Effects of Metformin on JNK Signaling Pathway and PD-L1 Expression in Triple Negative Breast Cancer.

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. Metformin has been shown to have the potential to inhibit the proliferation of malignant cells. This study aimed to investigate the regulatory effect of metformin on the expression of programmed death protein ligand 1(PD-L1) and mechanisms in TNBC. Methods: Mouse breast cancer cell line 4T1 was co-cultured with metformin, and the effect of metformin on cell proliferation was detected by MTT assay. The effect of metformin on the expression of JNK, RSK2 and CREB was detected by MAPK pathway protein chip. BALB/c mice were inoculated with 4T1 cells with knockdown/overexpression of C-Jun N-terminal kinase (JNK), and administered with metformin. The weight of tumor tissue was observed at the end of the experiment. The expression of PD-L1 in tumor cells was observed by immunofluorescence staining and the level of INF-γwas quantitatively determined by ELISA. Results: Metformin inhibited the viability of 4T1 cells and increased the phosphorylation of JNK to reduce the phosphorylation of RSK2 and CREB. Metformin and JNK knockdown reduced the expression of PD-L1 in tumor cells, but there was no significant difference in the weight of tumor tissue. Metformin can reduce the level of INF-γ in tumor tissues, but JNK has no effect. Conclusion: Metformin can inhibit the expression of PD-L1 in triple-negative breast cancer mice and improve the tumor microenvironment, but does not reduce the size of the tumor.

[Modulation of autophagy in the rats with acute intracerebral hemorrhage by acupuncture based on JNK pathway].

OBJECTIVE: To investigate the effects of acupuncture on JNK pathway and autophagy level in rats with intracerebral hemorrhage （ICH） and explore the partial mechanism of acupuncture against ICH. METHODS: SD rats were randomly divided into blank group, model group and acupuncture group. Each group was divided into Day 1, Day 3 and Day 7 subgroups respectively, with 5 rats in each group. The autologous blood injection was adopted to duplicate rat model of ICH. In the acupuncture group, the needle was inserted from "Baihui" （GV20） towards "Qubin" （GB7） on the affected side, stimulating for 30 min each time, once daily； the same acupuncture technique was opera-ted in each subgroup for 1, 3 and 7 days, separately. Using Bederson scale, the neurological deficit was evaluated in each group. Western blot was adopted to detect the protein expression levels of Beclin1, LC3Ⅰ/Ⅱ, phosphorylated c-Jun amino-terminal kinase （p-JNK） and the phosphorylated （p）-c-Jun around hematoma lesion of the brain tissue of rats in each group. RESULTS: After treatment, the neurological deficit score of rats in the model group was higher than that of the blank group at each time point （P<0.05）, and the score of the acupuncture group started declining since the 3rd day of treatment when compared with the model group （P<0.05）. At each time point, compared with the blank group, the protein expression levels of LC3Ⅰ/Ⅱ, Beclin1, p-c-Jun and p-JNK was increased （P<0.01）. Compared with the model group, the protein expression level of LC3Ⅰ/Ⅱ was reduced （P<0.05）； the protein expression levels of Beclin1, p-c-Jun and p-JNK was increased （P<0.05, P<0.01） on day 3 and 7 in the acupuncture group. CONCLUSION: Acupuncture can activate the JNK pathway in the brain tissue of rats with ICH and increase the level of autophagy, thereby improving the neurological function of the rats with ICH.

