Chinese Medicines Regulate p38 MAPK， ERK1/2， and JNK in Treatment of Osteoporosis： A Review / 中国实验方剂学杂志

With the aging of population， osteoporosis has become one of the main diseases endangering the health of the elderly in China. Therefore， the research on osteoporosis has become a hot spot. Since Chinese medicines demonstrate significant therapeutic effects on osteoporosis， this issue is attracting increasing attention from researchers， especially in the deciphering of the molecular mechanism. This paper introduces the mechanism of the prevention and treatment of osteoporosis by Chinese medicines via the mitogen-activated protein kinase （MAPK） signaling pathway， aiming to provide a theoretical basis for deciphering the mechanism of Chinese medicines in the treatment of osteoporosis and promoting their clinical application. MAPK signaling pathway mainly involves p38 MAPK， extracellular signal-regulated kinase 1/2 （ERK1/2）， c-Jun N-terminal kinase （JNK）， and extracellular signal-regulated kinase 5 （ERK5）. Studies have shown that these proteins play a role in the progression of osteoporosis by regulating cell proliferation， differentiation， and apoptosis. Chinese medicines as a unique therapy with Chinese characteristics has definite efficacy， high safety， and mild side effects. Researchers have proved by experiments that the extracts or compounds of Chinese medicines can significantly mitigate osteoporosis by regulating the proteins involved in the MAPK signaling pathway. Therefore， this article reviews the relevant studies with focus on these proteins.

Effects of lead and manganese combined exposure on neurodevelopmental toxicity and JNK expression in zebrafish / 环境与职业医学

Background Lead and manganese are heavy metal pollutants widely existing in the environment, which can accumulate in the human body through the food chain, exert neurotoxicity, and cause neurodegenerative disorders. Especially in early childhood, the developing blood-brain barrier and nervous system are highly susceptible to environmental chemical pollutants. Most of the previous studies focused on the toxic effects of single heavy metal such as lead or manganese, while the studies on combined toxic effect are still scarce, and involved mechanisms are still unclear. c-Jun N-terminal kinase (JNK) is involved in neuronal development and regeneration, and some studies have found that JNK is involved in lead or manganese induced neurotoxicity. Its role in the toxicity of combined lead and manganese is unknown. Objective To understand the neurodevelopmental toxicity mechanism and to observe changes of JNK expression in zebrafish induced by combined lead and manganese exposure at environmentlly low concentrations. Methods Zebrafish embryos within 2 h post fertilization (hpf) were divided into four groups: control group, lead exposure group (0.1 mg·L−1 lead acetate), manganese exposure group (0.3 mg·L−1 manganous chloride), and lead-manganese combined exposure group (0.1 mg·L−1 lead acetate +0.3 mg·L−1 manganous chloride) and exposed to lead or/and manganese at designed levels for 7 d. Spontaneous movements and motor locomotion were observed, and mortality rate were calculated. The changes of JNK mRNA expression in zebrafish were evaluated. Results The experimental results showed that no significant effect of lead or/and manganese on spontaneous movements and mortality rate was found in zebrafish compared with the control group (P＞0.05). The results of locomotion analysis showed that compared with the control group, the activity counts and activity distance of zebrafish in the manganese exposure group were slightly increased (P＜0.01); the activity counts and activity distance of zebrafish in the lead exposure group were reduced by 50% and those in the lead-manganese exposure group were reduced by 80% (P＜0.01). Compared with the lead exposure group, the activity counts and activity distance of zebrafish in the lead-manganese combined exposure group decreased significantly by 60% (P＜0.05). The real-time quantitative PCR results showed that the JNK mRNA expression level was significantly increased in the lead-manganese combined exposure group compared with the control group(P＜0.01). Conclusion Lead exposure combined with manganese exposure at environmentlly low concentration can induce neurodevelopmental toxicity to zebrafish. JNK may be involved in neurodevelopmental toxicity induced by the combined exposure to lead and manganese.

