Heat shock protein 72 confers protection in retinal ganglion cells and lateral geniculate nucleus neurons via blockade of the SAPK/JNK pathway in a chronic ocular-hypertensive rat model

Optic nerve transection increased the expression of heat shock protein 72 （HSP72） in the lateral geniculate body, indicating that this protein is involved in the prevention of neuronal injury. Zinc sulfate and quercetin induced and inhibited the expression of HSP72, respectively. Intraperitoneal injections of zinc sulfate, SP600125 （c-Jun N-terminal kinase inhibitor）, or quercetin were performed on retinal ganglion cells in a Wistar rat model of chronic ocular hypertension. Our results showed that compared with the control group, the expression of HSP72 in retinal ganglion cells and the lateral geniculate body was increased after the injection of zinc sulfate, but was decreased after the injection of quercetin. The expression of phosphorylated c-Jun N-terminal kinases and phosphorylated c-Jun were visible 3 days after injection in the control group, and reached apeak at 7 days. Zinc sulfate and SP600125 significantly decreased the expression of p-c-Jun, whereas quercetin significantly enhanced the expression of this protein. These results suggest that HSP72 protects retinal ganglion cells and lateral geniculate body in a rat model of chronic ocular hypertension from injury by blocking the activation of the stress-activated kinase/c-Jun N-terminal kinase apoptotic pathway.

Jian-Gan-Xiao-Zhi decoction ameliorates high-fat high-carbohydrate diet-induced non-alcoholic fatty liver disease and insulin resistance by regulating the AMPK/JNK pathway

Background:Non-alcoholic fatty liver disease(NAFLD)can cause insulin resistance(IR)and diabetes.Our previous studies have demonstrated that Jian-Gan-Xiao-Zhi decoction(JGXZ)could be effective for the treatment of NAFLD and IR.However,the possible mechanism underlying the effects of JGXZ on NAFLD and IR remains unknown.Methods:Fifty rats received a high-fat high-carbohydrate(HFHC)diet for 12 weeks to induce NAFLD.After 4 weeks of HFHC treatment,rats were orally treated with JGXZ(8,16,and 32 g/kg weight)for 8 weeks.Ten rats in the control group received standard chow.In the positive control group,rats were orally treated with metformin(90 mg/kg weight)for 8 weeks.After JGXZ and metformin treatment,H&E staining was conducted on rat livers and serum biochemical markers,including alanine aminotransferase(ALT),aspartate aminotransferase(AST),triglyceride(TG),and total cholesterol(TC),were measured using test kits.Moreover,a fasting blood glucose test and an oral glucose tolerance test(OGTT)were conducted.Serum levels of insulin were determined using ELISA kit,and the homeostatic model assessment of insulin resistance(HOMA-IR)was calculated.The levels of total insulin receptor substrate-1(IRS1),AMP-activated protein kinase-α(AMPKα)and c-Jun N-terminal kinase(JNK)as well as the levels of phosphorylation of IRS1(p-IRS1),phosphorylation of AMPK(p-AMPK)and phosphorylation of JNK(p-JNK)were measured using western blotting.Results:The body weights in JGXZ low-,middle-,and high-dose groups were lower than those in the model group(P<0.05,P<0.01,P<0.01,respectively).The serum levels of AST(P<0.05 in JGXZ middle-and high-dose groups),ALT(P<0.01 in JGXZ middle-dose group and P<0.05 in JGXZ high-dose group),TG(P<0.01 in JGXZ middle-and high-dose groups),and TC(P<0.01)upon JGXZ treatment were lower those than in NAFLD model rats.H&E staining showed that JGXZ treatment reduced steatosis of the hepatocytes in NAFLD model rats.JGXZ decreased the levels of fasting blood glucose(P<0.01),HOMA-IR(P<0.01),AUC(area under the curve)of the OGTT(P<0.05)and p-IRS1(P<0.01 in JGXZ middle-and high-dose groups,P<0.05 in JGXZ low-dose groups).Moreover,JGXZ regulated the hepatic AMPKα/JNK pathway in NAFLD model rats,which reflected the induction of p-AMPKαand inhibition of p-JNK.Conclusion:This study showed that JGXZ improved liver function and reduced steatosis of the hepatocytes in NAFLD model rats.Moreover,JGXZ improved IR in NAFLD model rats.The possible mechanism underlying the effects of JGXZ on NAFLD and IR involves the modulation of the AMPK/JNK pathway.