[Effects of electroacupuncture on neurological function and expressions of p-JNK and Beclin-1 in traumatic brain injury rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on neurological function, the expressions of phosphorylated c-Jun amino terminal kinase (p-JNK) and Beclin-1 in rats with traumatic brain injury (TBI), so as to explore the underlying mechanism of EA in the treatment of TBI. METHODS: A total of 64 SD rats were randomly divided into blank, sham, modeling groups, with 8 rats in the blank group and the sham group and 48 rats in the modeling group. The modified Feeney free-fall impact method was used to establish the TBI rat model. After modeling, rats of the modeling group were randomly divided into model and EA groups, which were further divided into 3 d, 7 d and 14 d subgroups with 8 rats in each group. Rats in the EA group were treated with acupuncture at "Baihui" (GV20, retained for 15 min), "Shuigou" (GV26, stabbed for 20 s), "Neiguan" (PC6) and "Zusanli" (ST36) of the right side. EA (2 Hz, 1 mA) was applied to PC6 and ST36 for 15 min. The above treatments were performed once a day, and different subgroups were continuously stimulated for 3, 7 and 14 days, respectively. The neurological impairment was evaluated by modified neurological severity score(mNSS). The pathological morphological changes and the protein expressions of p-JNK and Beclin-1 in the injured area of the brain were detected by Nissl staining and immunohistochemistry, separately. RESULTS: After modeling, the mNSS and the protein expressions of p-JNK and Beclin-1 were increased (P< 0.05) on day 3, 7 and 14 in the model group relative to the sham group. The Nissl bodies were reduced or even dissolved and neurons were seriously damaged in the model group on the 3rd day, which were mildly repaired on day 7 and 14. Following acupuncture interventions, compared with the model group, the mNSS on day 7 and 14 and the protein expressions of p-JNK and Beclin-1 on day 3, 7 and 14 were decreased (P< 0.05)in the EA group. The status of Nissl bodies and neurons in the EA group was better at all time points than that in the model group. There were no significant differences in the above indicators between the blank group and the sham group. CONCLUSION: EA can significantly improve the neurological function of TBI model rats, which may be related to its effects in down-regulating the protein expressions of p-JNK and Beclin-1 in the injured area of the brain.

Protective Effect of Jianpi Huogu Prescription on Functional Injury of Vascular Endothelial Cells Caused by Alcohol Based on Akt/JNK/p38 MAPK Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the protective effect of Jianpi Huogu prescription （JPHGP） on the functional injury of vascular endothelial cells caused by alcohol and explore its mechanism based on protein kinase B/c-Jun amino-terminal kinase/p38 MAPK （Akt/JNK/p38 MAPK） signaling pathway. MethodThrough chick embryo allantoic membrane， thoracic aortic ring， and migration， invasion， adhesion， and lumen formation of human umbilical vein endothelial cells （HUVEC）， the effect of JPHGP with different concentrations （8， 16 and 32 μg·L-1） on angiogenesis was observed in the presence or absence of alcohol. The expression levels of phosphorylation of Akt， JNK， and p38 MAPK were determined by Western blot. ResultAs compared with the normal group， the number and length of capillaries around the arterial ring in the model group were decreased， and the migration， invasion， and lumen formation capacity of HUVEC were decreased （P<0.05， P<0.01）. After treatment with 16 and 32 μg·L-1 JPHGP， the length of neovascularization in chick embryo allantoic membrane was significantly increased （P<0.05， P<0.01）. Compared with the model group， the 8， 16， and 32 μg·L-1 JPHGP groups increased the number of capillaries around the thoracic aortic ring in a concentration-dependent manner （P<0.05， P<0.01）， and the 32 μg·L-1 JPHGP group increased the length of capillaries around the thoracic aortic ring （P<0.05）. The 16 and 32 μg·L-1 JPHGP groups enhanced the migration， invasion， and lumen formation capacity of HUVEC. The results of Western blot showed that， as compared with the normal group， the protein expression levels of p-JNK/JNK， p-p38 MAPK/p38 MAPK， and p-Akt/Akt were significantly decreased in the model group （P<0.01）， and as compared with the model group， the protein expression levels of p-p38 MAPK/p38 MAPK and p-Akt/Akt were significantly increased in the 8， 16， and 32 μg·L-1 JPHGP groups （P<0.01） and the protein expression level of p-JNK/JNK was increased significantly in the 16 and 32 μg·L-1 JPHGP groups （P<0.01）. ConclusionJPHGP has a protective effect on the functional injury of vascular endothelial cells caused by alcohol， and its mechanism may be related to the activation of Akt/JNK/p38 MAPK signaling pathway. Relevant research results will provide certain scientific basis for clarifying the effect of JPHGP on 'invigorating spleen and promoting blood circulation'.

Corrigendum: c-Jun amino terminal kinase signaling promotes aristolochic acid-induced acute kidney injury.