Protective Effect of Huazhuo Jiedu Huoxue Tongluo Prescription on Cerebral Ischemia-reperfusion Injury via Regulating Autophagy Based on JNK Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Huazhuo Jiedu Huoxue Tongluo prescription in alleviating cerebral ischemia-reperfusion injury via regulating nerve cell autophagy based on c-Jun N-terminal kinase（JNK）signaling pathway . MethodSixty SD rats were randomly divided into 6 groups： sham group， middle cerebral artery occlusion/reperfusion （MCAO/R） group （model group）， Huazhuo Jiedu Huoxue Tongluo prescription group ［traditional Chinese medicine （TCM） group（25.0 g·kg-1）］， JNK inhibitor SP600125 （SP） group（5 mg·kg-1）， TCM+SP group and JNK agonist Anisomycin （Ani） group（15 mg·kg-1）. After 24 h of modeling， TCM group and TCM+SP group were given TCM decoction （ig） for 3 consecutive days， and the other groups were given equal volume of normal saline （ig）. Neurological deficit was evaluated by neurological function score and cerebral infarct volume was determined by 2，3，5-triphenyltetrazole chloride （TTC） staining. Hematoxylin-eosin （HE） staining and Nissl staining were used to observe the structural changes of brain tissue and the damage of neurons， respectively. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay （TUNEL） was performed to detect cell apoptosis. The ultrastructure of autophagosomes was observed by transmission electron microscope. Western blot was employed to detect the protein expressions of microtubule-associated protein 1 light chain 3A/B （LC3A/B）， autophagy related 5 （Atg5）， the ortholog of yeast Atg6 （Beclin1）， p62， B-cell lymphoma 2 （Bcl-2）， JNK， phosphorylated （p）-JNK and c-Jun in brain tissue. The mRNA expressions of LC3A/B， Beclin1， p62， Atg5， Bcl-2， JNK and c-Jun were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the sham group， the model group had elevated neurological deficit score （P<0.05）， enlarged cerebral infarct volume （P<0.05）and typical infarction manifestations formed in hippocampal region and its surrounding brain tissue. In addition， there were a large number of neuronal cell degeneration， necrosis， liquefaction， nucleus pyknosis and deep staining， and inflammatory cell infiltration in the cortex in the model group， and severe swelling of mitochondria， lysosomes， autophagosomes and autophagolysosomes were clearly seen under electron microscope. TUNEL positive cells were increased （P<0.05）， and cell apoptosis was severe. The nuclear membrane and nucleolus of neurons in brain tissue were blurred with discontinuous processes， and Nissl bodies in cytoplasm were stained light with reduced number. Western blot revealed that the model group had up-regulated protein expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK and c-Jun in brain tissue （P<0.05）， while down-regulated protein expressions of p62 and Bcl-2 （P<0.05）as compared with the sham group. Real-time PCR indicated that the mRNA expressions of LC3A/B， Beclin1， Atg5， JNK and c-Jun in the model group were higher （P<0.05） while the mRNA expressions of p62 and Bcl-2 were lower （P<0.05） than those in the sham group. Compared with the conditions in model group， the neurological deficit scores of TCM， SP and TCM+SP groups were lowered （P<0.05）， and the cerebral infarct volume was reduced （P<0.05）， with improved pathological status of brain tissue， especially in the TCM group. Furthermore， there were abundant and basically normal mitochondrial cristae， slightly dilated endoplasmic reticulum， slightly swollen golgi apparatus， slightly fused nuclear membrane， and few visible lysosomes， autophagosomes and autophagolysosomes. TUNEL positive cells were decreased （P<0.05）， displaying reduced apoptosis， especially in the TCM group. The nucleolus and nuclear membrane of neurons in brain tissue were discernible， and Nissl bodies in cytoplasm was reduced to a certain degree as compared with those in the model group. Western blot showed a decrease in the protein expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK and c-Jun in the TCM group ，the SP group，and the TCM+SP group（P<0.05），while an increase in the protein expressions of p62 in the TCM group and SP group（P<0.05），and an increase in the protein expressions of Bcl-2 in the TCM group and TCM+SP group. By Real-time PCR， the mRNA expressions of LC3A， LC3B， Beclin1， Atg5， JNK and c-Jun had a down-regulation（P<0.05） while the mRNA expression of p62 a up-regulation in the TCM group ，the SP group，and the TCM+SP group（P<0.05），and the mRNA expression of Bcl-2 a up-regulation in the TCM group and the TCM+SP group（P<0.05）.Scores of the Ani group were raised （P<0.05）， and infarct volume was increased significantly， with aggravated neuronal cell necrosis and obvious inflammatory infiltration. Moreover， there were neuronal nuclear membrane fusion with abnormal protrusion， increased heterochromatin aggregation in edge， severe mitochondrial swelling， endoplasmic reticulum expansion， increased lysosomes， increased intracytoplasmic vesicles， and visible autophagosomes and autophagolysosomes. TUNEL positive cells were increased （P<0.05）， representing severe apoptosis. The number of Nissl bodies dropped with light staining， and the nucleolus and nuclear membrane were blurred. Real-time PCR found that the mNRA expressions of Atg5， c-Jun， JNK were up-regulated （P<0.05），while Beclin1， p62， Bcl-2 were were down-regulated in the Ani group （P<0.05）. Compared with the TCM group and SP group，the protein expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK， c-Jun were decreased，and p62， Bcl-2 were increased in the Ani group（P<0.05）. Compared with the TCM group，the mRNA expressions of JNK mRNA had a down-regulation in the SP group and TCM+SP group，while LC3A， LC3B， Atg5， c-Jun， JNK had an up-regulation（P<0.05） and Bcl-2 had a down-regulation in the Ani group（P<0.05）. Compared with the SP group，the mRNA expressions of Atg5， c-Jun， JNK had an up-regulation（P<0.05）， and Beclin1， p62， Bcl-2 had a down-regulation in the Ani group（P<0.05）. ConclusionHuazhuo Jiedu Huoxue Tongluo prescription significantly up-regulates the protein and mRNA expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK and c-Jun， and down-regulates the protein and mRNA expressions of p62 and Bcl-2， suggesting that the prescription can inhibit autophagy through JNK signaling pathway to reduce ischemia/reperfusion injury in rats.

Effect of Stemona tuberosa Alkaloids on Apoptosis and PI3K/Akt and JNK/MAPK Signaling Pathways of Human Lung Cancer A549 Cells / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Stemona tuberosa alkaloids （STA） on apoptosis and phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） and c-Jun N-terminal kinase/p38 mitogen-activated protein kinase （JNK/p38 MAPK） signaling pathways in human lung cancer A549 cells. MethodA549 cells were classified into blank group and STA groups （100， 150， 200， 250， 300 mg⋅L-1）. Thiazole blue （MTT） assay and colony formation assay were used to evaluate the proliferation of A549 cells. Apoptosis was observed based on Hoechst 33258 staining， flow cytometry， and Annexin V-FITC/PI staining. Western blot was employed to detect the expression of apoptosis-related proteins cysteine-aspartic acid protease-3 （Caspase-3）， B-cell lymphoma-2 （Bcl-2）-associated X protein （Bax）， and Bcl-2， and the expression of PI3K， phosphorylated （p）-PI3K， Akt， p-Akt， JNK， p-JNK， p38 MAPK， and p-p38 MAPK. ResultCompared with the blank group， STA groups （150， 200， 250， 300 mg⋅L-1） demonstrated the increase in inhibition rate of cell proliferation （P<0.01） and cell clone inhibition rate， and decrease in cell clone formation rate （P<0.01）. In comparison with the blank group， STA groups showed typical characteristics of apoptosis， such as chromatin condensation and enhanced fluorescence reaction. The apoptosis rate of STA groups was significantly higher than that of the blank group （P<0.01）. Compared with the blank group， STA （150， 200， 250， 300 mg⋅L-1） significantly up-regulated the protein expression of Caspase-3 and Bax （P<0.05， P<0.01） and down-regulated the expression of Bcl-2 protein （P<0.01）. Compared with the blank group， STA had no significant influence on the total protein expression of PI3K， Akt， JNK， and p38 MAPK. However， STA （150， 200， 250， 300 mg⋅L-1） significantly decreased the levels of p-PI3K and p-Akt （P<0.05， P<0.01） and increased the level of p-p38 MAPK （P<0.05， P<0.01）. Compared with the blank group， STA （200， 250， 300 mg⋅L-1） significantly raised the level of p-JNK （P<0.05， P<0.01）. ConclusionSTA can inhibit the proliferation and induce the apoptosis of A549 cells by inhibiting PI3K/Akt signaling pathway and activating JNK/p38 MAPK signaling pathway.

