibitor antagonized CA induced tau phosphorylation at Ser422 site and JNK phosphorylation. CONCLUSlON lnhibiton of JNK phosphorylation is possibly involved in the protection of melatonin on CA-induced tau hyperphosphorylation at Ser422 site.

MicroRNA-21-5p regulating p38 mitogen-activated protein kinases signaling pathway / 中华实用儿科临床杂志

Objective To verify the targeting regulatory relationship between microRNA (miR)-21-5p and mitogen-activated protein kinase kinase 3 (MKK3).Methods miR-21-5p and MKK3 targeted relationship was analyzed by bioinformatics database predict.Constructing MKK3 3'UTR reporter vector (MKK3-3U) and mutation reporter vector (MKK3-3 U-M) was constructed by synthesizing miR-21-5p mimics (mimic),miR-2 1-5p inhibitor (inhibitor),nonsense miR (NC).NC (NC1 group),mimics (mimic1 group),inhibitor (inhibitor1 group) together with MKK3-3U cotransfected human embryonic kidney (HEK293) cells,respectively.Empty vector-pYr-MirTarget (NC2 group),MKK33U (Mt group),M KK3-3U-M (Mut group) together with mimic co-transfected HEK293,respectively.The fluorescence ratio was detected by dual luciferase assay.NC,mimics,inhibitor intervened human renal tubular epithelial (HK-2) cells,respectively.RT-PCR and Western blot were used to test cell MKK3 expression level in HK-2.Results 1.The fluorescence ratios of NC1 group,mimicsl group,and inhibitor1 group were 100.00％,67.99％,133.17％,and there was difference between mimic1 group and NC1 group(t =19.93,P ＜ 0.05).2.The fluorescence ratios of NC2 group,Mt group and Mut group were 100.00％,65.30％,98.48％,and there was difference between Mt group and NC2 group (t =14.39,P ＜ 0.05).There was no significant difference between NC2 and Mut group (t =0.63,P ＞ 0.05).3.MKK3 mRNA relative expression levels of the control group mimic2 group and inhibitor2 group were 1.36 ± 0.02,1.01 ± 0.04,1.43 ± 0.06 ; relative protein expression levels were 0.97 ± 0.05,0.62 ± 0.06,1.36 ± 0.32.MKK3 mRNA and protein levels of mimic2 group were significantly lower compared with the control group (F =85.98,118.26,all P ＜ 0.01).Conclusions MKK3 is the target gene of miR-21-5p.miR-21-5p can regulate the expression of MKK3 both in mRNA and protein levels.miR-21-5p may be an important regulatory molecule in p38 mitogen-activated protein kinase signaling pathway.

Mutation analysis of mitogen activated protein kinase 1 gene in Indian cases of 46,XY disorder of sex development.

BACKGROUND: Determination of sex is the result of cascade of molecular events that cause undifferentiated bipotential gonad to develop as a testis or an ovary. A series of genes such as SRY, steroidogenic factor‑1 (SF1), AR, SRD5 α, Desert hedgehog (DHH) etc., have been reported to have a significant role in development of sex in the fetus and secondary sexual characteristics at the time of puberty. Recently, mitogen activated protein kinase kinase kinase 1 (MAP3K1) gene was found to be associated with 46, XY disorders of sex development (DSD). AIM: The present study is focused to identify mutations in MAP3K1 gene in the cohort of 10 Indian patients with 46,XY DSD including one family with two affected sisters. These patients were already screened for SRY, SF1 and DHH gene, but no mutation was observed in any of these genes. MATERIALS AND METHODS: The entire coding regions of MAP3K1 were amplified and sequenced using the gene specific primers. RESULTS AND DISCUSSIONS: Sequence analysis of MAP3K1 gene has revealed four variants including one missense, two silent and one deletion mutation. The missense mutation p.D806N was observed in four patients with hypospadias. Two patients showed the presence of silent mutation p.Q1028Q present in exon 14. Another silent mutation p.T428T was observed in a patient with gonadal dysgenesis. We have also observed one deletion mutation p. 942insT present in two patients. The pathogenicity of the missense mutation p.D806N was carried out using in‑silico approach. Sequence homology analysis has revealed that the aspartate at 806 was found to be well‑conserved across species, indicated the importance of this residue. The score for polyphen analysis of this mutation was found to be 0.999 indicating to be pathogenic mutation. Since, p.D806N mutation was found to be important residue; it might contribute to sexual development. We have reported the presence of mutations/polymorphism in MAP3K1 gene. All the mutations were found to be polymorphism upon comparing to single nucleotide polymorphism database. However, in‑silico analysis of the missense mutation revealed to be a pathogenic mutation.