[This corrects the article DOI: 10.3389/fphys.2021.599114.].

c-Jun Amino Terminal Kinase Signaling Promotes Aristolochic Acid-Induced Acute Kidney Injury.

Aristolochic acid (AA) is a toxin that induces DNA damage in tubular epithelial cells of the kidney and is the cause of Balkan Nephropathy and Chinese Herb Nephropathy. In cultured tubular epithelial cells, AA induces a pro-fibrotic response via the c-Jun amino terminal kinase (JNK) signaling pathway. This study investigated the in vivo role of JNK signaling with a JNK inhibitor (CC-930) in mouse models of acute high dose AA-induced kidney injury (day 3) and renal fibrosis induced by chronic low dose AA exposure (day 22). CC-930 treatment inhibited JNK signaling and protected from acute AA-induced renal function impairment and severe tubular cell damage on day 3, with reduced macrophage infiltration and expression of pro-inflammatory molecules. In the chronic model, CC-930 treatment inhibited JNK signaling but did not affect AA-induced renal function impairment, tubular cell damage including the DNA damage response and induction of senescence, or renal fibrosis; despite a reduction in the macrophage pro-inflammatory response. In conclusion, JNK signaling contributes to acute high dose AA-induced tubular cell damage, presumably via an oxidative stress-dependent mechanism, but is not involved in tubular atrophy and senescence that promote chronic kidney disease caused by ongoing DNA damage in chronic low dose AA exposure.

HIPK3 Mediates Inflammatory Cytokines and Oxidative Stress Markers in Monocytes in a Rat Model of Sepsis Through the JNK/c-Jun Signaling Pathway.

Sepsis is a fetal immunological disorder and its complication worsens in the patients with hemodialysis which may increase the risk of death. In the present study, we aimed to investigate the effect of homeodomain-interacting protein kinase 3 (HIPK3) on inflammatory factors and oxidative stress markers in monocytes of rats with sepsis by regulating the c-Jun amino-terminal kinase (JNK)/c-Jun signaling pathway. A rat model of sepsis was initially established using cecal ligation and puncture (CLP) and was further identified by enlarged spleen tissues, inflammation, and oxidative stress. Monocytes were isolated from rats with CLP-induced sepsis. HIPK3 was observed to be downregulated while JUN was upregulated in monocytes from rats with CLP-induced sepsis. Furthermore, isolated monocytes were transduced with lentiviral vectors expressing HIPK3 or shRNA against HIPK3 to explore the effect of HIPK3 on viability and apoptosis of monocytes as well as inflammatory factors and oxidative stress markers. The obtained data exhibited that overexpression of HIPK3 or inhibition of the JNK signaling pathway enhanced proliferation, reduced apoptosis of monocytes, alleviated inflammation, and oxidative stress injury. Consistently, our results may provide evidence that HIPK3 could inhibit the JNK/c-Jun signaling pathway, thereby potentially retarding the progression of sepsis.

A Positive Feedback Loop of Hippo- and c-Jun-Amino-Terminal Kinase Signaling Pathways Regulates Amyloid-Beta-Mediated Neurodegeneration.

Alzheimer's disease (AD, OMIM: 104300) is an age-related disorder that affects millions of people. One of the underlying causes of AD is generation of hydrophobic amyloid-beta 42 (Aß42) peptides that accumulate to form amyloid plaques. These plaques induce oxidative stress and aberrant signaling, which result in the death of neurons and other pathologies linked to neurodegeneration. We have developed a Drosophila eye model of AD by targeted misexpression of human Aß42 in the differentiating retinal neurons, where an accumulation of Aß42 triggers a characteristic neurodegenerative phenotype. In a forward deficiency screen to look for genetic modifiers, we identified a molecularly defined deficiency, which suppresses Aß42-mediated neurodegeneration. This deficiency uncovers hippo (hpo) gene, a member of evolutionarily conserved Hippo signaling pathway that regulates growth. Activation of Hippo signaling causes cell death, whereas downregulation of Hippo signaling triggers cell proliferation. We found that Hippo signaling is activated in Aß42-mediated neurodegeneration. Downregulation of Hippo signaling rescues the Aß42-mediated neurodegeneration, whereas upregulation of Hippo signaling enhances the Aß42-mediated neurodegeneration phenotypes. It is known that c-Jun-amino-terminal kinase (JNK) signaling pathway is upregulated in AD. We found that activation of JNK signaling enhances the Aß42-mediated neurodegeneration, whereas downregulation of JNK signaling rescues the Aß42-mediated neurodegeneration. We tested the nature of interactions between Hippo signaling and JNK signaling in Aß42-mediated neurodegeneration using genetic epistasis approach. Our data suggest that Hippo signaling and JNK signaling, two independent signaling pathways, act synergistically upon accumulation of Aß42 plaques to trigger cell death. Our studies demonstrate a novel role of Hippo signaling pathway in Aß42-mediated neurodegeneration.