Effect of Xiaoyaosan on JNK Pathway in LPS-induced Depressive-like Behavior in Mice / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Xiaoyaosan in alleviating lipopolysaccharide （LPS）-induced depressive-like behavior in mice based on the c-Jun N-terminal kinase （JNK） pathway. MethodAfter adaptive feeding， C57BL/6J mice were randomly divided into normal group， model group， minocycline group （intrabitoneal injection， 50 mg·kg-1）， fluoxetine group （intragastric administration， 2.6 mg·kg-1）， and low-， medium-， and high-dose Xiaoyaosan groups （intragastric administration，6.012 5， 12.025， and 24.050 g·kg-1）. After 14 days of administration， the model group and each administration group were intraperitoneally injected with 2 mg·kg-1 LPS， and the normal group was intraperitoneally injected with equal volume of normal saline. Depressive-like behavior in mice was assessed using the open field test and the elevated zero maze test. High-performance liquid chromatography （HPLC） was used to measure the levels of norepinephrine （NE） and epinephrine （E） in the mouse hippocampus. Enzyme-linked immunosorbent assay （ELISA） was performed to determine serum interleukin-1β （IL-1β） levels. Immunohistochemistry was used to measure the protein expression levels of ionized calcium-binding adapter molecule-1 （Iba-1）， c-Fos， and c-Jun. Real-time polymerase chain reaction （Real-time PCR） was used to measure mRNA expression levels of IL-1β， c-Jun， c-Fos， and JNK3 in the mouse hippocampus. Protein expression levels of JNK and phosphorylated （p）-JNK in the mouse hippocampus were measured using capillary protein automated protein expression analysis system （Western）. ResultCompared with the normal group， the model group exhibited significantly reduced central area residence time， crossing times， and travel distance in the open field （P<0.01）， significantly increased serum IL-1β levels （P<0.01）， significantly decreased NE and E levels （P<0.05）， upregulated mRNA expression of IL-1β， JNK3， and c-Fos， and increased protein expression of Iba-1， c-Fos， and c-Jun （P<0.05， P<0.01）. Compared with the model group， the Xiaoyaosan groups showed increased central area residence time and open arm residence time （P<0.05）， increased NE and E levels （P<0.01）， decreased mRNA expression of IL-1β， JNK3， c-Jun， and c-Fos， and decreased protein expression of Iba-1， c-Fos， JNK， and p-JNK （P<0.05， P<0.01）. The minocycline group and the fluoxetine group showed decreased mRNA expression of JNK3， c-Jun， and c-Fos （P<0.05， P<0.01）. The minocycline group showed decreased serum IL-1β and p-JNK protein expression （P<0.01）. The fluoxetine group exhibited increased NE and E levels and decreased c-Fos protein expression （P<0.01）. ConclusionXiaoyaosan can improve depressive-like behavior induced by LPS in mice， and its mechanism may be related to the inhibition of neuroinflammatory responses and the JNK pathway.

Mechanism of Stemona tuberosa Alkaloids in Inhibiting Proliferation and Inducing Apoptosis of Non-small Cell Lung Cancer NCI-H460 Cells / 中国实验方剂学杂志

ObjectiveTo study the effect and underlying mechanism of Stemona tuberosa alkaloids on the proliferation and apoptosis of human non-small cell lung cancer NCI-H460 cells. MethodNon-small cell lung cancer NCI-H460 cells were divided into a blank group and S. tuberosa alkaloids groups （50， 100， 150， 200， and 250 mg·L-1）. The effect of S. tuberosa alkaloids on the proliferation of human NCI-H460 cells was observed by thiazolyl blue tetrazolium bromide （MTT） assay and colony formation assay. Cell apoptosis was observed by Hoechst 33258 staining and flow cytometry. Real-time fluorescence-based polymerase chain reaction （Real-time PCR） was used to detect the effect of S. tuberosa alkaloids on the mRNA expression of cysteinyl aspartate-specific protease 3 （Caspase-3）， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， and epidermal growth factor receptor （EGFR）. The protein expression levels of Caspase-3， Bax， Bcl-2， protein kinase B （Akt）， phosphorylated （p-）Akt， EGFR， c-Jun N-terminal kinase （JNK）， p-JNK， p38 mitogen-activated protein kinase （p38 MAPK）， and p-p38 MAPK were measured by Western blot. ResultCompared with the blank group， the S. tuberosa alkaloids groups showed increased inhibition rate on cell proliferation （P<0.01）， reduced number of cell clones formed and the rate of cell clonal formation （P<0.05， P<0.01）， and increased karyopyknosis， cytoplasmic aggregation， and cell apoptosis rate （P<0.01）. The S. tuberosa alkaloids groups at 100， 150， 200， and 250 mg·L-1 showed increased Caspase-3 mRNA expression （P<0.05）， decreased EGFR mRNA expression （P<0.05， P<0.01）， up-regulated protein expression of Caspase-3 and p-JNK （P<0.01）， and down-regulated protein expression of EGFR and p-Akt （P<0.05， P<0.01）. Additionally， compared with the blank group， the S. tuberosa alkaloids groups showed increased expression of Bax mRNA （P<0.01）， decreased expression of Bcl-2 mRNA （P<0.01）， up-regulated protein expression of Bax and p-p38 MAPK （P<0.01）， and down-regulated protein expression of Bcl-2 （P<0.01）. ConclusionsS. tuberosa alkaloids can inhibit proliferation and induce apoptosis of human non-small cell lung cancer NCI-H460 cells， and the mechanism may be related to the inhibition of EGFR protein expression and phosphorylation of Akt protein， as well as the activation of the JNK/p38 MAPK signaling pathway.