Ultraviolet A Enhances Cathepsin L Expression and Activity via JNK Pathway in Human Dermal Fibroblasts

Background： Cathepsin L （CatL） is a cysteine protease with strong matrix degradation activity that contributes to photoaging. Mannose phosphate-independent sorting pathways mediate ultraviolet A （UVA）-induced alternate trafficking of CatL. Little is known about signaling pathways involved in the regulation of UVA-induced CatL expression and activity. This study aims to investigate whether a single UVA irradiation affects CatL expression and activity and whether mitogen-activated protein kinase （MAPK）/activator protein- 1 （AP- 1 ） pathway is involved in the regulation of UVA-induced CatL expression and activity in human dermal fibroblasts （HDFs）. Methods： Primary HDFs were exposed to UVA. Cell proliferation was determined by a cell counting kit. UVA-induced CatL production and activity were studied with quantitative real-time reverse transcription polymerase chain reaction （RT-PCR）, Western blotting, and fluorimetric assay in cell lysates collected on three consecutive days after irradiation. Time courses of UVA-activated JNK and p38MAPK signaling were examined by Western blotting. Effects ofMAPK inhibitors and knockdown of dun and Fos on UVA-induced CatL expression and activity were investigated by RT-PCR, Western blotting, and fluorimetric assay. Data were analyzed by one-way analysis of variance. Results： UVA significantly increased CatL gene expression, protein abundance, and enzymatic activity for three consecutive days after irradiation （F = 83.11, 56.14, and 71. 19, respectively; all P 〈 0.05）. Further investigation demonstrated phosphorylation of JNK and p38MAPK activated by UVA. Importantly, inactivation of JNK pathway significantly decreased UVA-induced CatL expression and activity, which were not affected by p38MAPK inhibition. Moreover, knockdown of dun and Fos significantly attenuated basal and UVA-induced CatL expression and activity. Conclusions： UVA enhances CatL production and activity in HDFs, probably by activating JNK and downstreaming AP- 1. These findings provide a new possible molecular approach for antiphotoaging therapy.

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

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

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

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

Luteolin delays photoreceptor degeneration in a mouse model of retinitis pigmentosa

Luteolin is neuroprotective for retinal ganglion cells and retinal pigment epithelial cells after oxidative injury,whereby it can inhibit microglial neurotoxicity.Therefore,luteolin holds the potential to be useful for treatment of retinal diseases.The purpose of this study was to investigate whether luteolin exhibits neuroprotective effects on rod cells in rd10 mice,a slow photoreceptor-degenerative model of retinitis pigmentosa.Luteolin(100 mg/kg)intraperitoneally injected daily from postnatal day 14(P14)to P25 significantly enhanced the visual performance and retinal light responses of rd10 mice at P25.Moreover,it increased the survival of photoreceptors and improved retinal structure.Mechanistically,luteolin treatment attenuated increases in reactive oxygen species,photoreceptor apoptosis,and reactive gliosis;increased mRNA levels of anti-inflammatory cytokines while lowering that of pro-inflammatory and chemoattractant cytokines;and lowered the ratio of phospho-JNK/JNK.Application of the JNK inhibitor SP600125 exerted a similar protective effect to luteolin,suggesting that luteolin delays photoreceptor degeneration and functional deterioration in rd10 mice through regulation of retinal oxidation and inflammation by inhibiting the JNK pathway.Therefore,luteolin may be useful as a supplementary treatment for retinitis pigmentosa.This study was approved by the Qualified Ethics Committee of Jinan University,China(approval No.IACUC-20181217-02)on December 17,2018.

miR-202 contributes to sensitizing MM cells to drug significantly via activing JNK/SAPK signaling pathway

Objective To explore the role of miR-202 in multiple myeloma(MM)cells,and study the regulation of miR-202 on drug sensitivity of MM cells.Methods miR-202 and BAFF mRNA levels were detected by real-time PCR.U266 cells were transfected with miR-202-mimics,miR-202-inhibitor,siB AFF and their negative controls.