Protective Effect and Mechanism of Ginkgolide B on Amyotrophic Lateral Sclerosis Cell Model / 中国实验方剂学杂志

Objective:To investigate the effect of ginkgolide B （GB） on the activation of c-Jun aminoterminal kinase（JNK） signaling pathway and apoptosis in amyotrophic lateral sclerosis cell model. Method:NSC34 cells were infected by slow virus containing expression superoxide dismutase1（SOD1）WT and hSOD1G93A and empty plasmid， and screened with a certain concentration of puromycin， so as to observe the transfection efficiency of slow virus and cell morphology under inverted fluorescence microscope. Western blot method was used to verify whether infected cells were over-expressing SOD1 target proteins. The hSOD1G93A-NSC34 cell lines were established and given GB. Cell cultures were divided into normal group， model group and different concentrations of ginkgolide B groups （25， 50， 75， 100 mg∙L-1）. After 48 h， methyl thiazolyl tetrazolium （MTT） was used to detect cell survival rates， and select the best drug concentration. Subsequent experimental groups were divided into normal group， model group， 75 mg∙L-1 GB group， SP600125 group， and 75 mg∙L-1 GB + SP600125 group. Flow cytometry was used to detect the apoptosis of each group of cells. Western blot was used to detect the expressions of phosphorylation（p）-JNK， c-Jun， p-c-Jun， and cysteine aspartic acid protease -3（Caspase-3） proteins. Result:Compared with normal NSC34 cells， hSOD1G93A-NSC34 cell body became round， the synapses decreased and shortened， but the cell morphology of hSODWT-NSC34 cell and empty plasmid group did not change significantly. Western blot showed that hSOD1G93A-NSC34， hSOD1WT-NSC3 intracellular SOD1 protein levels increased significantly （P<0.01）， and the amyotrophic lateral sclerosis cell model was established. Compared with the normal group， the cell activity in the model group was significantly reduced （P<0.01）. Compared with the model group， the cell activity increased at different concentrations of GB， especially when the drug concentration was 75 mg∙L-1 （P<0.01）. In subsequent experiments， compared with the normal group， the apoptosis， and expressions of p-JNK， p-c-Jun， and cleaved Caspase-3 proteins in the model group increased significantly （P<0.01）. Compared with the model group， the apoptosis and p-JNK， p-c-Jun， released Caspase-3 protein expressions of 75 mg∙L-1 GB group， SP600125 group， 75 mg∙L-1 GB + SP600125 group decreased significantly （P<0.05， P<0.01）. Conclusion:GB has a protective effect on the cell model of atrophy lateral sclerosis， which may be realized by JNK signal pathway.

Roles of the MST1-JNK signaling pathway in apoptosis of colorectal cancer cells induced by Taurine.