Inhibitory effect of sodium ferulate on inflammatory response in migraine rats based on c-Jun N-terminal kinase / p38 mitogen-activated protein kinase signaling pathway / 解剖学报

[Abstract] Objective To explore the inhibitory effect of sodium ferulate (SF) on the inflammatory response in migraine rats by regulating the c-Jun N-terminal kinase (JNK) / p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway. Methods The migraine rat model was prepared by intraperitoneal injection of nitroglycerin. After successful modeling, the rats were randomly grouped into model group, SF low dose (SF-L) group (50 mg/ kg), SF high dose (SF-H) group (100 mg/ kg), SF+JNK inhibitor (SF + SP600125) group (SF 100 mg/ kg +SP600125 10 mg/ kg), and SF+JNK activator [SF + anisomycin(AN)] group (SF 100 mg/ kg +AN 5 mg/ kg), 12 in each group, another 12 SD rats without treatment were taken as blank group. The behavioral changes of the rats in each group were observed 24 hours after the administration, the levels of 5-hydroxytryptamine (5-HT), nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in serum were detected by ELISA, the neuronal apoptosis in brain tissue was observed by TUNEL staining, immunohistochemistry was used to evaluate the expressions of TNF-α, IL-6 and calcitonin gene-related peptide (CGRP) in brain tissue, Western blotting was used to detect the expressions of JNK/ p38 MAPK pathway-related proteins in brain tissue. Results Compared with the blank group, the number of times of scratching the head and climbing the cage of the rats in the model group increased significantly, and the apoptosis rate of neurons increased significantly; the content of 5-HT in serum decreased significantly, and the levels of NO, TNF-α and IL-6 increased significantly; the expressions of TNF-α, IL-6 and CGRP, and the ratios of phosphorylated JNK (p-JNK) / JNK and phosphorylated p38 MAPK(p-p38 MAPK) / p38 MAPK in brain tissue obviously increased (all P<0. 05). Compared with the model group, the number of times of scratching the head and the times of climbing the cage of the rats in the SF-L group and the SF-H group reduced significantly, and the neuron apoptosis rate reduced significantly; the content of 5-HT in serum increased significantly, and the levels of NO, TNF-α and IL-6 decreased significantly; the expressions of TNF-α, IL-6 and CGRP, and the ratios of p-JNK/ JNK and p-p38 MAPK/ p38 MAPK in brain tissue obviously decreased (all P<0. 05). Compared with SF-H group, the protective effect of SF on migraine rats in SF+SP600125 group enhanced significantly; the protective effect of SF on migraine rats in the SF+AN group reversed significantly. Conclusion SF may inhibit the expression of JNK/ p38 MAPK signaling pathway, effectively inhibit neurogenic inflammatory response in migraine rats, reduce neuronal apoptosis, and achieve a protective effect on migraine rats.

Effects of volatile oil from Acori graminei Rhizoma on glial fibrillary acidic protein, c-Jun N-terminal protein kinase and tumor necrosis factor-α expressions in the spinal cord dorsal horn of the inflammatory pain rats / 解剖学报

Objective To investigate the influence of volatile oil from Acori graminei Rhizoma (VOA) on expressions of glial fibrillary acidic protein (GFAP), c-Jun N-terminal protein kainse (JNK) and tumour necrosis factor-α (TNF-α) in the spinal cord dorsal horn of imflammatory pain rats. Methods Totally 36 male SD rats were randomly divided into control group (control), sham-operated group (sham), complete Freund' s adjuvant group (CFA), 5 g/(kg·d) low dose VOA+CFA group (VOA-L+CFA), 10 g/(kg·d) medium dose VOA + CFA group (VOA-M+CFA) and 20 g/(kg·d) high dose VOA + CFA group (VOA-H+CFA). All animals were sacrificed immediately after continuous gavage administration for 22 days. The expressions of GFAP, JNK and TNF-α in the spinal cord dorsal horn of rats in each group were detected by immunofluorescence and Western blotting methods. Results The present results showed that the positive expressions of GFAP, JNK and TNF-α in the spinal cord dorsal horn of rats increased significantly in the CFA group, when compared to the control and sham groups (P < 0. 01). The expressions of GFAP, JNK and TNF-α in the spinal cord dorsal horn of rats with VOA treatment reduced in the dose-dependent manner, when compared to the CFA group, the positive expressions of GFAP, JNK and TNF-α reduced significantly in the dorsal horn of the spinal cord of the VOA-H+CFA group (P<0. 05, P<0. 01). Conclusion VOA reduces the expressions of GFAP, JNK and TNF-α in the spinal cord dorsal horn of rats of CFA-induced inflammatory pain.