Influences of extracellular matrices secreted by ultraviolet B-induced senescent fibroblasts on ERK signaling in HaCaT cells / 中华皮肤科杂志

Objective To explore the influences of extracellular matrices (ECM) secreted by ultraviolet B (UVB)-induced senescent fibroblasts on the proliferation of and extracellular signal-regulated kinase (ERK) signaling in HaCaT cells. Methods Fibroblasts were irradiated with UVB of 15 mJ/cm2 once daily for 5 days to induce premature senescence, which was identified by SA-β-gal staining 72 hours after the last irradiation.HaCaT cells were divided into 3 groups and inoculated into plates coated with extracellular matrices secreted by non-senescent (PRE-ECM) or senescent fibroblasts (SIPS-ECM) or into uncoated plates (NON-ECM), fol-lowed by additional culture. U0126, an inhibitor of ERK1/2, was used to treat the HaCaT cells 1 hour before inoculation. Then, MTT assay was carried out to detect the proliferation of HaCaT cells after a 3-day culture,Western blot to assess the phosphorylation of ERK at 0.5, 1, 2 and 4 hours after the inoculation, flow cytometry to analyse cell cycle and apoptosis after 24 hours of culture. Results The most rapid and intense phosphory-lation of ERK was observed in SIPS-ECM group. Inhibiting the activation of ERK pathway with U0126 could completely suppress the promoting effect of ECM from senescent fibroblasts on the proliferation of HaCaT cells.After the blocking of ERK activation, the proportion of HaCaT cells in S and G2/M phase decreased from 37.40%, 41.34% and 43.31% to 29.41%, 36.48% and 39.96%, respectively, in NON-ECM, PRE-ECM and SCIP-ECM group. Conclusion The ECM produced by UVB-induced senescent fibroblasts promote the prolifera-tion of HaCaT cells via inducing the phosphorylation of ERK.

Effects of tempol on p38MAPK activation of gastric mucosa ulceration in rat after water-immersion restraint stress / 解放军医学杂志

Objective To investigate the temporal activation of p38MAPK signaling transduction cascade,and the relationship between the activation and reactive oxygen species(ROS)in a well-defined experimental model of water-immersion restraint(WIR)stress-induced gastric mucosa ulceration.Methods Male Sprague-Dawley rats were randomly divided into control group,WIR group and tempol treated group.Animals were restrained and immersed in water bath to induce gastric mucosal lesions,with or without pretreatment with the free radical scavenger,tempol,and the WIR group animals were killed at different time points after WIR stress.Tempol treated animals underwent the same protocol followed 30min or 360min of stress,then the gastric mucosa was harvested,and the activity of gastric mucosal p38 was analyzed by Western blot.Malondialdehyde(MDA)activity and pro-inflammatory cytokine expression were detected.Results A rapid activation of p38MAPK in gastric mucosa occurred as early as 15 min after stress,and this activation was maximal after 90min of stress and still persisted until 360min after stress.Pretreatment with tempol prevented stress-induced p38MAPK activation at 30 min and 360min time points(0.77?0.24 and 0.58?0.12,respectively)compared with that in model groups(1.22?0.16 and 1.73?0.09,respectively,P