The aim of this study was to observe the impact of the mammalian sterile 20-like kinase 1-c-Jun N-terminal kinase (MST1-JNK) signaling pathway on apoptosis in colorectal cancer (CRC) cells induced by Taurine (Tau). Caco-2 and SW620 cells transfected with p-enhanced green fluorescent protein (EGFP)-MST1 or short interfering RNA (siRNA)-MST1 were treated with Tau for 48 h. Apoptosis was detected by flow cytometry, and the levels of MST1 and JNK were detected by western blotting. Compared with the control group, 80 mM Tau could significantly induce apoptosis of CRC cells, and the apoptotic rate increased with increasing Tau concentration (P < 0.01). Meanwhile, the protein levels of MST1 and phosphorylated (p)-JNK in Caco-2 cells increased significantly (P < 0.01). The apoptotic rate of the p-EGFP-MST1 plasmid-transfected cancer cells was significantly higher than that of the control group (P < 0.05); however, the apoptotic rate of the p-EGFP-MST1+Tau group was increased further (P < 0.01). Silencing the MST1 gene could decrease the apoptotic rate of cancer cells, and Tau treatment could reverse this decrease. Blocking the JNK signaling pathway significantly reduced the Tau-induced apoptotic rate of CRC cells. Thus, the MST1-JNK pathway plays an important role in Tau-induced apoptosis of CRC cells.

Abnormal expression of TFIIIB subunits and RNA Pol III genes is associated with hepatocellular carcinoma.

The levels of the products of RNA polymerase III-dependent genes (Pol III genes), including tRNAs and 5S rRNA, are elevated in transformed and tumor cells, which potentiate tumorigenesis. TFIIB-related factor 1 (Brf1) is a key transcription factor and specifically regulates the transcription of Pol III genes. In vivo and in vitro studies have demonstrated that a decrease in Brf1 reduces Pol III gene transcription and is sufficient for inhibiting cell transformation and tumor formation. Emerging evidence indicates that dysregulation of Brf1 and Pol III genes is linked to the development of hepatocellular carcinoma (HCC) in humans and animals. We have reported that Brf1 is overexpressed in human liver cancer patients and that those with high Brf1 levels have shorter survivals. This review summarizes the effects of dysregulation of these genes on HCC and their regulation by signaling pathways and epigenetics. These novel data should help us determine the molecular mechanisms of HCC from a different perspective and guide the development of therapeutic approaches for HCC patients.

Class I HDACs control a JIP1-dependent pathway for kinesin-microtubule binding in cardiomyocytes.

Class I histone deacetylase (HDAC) inhibitors block hypertrophy and fibrosis of the heart by suppressing pathological signaling and gene expression programs in cardiac myocytes and fibroblasts. The impact of HDAC inhibition in unstressed cardiac cells remains poorly understood. Here, we demonstrate that treatment of cultured cardiomyocytes with small molecule HDAC inhibitors leads to dramatic induction of c-Jun amino-terminal kinase (JNK)-interacting protein-1 (JIP1) mRNA and protein expression. In contrast to prior findings, elevated levels of endogenous JIP1 in cardiomyocytes failed to significantly alter JNK signaling or cardiomyocyte hypertrophy. Instead, HDAC inhibitor-mediated induction of JIP1 was required to stimulate expression of the kinesin heavy chain family member, KIF5A. We provide evidence for an HDAC-dependent regulatory circuit that promotes formation of JIP1:KIF5A:microtubule complexes that regulate intracellular transport of cargo such as autophagosomes. These findings define a novel role for class I HDACs in the control of the JIP1/kinesin axis in cardiomyocytes, and suggest that HDAC inhibitors could be used to alter microtubule transport in the heart.

Apoptosis and Compensatory Proliferation Signaling Are Coupled by CrkI-Containing Microvesicles.

Apoptosis has been implicated in compensatory proliferation signaling (CPS), whereby dying cells induce proliferation in neighboring cells as a means to restore homeostasis. The nature of signaling between apoptotic cells and their neighboring cells remains largely unknown. Here we show that a fraction of apoptotic cells produce and release CrkI-containing microvesicles (distinct from exosomes and apoptotic bodies), which induce proliferation in neighboring cells upon contact. We provide visual evidence of CPS by videomicroscopy. We show that purified vesicles in vitro and in vivo are sufficient to stimulate proliferation in other cells. Our data demonstrate that CrkI inactivation by ExoT bacterial toxin or by mutagenesis blocks vesicle formation in apoptotic cells and inhibits CPS, thus uncoupling apoptosis from CPS. We further show that c-Jun amino-terminal kinase (JNK) plays a pivotal role in mediating vesicle-induced CPS in recipient cells. CPS could have important ramifications in diseases that involve apoptotic cell death.