Effects of different doses of fluoride on expression of IRE1α-ASK1-JNK proteins in rat renal cortex / 中华地方病学杂志

Objective:To investigate the expression and significance of endoplasmic reticulum stress apoptosis pathway related proteins in renal cortex of rats with chronic fluorosis.Methods:Twenty four healthy SD rats were divided into 4 groups (6 rats/group, half male and half female) according to their body mass (100 - 120 g) by random number table method, rats in control group drank tap water (fluoride content < 0.5 mg/L), and in low, medium and high fluoride groups drank tap water with fluoride content (sodium fluoride) of 10, 50 and 100 mg/L, respectively. After 180 days of feeding, dental fluorosis was examined, 24-hour urine sample was collected and the content of fluoride in urine was detected by fluoride ion selective electrode method. Renal tissue was taken after anesthesia, and the pathological changes of renal cortex were observed by hematoxylin-eosin (HE) staining. The expressions of endoplasmic reticulum stress apoptosis pathway related proteins [inositol-requiring enzyme 1α (IRE1α), apoptosis signal-regulating kinase 1 (ASK1), C-Jun N-terminal kinase (JNK) and phosphorylated JNK (P-JNK)] were determined by immunohistochemical staining in rat renal cortex.Results:No dental fluorosis was found in control group. The incidence of dental fluorosis in low, medium and high fluoride groups were 2/6, 5/6 and 6/6, respectively. Compared with control group [(5.707 ± 1.190) mg/L], the urinary fluoride in low, medium and high fluoride groups [(17.028 ± 3.006), (34.378 ± 12.045), (94.759 ± 31.773) mg/L] was significantly higher ( P < 0.05), and the urinary fluoride in high fluoride group was higher than that in low and medium fluoride groups ( P < 0.05). HE staining showed that, compared with control group, the cell volume of renal tubules and glomeruli in medium and high fluoride groups increased, the cells arranged closely, and the eosinophilia of the cytoplasm increased. The immunohistochemical staining results showed that there was no significant difference in the expression of JNK protein in rat renal cortex between control group and low, medium and high fluoride groups ( F = 0.07, P > 0.05). The expressions of IRE1α, ASK1 and P-JNK proteins in rat renal cortex in high fluoride group were higher than those of control, low and medium fluoride groups ( P < 0.05), and the expressions of IRE1α and ASK1 proteins in medium fluoride group were significantly higher than those in control and low fluoride groups ( P < 0.05). Conclusion:Long-term excessive fluoride intake can lead to renal cortex injury in rats, and the mechanism of injury may be related to the activation of IRE1α-ASK1-JNK endoplasmic reticulum stress apoptosis pathway.

Exosomes from PM 2.5-treated Human Bronchial Epithelial Cells Increase Lung Cancer Metastatic Potential / 生物医学与环境科学（英文）

Objective@#Fine particulate matter (PM 2.5) is an air pollutant that has become of great concern in recent years. Numerous studies have found that PM 2.5 may contribute to lung cancer, but the pathogenesis has not yet been fully elucidated. In this study, we explored the roles of exosomes from bronchial epithelial cells in PM 2.5-promoted lung cancer metastasis.@*Methods@#Exosomes were isolated from cell supernatants. An animal model of lung metastasis (established by tail vein injection of A549-luc) and in vitro studies with lung cancer cell lines were used to investigate the effects of exosomes derived from PM 2.5-treated human bronchial epithelial cells (PHBE-exo).@*Results@#The animal experiments revealed that PHBE-exo-treated mice showed stronger luciferase activity and a larger relative metastatic region in the lungs, thus indicating that PHBE-exo promoted the metastatic potential of lung cancer. Additionally, PHBE-exo promoted the migration, invasion and epithelial-to-mesenchymal transition of lung cancer cells, in a manner mediated by activation of c-Jun N-terminal kinase.@*Conclusion@#These results implied that PM 2.5 may promote the development of lung cancer through exosomes derived from bronchial epithelial cells, thus providing a potential interventional target for lung cancer. These findings broadened our understanding of cancer-promoting mechanisms of environmental pollutants.

Effect of Jingangwan on p38 MAPK，JNK，and IL-1 Content in Osteoporosis Model Rats / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Jingangwan on the expression of osteoclast， c-Jun N-terminal kinase（JNK）， p38 mitogen-activated protein kinase（p38 MAPK）， and interleukin-1（IL-1） in the osteoporosis model rats， explore the mechanism of Jingangwan in the treatment of osteoporosis， and determine the optimal dosing concentration of Jingangwan. MethodFifty-six rats of SPF grade were randomized into a blank group，a sham operation group，a model group， model group，high-， medium-， and low-dose Jingangwan groups （0.72， 0.36， 0.18 g·kg-1·d-1， ig），and an estradiol valerate group （0.009 g·kg-1·d-1， ig）， with eight rats in each group. The rats in the model group， the blank group， and the sham operation group received 3 mL of normal saline， respectively. Samples were collected 12 weeks after drug administration. The number of osteoclasts was observed by tartrate-resistant acid phosphatase （TRAP） staining. Serum levels of JNK， p38 MAPK， and IL-1 were detected by enzyme-linked immunosorbent assay （ELISA）. The mRNA expression levels of p38 MAPK and JNK were detected by real-time quantitative polymerase chain reaction （Real-time PCR）. ResultThe TRAP staining results showed that compared with the model group， the estradiol valerate group and the Jingangwan groups could inhibit the formation of osteoclasts to different degrees. As revealed by ELISA results， compared with the model group and the sham operation group， the model group showed increased serum levels of p38 MAPK， JNK， and IL-1 （P<0.01）， while compared with the model group， all the groups with drug intervention showed decreased levels of p38 MAPK， JNK， and IL-1 （P<0.01）. The serum levels of JNK and IL-1 in the high-dose Jingangwan group were lower than those in the estradiol valerate group （P<0.05）. Real-time PCR results showed that compared with the blank group， the model group showed increased relative mRNA expression of p38 MAPK and JNK in the thighbone （P<0.01）， while compared with the model group， all the groups with drug intervention showed decreased relative mRNA expression of p38 MAPK and JNK in the thighbone （P<0.01）. ConclusionJingangwan can inhibit the formation of osteoblasts，reduce the diameter of the bone marrow cavity，improve bone quality，suppress the production of inflammatory factors，affect the metabolism of the MAPK signaling pathway，and blunt p38 MAPK and JNK activities to inhibit the differentiation and proliferation of osteoblasts and regulate bone metabolism， thereby preventing osteoporosis. Therefore，Jingangwan may be of application value in maintaining bone health and treating osteoporosis.