Loss of GRB2 associated binding protein 1 in arteriosclerosis obliterans promotes host autophagy

Background:Arteriosclerosis obliterans(ASO)is a major cause of adult limb loss worldwide.Autophagy of vascular endothelial cell(VEC)contributes to the ASO progression.However,the molecular mechanism that controls VEC autophagy remains unclear.In this study,we aimed to explore the role of the GRB2 associated binding protein 1(GAB1)in regulating VEC autophagy.Methods:In vivo and in vitro studies were applied to determine the loss of adapt protein GAB1 in association with ASO progression.Histological GAB1 expression was measured in sclerotic vascular intima and normal vascular intima.Gain-and loss-of-function of GAB1 were applied in VEC to determine the effect and potential downstream signaling of GAB1.Results:The autophagy repressor p62 was significantly downregulated in ASO intima as compared to that in healthy donor(0.80 vs.0.20,t=6.43,P<0.05).The expression level of GAB1 mRNA(1.00 vs.0.24,t=7.41,P<0.05)and protein(0.72 vs.0.21,t=5.97,P<0.05)was significantly decreased in ASO group as compared with the control group.Loss of GAB1 led to a remarkable decrease in LC3II(1.19 vs.0.68,t=5.99,P<0.05),whereas overexpression of GAB1 significantly led to a decrease in LC3II level(0.41 vs.0.93,t=7.12,P<0.05).Phosphorylation levels of JNK and p38 were significantly associated with gain-and loss-of-function of GAB1 protein.Conclusion:Loss of GAB1 promotes VEC autophagy which is associated with ASO.GAB1 and its downstream signaling might be potential therapeutic targets for ASO treatment.

Molecular vulnerabilities and therapeutic resistance in hormone receptor positive and HER2 dependent breast cancer tumours

Over the past two decades,high sensitivity to HER2-amplified primary breast cancers has been achieved with HER2-targeted therapies.CDK4/6 inhibitors have long been identified as a potential treatment option for advanced breast cancer patients.However,acquired HER2 heterogeneity leading to resistance during the treatment has been identified as a bottleneck.This review focuses on the recent resistance mechanisms identified and potential therapeutic targets for conventional and combination endocrine therapies with CDK4/6 inhibitors by various breast cancer clinical trials and research groups in HER amplified and/or mutated breast cancer tumour.Activating HER2 alterations,JNK pathway,hyperactivated TORC1,co-mutations in HER2 and HER3,phenotypic changes of HER2,and few other advanced findings are identified as potential therapeutic targets in treating current HER2 endocrine therapy-resistant tumour.Along with the HER2-focused resistance mechanisms,we also describe how the microbiome may play a role in breast cancer therapy and its potential for new therapeutic strategies to overcome drug resistance in breast cancers.

Regulation of the protein stability of POSH and MLK family

Sequential activation of the JNK pathway components,including Rac1/Cdc42,MLKs(mixed-lineage kinases),MKK4/7 and JNKs,plays a required role in many cell death paradigms.Those components are organized by a scaffold protein,POSH(Plenty of SH3’s),to ensure the effective activation of the JNK pathway and cell death upon apoptotic stimuli.We have shown recently that the expression of POSH and MLK family proteins are regulated through protein stability.By generating a variety of mutants,we provide evidence here that the Nterminal half of POSH is accountable for its stability regulation and its over-expression-induced cell death.In addition,POSH’s ability to induce apoptosis is correlated with its stability as well as its MLK binding ability.MLK family’s stability,like that of POSH,requires activation of JNKs.However,we were surprised to find out that the widely used dominant negative(d/n)form of c-Jun could down-regulate MLK’s stability,indicating that peptide from d/n c-Jun can be potentially developed into a therapeutical drug.