A molecular dynamics study of the binary complexes of APP, JIP1, and the cargo binding domain of KLC.

Mutations in the amyloid precursor protein (APP) are responsible for the formation of amyloid-ß peptides. These peptides play a role in Alzheimer's and other dementia-related diseases. The cargo binding domain of the kinesin-1 light chain motor protein (KLC1) may be responsible for transporting APP either directly or via interaction with C-jun N-terminal kinase-interacting protein 1 (JIP1). However, to date there has been no direct experimental or computational assessment of such binding at the atomistic level. We used molecular dynamics and free energy estimations to gauge the affinity for the binary complexes of KLC1, APP, and JIP1. We find that all binary complexes (KLC1:APP, KLC1:JIP1, and APP:JIP1) contain conformations with favorable binding free energies. For KLC1:APP the inclusion of approximate entropies reduces the favorability. This is likely due to the flexibility of the 42-residue APP protein. In all cases we analyze atomistic/residue driving forces for favorable interactions. Proteins 2017; 85:221-234. © 2016 Wiley Periodicals, Inc.

In vitro metabolism of a novel JNK inhibitor tanzisertib: interspecies differences in oxido-reduction and characterization of enzymes involved in metabolism.

1. In vitro metabolism of Tanzisertib [(1S,4R)-4-(9-((S)tetrahydrofuran-3-yl)-8-(2,4,6-trifluorophenylamino)-9H-purin-2-ylamino) cyclohexanol], a potent, selective c-Jun amino-terminal kinase (JNK) inhibitor, was investigated in mouse, rat, rabbit, dog, monkey and human hepatocytes over 4 h. The extent of metabolism of [(14)C]tanzisertib was variable, with <10% metabolized in dog and human, <20% metabolized in rabbit and monkey and >75% metabolized in rat and mouse. Primary metabolic pathways in human and dog hepatocytes, were direct glucuronidation and oxidation of cyclohexanol to a keto metabolite, which was subsequently reduced to parent or cis-isomer, followed by glucuronidation. Rat and mouse produced oxidative metabolites and cis-isomer, including direct glucuronides and sulfates of tanzisertib and cis-isomer. 2. Enzymology of oxido-reductive pathways revealed that human aldo-keto reductases AKR1C1, 1C2, 1C3 and 1C4 were responsible for oxido-reduction of tanzisertib, CC-418424 and keto tanzisertib. Characterizations of enzyme kinetics revealed that AKR1C4 had a high affinity for reduction of keto tanzisertib to tanzisertib compared to other isoforms. These results demonstrate unique stereoselectivity of the reductive properties documented by human AKR1C enzymes for the same substrate. 3. Characterization of UGT isoenzymes in glucuronidation of tanzisertib and CC-418424 revealed that, tanzisertib glucuronide was catalyzed by: UGT1A1, 1A4, 1A10 and 2B4, while CC-418424 glucuronidation was catalyzed by UGT2B4 and 2B7.

Metabolism and disposition of a potent and selective JNK inhibitor [14C]tanzisertib following oral administration to rats, dogs and humans.

1. The disposition of tanzisertib [(1S,4R)-4-(9-((S)tetrahydrofuran-3-yl)-8-(2,4,6-trifluorophenylamino)-9H-purin-2-ylamino) cyclohexanol], a potent, orally active c-Jun amino-terminal kinase inhibitor intended for treatment of fibrotic diseases was studied in rats, dogs and humans following a single oral dose of [(14)C]tanzisertib (Independent Investigational Review Board Inc., Plantation, FL). 2. Administered dose was quantitatively recovered in all species and feces/bile was the major route of elimination. Tanzisertib was rapidly absorbed (Tmax: 1-2 h) across all species with unchanged tanzisertib representing >83% of plasma radioactivity in dogs and humans, whereas <34% was observed in rats. Variable amounts of unchanged tanzisertib (1.5-32% of dose) was recovered in urine/feces across all species, the highest in human feces. 3. Metabolic profiling revealed that tanzisertib was primarily metabolized via oxidation and conjugation pathways, but extensively metabolized in rats relative to dogs/humans. CC-418424 (S-cis isomer of tanzisertib) was the major plasma metabolite in rats (38.4-46.4% of plasma radioactivity), while the predominant plasma metabolite in humans and dogs was M18 (tanzisertib-/CC-418424 glucuronide), representing 7.7 and 3.2% of plasma radioactivity, respectively. Prevalent biliary metabolite in rats and dogs, M18 represented 16.8 and 17.1% of dose, respectively. 4. In vitro studies using liver subcellular fractions and expressed enzymes characterized involvement of novel human aldo-keto reductases for oxido-reduction and UDP-glucuronosyltransferases for conjugation pathways.