Differences in Protective Effect of Five Formulas for Promoting Blood Circulation and Removing Blood Stasis on Endothelial Cells of New Zealand Rabbits with Heart Blood Stasis Syndrome Through JNK Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the differences in the protective effects of five formulas for promoting blood circulation and removing blood stasis on the aortic endothelial cells of New Zealand rabbits with heart blood stasis syndrome. MethodEighty New Zealand rabbits were randomly divided into a normal group （n=10） and an experimental group （n=70）. The heart blood stasis syndrome model was induced by starvation combined with a high-fat diet and adrenaline in the rabbits of the experimental group. Subsequently， the model rabbits were randomly divided into a model group， a Xuefu Zhuyutang group （3.55 g·kg-1·d-1）， a Taohong Siwutang group （2.66 g·kg-1·d-1）， a Danshenyin group （1.962 g·kg-1·d-1）， a Huoluo Xiaolingdan group （2.80 g·kg-1·d-1）， a Shixiaosan group （0.56 g·kg-1·d-1）， and a c-Jun N-terminal kinase （JNK） inhibitor （SP600125， 5 μg·kg-1）group. The normal group and the model group received the same amount of distilled water. The rabbits in five Chinese medicine groups were treated correspondingly by gavage， and those in the SP600125 group were injected with 0.5 mL of SP600125-dimethyl sulfoxide diluent. After the treatment， the aorta was collected， and terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） assay was used to detect the apoptosis of aortic endothelial cells. The enzyme-linked immunosorbent assay （ELISA） was used to detect serum levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6）. Western blot was used to detect the protein expression of JNK， phosphorylated JNK （p-JNK）， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， cysteinyl aspartate-specific protease-9 （Caspase-9）， and cysteinyl aspartate-specific protease-3 （Caspase-3） in aortic tissues. Real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA levels of JNK， Bcl-2， Bax， Caspase-9， and Caspase-3 in aortic tissues. ResultFive formulas could improve the apoptosis of aortic endothelial cells to varying degrees. To be specific， Xuefu Zhuyutang and Taohong Siwutang were optimal in efficacy， followed by Huoluo Xiaolingdan， Shixiaosan， and Danshenyin， and SP600125 was the worst （P<0.05， P<0.01）. Five formulas could reduce the content of TNF-α and IL-6 （P<0.05， P<0.01）， down-regulate the protein expression levels of JNK， p-JNK， Bax， Caspase-9， and Caspase-3 （P<0.05， P<0.01）， decrease the mRNA expression levels of JNK， Bax， Caspase-9， and Caspase-3 （P<0.05， P<0.01）， and up-regulate the protein and mRNA expression levels of Bcl-2 （P<0.05， P<0.01）. ConclusionFive formulas can all reduce the apoptosis of aortic endothelial cells in New Zealand rabbits with heart blood stasis syndrome with different efficacies. It may be related to the different effects of five formulas on the JNK signaling pathway.

Intervention of Gandou Fumu Decoction on Liver Fibrosis in Mice with Wilson's Disease Through JNK Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo identify the protective effect and possible mechanism of Gandou Fumu decoction （GDFMD） on liver fibrosis in mice with Wilson's disease. MethodA total of 50 homozygous TXJ mice were randomly divided into five groups， with 10 mice in each group. Ten wild-type mice were selected as a normal group. The GDFMD high， medium， and low-dose groups were given 13.92， 6.96， 3.48 g·kg-1 of GDFMD， respectively. The penicillamine group were given 0.1 g·kg-1 of penicillamine. The model group and the normal group were given the same volume of 0.9% sodium chloride solution once a day for 4 consecutive weeks. The enzyme-linked immunosorbent assay （ELISA） method was performed to detect serum superoxide dismutase （SOD） activity and malondialdehyde （MDA） content. Corresponding kits were used to detect the mitochondrial adenine triphosphate （ATP） content and Na+-K+-ATPase activity in liver tissues. Hematoxylin-eosin （HE） and Masson staining were used to observe the pathological morphology of liver tissue， and transmission electron microscope was used to observe ultrastructural changes of liver tissues in mice. Western blot was used to detect the c-Jun N-terminal kinase， the phosphorylated protein， and the expressions of Caspase-3， B cell lymphoma-2 （Bcl-2）， and Bcl-2 associated X protein （Bax） in c-Jun N-terminal kinase （JNK） signaling pathway. ResultCompared with the normal group， MDA content increased and SOD activity decreased in the model group （P<0.05）. Compared with the model group， SOD activities in the GDFMD high-， medium-， and low-dose groups and the penicillamine group significantly increased （P<0.01）， and MDA content significantly decreased （P<0.05， P<0.01）. Compared with the normal group， ATP content and Na+-K+-ATPase activity significantly decrease in the model group （P<0.05）. Compared with the model group， ATP content and Na+-K+-ATPase activity in the GDFMD medium and high-dose groups and the penicillamine group significantly increased （P<0.05， P<0.01）. The results of the pathological morphology of liver tissue showed that a large number of liver cells degeneration and necrosis， inflammatory cell infiltration， unclear liver lobule structure， and collagen fiber deposition were observed in the model group. Transmission electron microscopy showed that the number of mitochondria in liver tissues significantly reduced， the mitochondria were locally damaged， and the cristae of mitochondria were broken even disappear in the model group. The pathological morphology of liver tissue and mitochondrial structure recovered to varying degrees after medicinal intervention. The results of Western blot suggested that， compared with the normal group， the expression levels of phosphorylation-JNK （p-JNK）， p-JNK/JNK， Caspase-3， and Bax in the liver tissues were up-regulated， while the expression of Bcl-2 was down-regulated in the model group （P<0.05）. The expression levels of p-JNK， p-JNK/JNK， Caspase-3 and Bax were down-regulated and the expression of Bcl-2 was up-regulated in the GDFMD high and medium-dose groups and the penicillamine group （P<0.01）. ConclusionGDFMD can alleviate oxidative stress damage and recover mitochondrial function of TXJ mice with liver fibrosis. The mechanism of GDFMD may be related to regulating the JNK signaling pathway and downstream factors and inhibiting cell apoptosis.

Effect of Huangqi Guizhi Wuwutang on Diabetic Peripheral Neuropathy in MKR Mice via Regulating Endoplasmic Reticulum Stress / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Huangqi Guizhi Wuwutang （HQGZWWT） in the treatment of diabetic peripheral neuropathy （DPN） in MKR mice via regulating endoplasmic reticulum （ER） stress. MethodThirty-two 8-week-old MKR mice （half were male and half were female） were fed with a high-fat diet for four weeks， and then 1% streptozotocin （STZ） was injected intraperitoneally for five days. After the blood glucose was stabilized， the mice were housed in the cage covered with ice bags for another one hour stimulation per day for four weeks. Mice with fasting blood glucose （FBG） value ≥11.1 mmol·L-1 were randomly divided into model group ， Huangqi Guizhi Wuwutang in original dosage group （30 g·kg-1·d-1）， Huangqi Guizhi Wuwutang in formula dosage group （6.25 g·kg-1·d-1）， and positive drug group （mecobalamin tablets， 0.17 mg·kg-1·d-1）. Another eight MKR mice of the same age were set as blank group and eight FVB mice were normal group. After four weeks of intragastric administration in each group， the change in FBG was tested， and hematoxylin and eosin （HE） staining and transmission electron microscope were used for observing the morphology of sciatic nerve tissue. In addition， the expression of c-Jun N-terminal kinase （JNK）， phosphorylated c-Jun N-terminal kinase （p-JNK） and inositol requiring enzyme 1α （IRE1α） proteins was determined by immunohistochemical test and Western blot （WB）. ResultCompared with the conditions in the normal group and blank group， the time of paw withdrawal， paw licking and tail flick in the model group was shortened （P<0.01）， and the conduction velocity of sciatic nerve was decreased （P<0.01）. Compared with the conditions in the model group， the behavioral and functional indicators were improved by HQGZWWT （P<0.05，P<0.01）. The immunohistochemical test revealed the JNK expression was elevated in the model group compared with the conditions in the normal group and blank group （P<0.05）， while that was lowered by HQGZWWT compared with the condition in the model group （P<0.05）. However， there was no difference among the treatment groups. According to the WB， the expression of IRE1α and p-JNK in the model group was enhanced compared with the conditions in the normal group and blank group （P<0.05，P<0.01）， while that was decreased by HQGZWWT compared with the condition in the model group （P<0.05，P<0.01）. No difference was observed between the HQGZWWTO and HQGZWWTF groups. ConclusionHQGZWWT can improve the neurophysiological function and pathological damage of sciatic nerve， which may be related to its delaying the ER stress response of sciatic nerve.

Effect of Koumine on Proliferation and Apoptosis of Colorectal Cancer Cells via ROS/JNK/FoxO3a Signaling Pathway / 中国实验方剂学杂志

Objective:To explore the effect and underlying mechanism of koumine （Kou） at different concentrations （0， 100， 200， 400 μmol·L^-1） on the proliferation and apoptosis of colorectal cancer HCT-116 cells. Method:After 24 hours of<italic> in vitro</italic> intervention with HCT-116 cells by Kou， cell counting kit-8 （CCK-8） assay was used to detect its effect on cell proliferation. Flow cytometry was used to detect cell cycle， apoptosis， and reactive oxygen species （ROS） expression. Real-time quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of forkhead box O3a （FoxO3a）. Cells were transfected with small interfering ribonucleic acid （siRNA）. Western blot was employed to detect the protein expression of the FoxO3a target gene. Result:Compared with the conditions in the blank group， Kou treatment reduced the proliferation rate of HCT-116 cells （<italic>P</italic><0.05， <italic>P</italic><0.01） in a dose-dependent manner， caused cell cycle arrest in the G₀/G₁ phase， and induced the apoptosis of HCT-116 cells （<italic>P</italic><0.05， <italic>P</italic><0.01）， which was positively correlated with the concentration of Kou. FoxO3a siRNA interference reduced the expression of FoxO3a and its downstream target genes cyclin-dependent kinase inhibitor 1A （p21）， cyclin-dependent kinase inhibitor 1B （p27）， and Bcl-2 interacting mediator of cell death （Bim） （<italic>P</italic><0.01）. Kou treatment induced the activation of c-Jun <italic>N</italic>-terminal kinase （JNK） in HCT116 cells. SP600125 （JNK specific inhibitor） treatment inhibited the Kou-induced FoxO3a activation and the expression of its downstream target genes. <italic>N</italic>-acetyl cysteine （NAC） treatment reduced Kou-induced ROS levels （<italic>P</italic><0.01） and JNK signal activation. The above results were significantly different from those in the blank group （<italic>P</italic><0.01）. Conclusion:Kou can effectively inhibit the proliferation of HCT-116 cells and promote apoptosis， and the mechanism may be related to the regulation of the ROS/JNK/FoxO3a pathway.