Antidepressant-like effects of Chaihu-Shugan-San via SAPK/JNK signal transduction in rat models of depression.

BACKGROUND: Chaihu-Shugan-San (CHSGS), a traditional Chinese medicinal herbal formula, registered in Jingyue Quanshu, has been indicated that oral administration of the extract from it can remit depressive disorder. C-Jun amino-terminal kinase (JNK/SAPK) signal transduction plays a key role in the apoptosis of nerve cells, be reported closely correlated with depression. This study was designed to investigate CHSGS antidepressant-like effects in rat models of depression and probe its possible mechanism. MATERIALS AND METHODS: The classical experimental depression model chronic mild unpredictable stress (CMUS) was used to evaluate the antidepressant-like effects of CHSGS. The extracts were administered orally for 14 days, while the parallel positive control was given at the same time using fluoxetine hydrochloride. The expressions of JNK in the hippocampus were detected by real-time fluorescent quantitation PCR and Western blot assay. RESULTS: Intragastric administration of CHSGS for 14 days caused a significant improvement of weight and locomotor activity in the open-field test. In addition, CHSGS treatment inhibited the expressions of JNK in the hippocampus tissue in CMUS rats. CONCLUSION: CHSGS could obviously improve the depressive state of the model rats and its mechanism may be correlated with regulating the expressions of JNK in the hippocampus.

Potential role of proteasome on c-jun related signaling in hypercholesterolemia induced atherosclerosis.

Atherosclerosis and its complications are major causes of death all over the world. One of the major risks of atherosclerosis is hypercholesterolemia. During atherosclerosis, oxidized low density lipoprotein (oxLDL) regulates CD36-mediated activation of c-jun amino terminal kinase-1 (JNK1) and modulates matrix metalloproteinase (MMP) induction which stimulates inflammation with an invasion of monocytes. Additionally, inhibition of proteasome leads to an accumulation of c-jun and phosphorylated c-jun and activation of activator protein-1 (AP-1) related increase of MMP expression. We have previously reported a significant increase in cluster of differentiation 36 (CD36) mRNA levels in hypercholesterolemic rabbits and shown that vitamin E treatment prevented the cholesterol induced increase in CD36 mRNA expression. In the present study, our aim is to identify the signaling molecules/transcription factors involved in the progression of atherosclerosis following CD36 activation in an in vivo model of hypercholesterolemic (induced by 2% cholesterol containing diet) rabbits. In this direction, proteasomal activities by fluorometry and c-jun, phospo c-jun, JNK1, MMP-9 expressions by quantitative RT-PCR and immunoblotting were tested in aortic tissues. The effects of vitamin E on these changes were also investigated in this model. As a result, c-jun was phosphorylated following decreased proteasomal degradation in hypercholesterolemic group. MMP-9 expression was also increased in cholesterol group rabbits contributing to the development of atherosclerosis. In addition, vitamin E showed its effect by decreasing MMP-9 levels and phosphorylation of c-jun.

Protection of granulocyte-colony stimulating factor to hemorrhagic brain injuries and its involved mechanisms: effects of vascular endothelial growth factor and aquaporin-4.