Effect of Danggui Shaoyaosan on TLR4/MyD88/JNK Signaling Pathway on Rats with Nonalcoholic Fatty Liver Disease / 中国实验方剂学杂志

Objective:To explore<italic> </italic>the efficacy and mechanism of Danggui Shaoyaosan on rats of nonalcoholic fatty liver disease （NAFLD）. Method:Sixrty SPF SD male rats were randomly divided into normal group， model group，essentiale （0.144 g·kg^-1） and low， middle and high-dose of Danggui Shaoyaosan groups （2.44， 4.88， 9.76 g·kg^-1）. High fat diet were fed to bulid the NAFLD model， and each treatment group was given corresponding drugs at the same time. After 8 weeks， the serum and liver tissue were collected to detect the contents or activities of total cholesterol （TC）， triglyceride （TG）， alanine aminotransferase （ALT）， aspartic acid aminotransferase （AST）， tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>） and interleukin-10 （IL-10） in serum， the contents of TC， TG and free fatty acid （FFA） in liver tissue， Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） and Western blot were used to observe the gene and protein expressions of Toll-like receptor 4 （TLR4）， myeloid different factory 88 （MyD88） and c-Jun n-terminal kinase （JNK） and the protein expression of phosphorylation JNK（p-JNK） in liver tissue. Hematoxylin-eosin （HE） staining and Oil red staining to observe the pathological morphological changes of liver. Result:Compared with control group， the contents or activities of TC， TG， ALT， AST and TNF-<italic>α</italic> in serum， the contents of TC， TG and FFA in liver and the gene and protein expressions of TLR4， MyD88， JNK， and the protein expression of p-JNK in liver tissue of model group were distinctly increased （<italic>P</italic><0.01）， the content of IL-10 was significantly decreased （<italic>P</italic><0.01）. Compared with model group， the contents or activities of TC， TG， ALT， AST and TNF-<italic>α</italic> in serum， the contents of TC， TG and FFA in liver and the mRNA and protein expressions of TLR4， MyD88 and JNK， and the protein expression of p-JNK in liver tissue of Danggui Shaoyaosan groups were significantly reduced （<italic>P</italic><0.05，<italic>P</italic><0.01）， the content of IL-10 in serum of Danggui Shaoyaosan groups was distinctly increased（<italic>P</italic><0.05，<italic>P</italic><0.01）， HE staining and Oil red staining show that the degree of liver steatosis was alleviated obviously by Danggui Shaoyaosan. Conclusion:Danggui Shaoyaosan has a better treatment on NAFLD by inhibiting TLR4/MyD88/JNK pathway and alleviating the inflammation response.

Cadmium promotes apoptosis of mouse spermatogenic cells via regulation of JNK/c-Jun signaling pathway / 公共卫生与预防医学

Objectives To study the effect of cadmium (Cd) on the proliferation and apoptosis of mouse spermatocyte (GC-2 spd) cells and explore the underlying molecular mechanism. Methods GC-2 spd cells were cultured with 0, 5, 10, 15, 20 and 30 μM CdCl2, respectively, for 24 hours. The cell viability and IC50 of Cd were estimated based on CCK-8 data. The apoptosis of GC-2 spd cells and cellular concentration of ROS were analyzed by flow cytometry after treatment of the cells with different concentrations of CdCl2 (0, 5, 10 μM) for 24 hours. The expression levels of JNK/c-Jun signaling pathway regulatory proteins, pro-apoptotic factor Bax and anti-apoptotic factor Bcl-2, were determined by Western blot. Results Cd inhibited the proliferation of GC-2 spd cells with IC50 value of 12.99 μM, 95% CI [11.95, 14.00]. Exposure to 5 and 10 μM CaCl2 resulted in increases in apoptosis and cellular ROS generation in a dose-dependent manner, which was statistically significant compared with the control (P 0.05), the phosphorylation level of JNK and c-Jun in Cd group was highly increased as compared to the control (P < 0.05). In addition, Cd exposure significantly increased the expression of Bax protein but decreased the expression Bcl-2 protein (P < 0.05). Conclusions Cadmium induces GC-2 spd cell apoptosis by increasing concentration of ROS and regulating the JNK/c-Jun signaling pathway.

Hydroxychavicol, a polyphenol from / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

This study aims to elucidate the antiproliferative mechanism of hydroxychavicol (HC). Its effects on cell cycle, apoptosis, and the expression of c-Jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinase (MAPK) in HT-29 colon cancer cells were investigated. HC was isolated from

Expressions of c-Jun N-terminal kinase, matrix metalloproteinase-9 and macrophage marker MAC387 in rats with vertebrobasilar dolichoectasia and intervention effect of rosuvastatin / 中华神经医学杂志

Objective:To observe the expressions of c-Jun N-terminal kinase (JNK), matrix metalloproteinase-9 (MMP-9) and macrophage marker MAC387 in the blood vessels of rats with vertebrobasilar dolichoectasia (VBD), and to study the therapeutic effect and mechanism of rosuvastatin on VBD.Methods:Sixty male SD rats were randomly divided into sham-operated group, VBD model group and rosuvastatin intervention group ( n=20). Elastase at 0.3 uL (1.5 U/μL) was injected into the cisterna magna of rats in the latter two groups to establish VBD models; while same volume of saline solution was given to rats in the sham-operated group. Twenty-four h after model making, 5 mg/(kg.d) rosuvastatin (once/d, for 4 weeks) was administered intragastrically into rats in the rosuvastatin intervention group, and same volume of distilled water was administered intragastrically into rats in the other 2 groups. The degrees of extension and expansion of vertebrobasilar artery were measured and compared among the 3 groups. Western blotting was used to detect the expressions of JNK and MMP-9, and immunofluorescent staining was used to detect the MAC387 expression in the vertebrobasilar artery. Results:Fourteen rats in the VBD model group and rosuvastatin intervention group, respectively, had successful model making. As compared with the sham-operated group, the VBD model group and rosuvastatin intervention group had statistically increased basilar artery diameter and basilar artery tortuosity index ( P<0.05). The expressions of JNK, MMP-9 and MAC387 in the VBD model group and rosuvastatin intervention group were significantly increased as compared with those in the sham-operated group ( P<0.05); as compared with those in the VBD model group, the expressions of JNK, MMP-9 and MAC387 in the vertebrobasilar artery of rosuvastatin intervention group were significantly decreased ( P<0.05). Conclusion:Rosuvastatin can delay VBD progress, whose mechanism may be related to the inhibition of inflammatory response.

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.