Granulocyte-colony stimulating factor (G-CSF) has protective effects on many neurological diseases. Here, we aimed to test G-CSF's effects on perihematomal tissue injuries following intracerebral hemorrhage (ICH) and examine whether the effects were functionally dependent on vascular endothelial growth factor (VEGF) and aquaporin-4 (AQP4). We detected the expression of perihematomal VEGF, VEGF receptors (VEGFRs) and AQP4 at 1, 3 and 7days after ICH. Also, we examined the effects of G-CSF on tissue injuries by ICH in wild type mice, and tested whether such effects were VEGF and AQP4 dependent by using VEGFR inhibitor - SU5416 and AQP4 knock-out (AQP4(-/-)) mice. Furthermore, we assessed the related signal transduction pathways via astrocyte cultures. We found G-CSF highly increased perihematomal VEGF, VEGFR-2 and AQP4. Importantly, G-CSF led to neurological functional improvement in both types of mice by associating with reduction of brain edema, blood-brain barrier (BBB) permeability and neuronal death and apoptosis and statistical analysis suggested AQP4 was required for these effects. Besides, except BBB leakage alleviation, the above effects were attenuated but not counteracted by SU5416, suggesting involvement of VEGF. G-CSF up-regulated phosphorylation of extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription 3 (STAT3) as well as VEGF and AQP4 proteins in cultured astrocytes. The latter was inhibited by ERK and STAT3 inhibitors respectively. Our data suggest the protective effects of G-CSF on perihematomal tissue injuries after ICH are highly associated with the increased levels of VEGF and AQP4, possibly act through C-Jun amino-terminal kinase and ERK pathways respectively.

Effect of electroacupuncture on c-Jun amino terminal kinase signaling pathway and inflammatory cytokines interferon-γ in substantia nigra cells of rotenone-induced rats model of Parkinson's disease / 中华物理医学与康复杂志

Objective To observe the effects of electroacupuncture (EA) on c-Jun amino terminal kinase (JNK) signaling pathway and inflammatory cytokines interferon-γ (IFN-γ) in substantia nigra (SN) cells of rotenone-induced rats model of Parkinson's disease (PD),and explore the underlying mechanism of EA on PD.Methods A total of 32 male Sprague-Dawley mice were randomly and evenly divided into a normal group,a shamoperation group,a model group and an EA group.Model group and EA group were injected intradermally with rotenone (1 mg/kg,dissolved in DMSO and saline,concentration:O.25 mg/ml) on the nape of neck.Sham-operation group was injected the same dose of DMSO and saline.Normal group had no intervention.EA group was applied to Fengfu (DU16) and Taichong (LR3) acupoints after the establishment of PD model in rats.Behavioral assessment was conducted after the treatments,the rats were sacrificed for sampling substantia nigra tissue to detect the expressions of tyrosine hydroxylase (TH),p-c-Jun amino terminal kinase (p-c-Jun) and interferon-γ(IFN-γ) protein with Western blotting (WB).Results Model rats showed significant PD syndrome characteristics,comparing with normal group and sham group,the difference was not statistically significant (P ＞ 0.05).The results of open box test showed that the scores of model group rats decreased significantly in terms of the horizontal movement [(19.12 ±2.34) points] and vertical locomotor activity [(5.27 ± 1.04) points] when compared with normal group and sham group,the difference was statistically significant (P ＜ 0.05).After EA treatment,locomotor activity of rats increased significantly when compared with model group (P ＜ 0.05),however,the normal group and sham group was not statistically and significantly different in locomotor activity (P ＞ 0.05).Compared with normal group,the expression of TH protein in (0.183 ± 0.0213) reduced significantly and the expressions of p-c-Jun (0.388 ± 0.0283) and IFN-γ protein(0.453 ± 0.0332) increased significantly in model group,the difference was statistically significant (P ＜0.05).Compared with normal group,the expression of TH protein(0.324 ± 0.0538) reduced and the expressions of p-c-Jun(0.207 ± 0.0592) and IFN-γ protein (0.239 ± 0.0215) increased in EA group,the difference was not statistically significant (P ＞ 0.05).Compared with model group,the expression of TH protein increased significantly in EA group(P ＜ 0.05),the expressions of p-c-Jun and IFN-γ protein reduced significantly in EA group(P ＜ 0.05).Conclusion EA therapy may reduce the expression of IFN-γ protein in SN of PD rats model by regulating the expression of JNK/mitogen-activated protein kinase (MAPK) pathway,which may delay the process of PD.