JNK Activation Research Articles

Resveratrol contributes to NK cell-mediated breast cancer cytotoxicity by upregulating ULBP2 through miR-17-5p downmodulation and activation of MINK1/JNK/c-Jun signaling

BackgroundsNatural killer (NK) cell mediated cytotoxicity is a crucial form of anti-cancer immune response. Natural killer group 2 member D (NKG2D) is a prominent activating receptor of NK cell. UL16-binding protein 2 (ULBP2), always expressed or elevated on cancer cells, functions as a key NKG2D ligand. ULBP2-NKG2D ligation initiates NK cell activation and subsequent targeted elimination of cancer cells. Enhanced expression of ULBP2 on cancer cells leads to more efficient elimination of these cells by NK cells. Resveratrol (RES) is known for its multiple health benefits, while current understanding of its role in regulating cancer immunogenicity remains limited. This study aims to investigate how RES affects the expression of ULBP2 and the sensitivity of breast cancer (BC) cells to NK cell cytotoxicity, along with the underlying mechanisms.MethodsThe effects of RES on ULBP2 expression were detected with qRT-PCR, western blot, flow cytometry analysis and immunohistochemistry. The effects of RES on sensitivity of BC cells to NK cell cytotoxicity were evaluated in vitro and in vivo. The target gene of miR-17-5p were predicted with different algorithms from five databases and further confirmed with dual-luciferase reporter assay. Overexpression and knockdown experiments of miR-17-5p and MINK1 were conducted to investigate their roles in regulating ULBP2 expression and subsequent JNK/c-Jun activation. The JNK inhibitor sp600125 was utilized to elucidate the specific role of JNK in modulating ULBP2 expression.ResultsRES increased ULBP2 expression on BC cells, thereby augmenting their vulnerability to NK cell-mediated cytotoxicity both in vitro and in vivo. RES administration led to a reduction in cellular miR-17-5p level. MiR-17-5p negatively regulated ULBP2 expression. Specifically, miR-17-5p directly targeted MINK1, leading to its suppression. MINK1 played a role in facilitating the activation of JNK and its downstream effector, c-Jun. Furthermore, treatment with sp600125, a JNK inhibitor, resulted in the suppression of ULBP2 expression.Conclusions：RES potentiates ULBP2-mediated immune eradication of BC cells by NK cells through the downregulation of miR-17-5p and concurrent activation of the MINK1/JNK/c-Jun cascade. This finding identifies RES as a potentially effective therapeutic agent for inhibiting BC progression and optimizing NK cell-based cancer immunotherapy.

RAGE-mediated intestinal pro-inflammatory responses triggered by Giardia duodenalis.

Giardia duodenalis is a waterborne zoonotic protozoan that causes gastrointestinal inflammation. Giardiasis and metabolic illnesses share features such as chronic inflammation and intestinal symptoms. Receptor for advanced glycation end products (RAGE) signaling plays a role in metabolic illnesses and intestinal inflammatory responses. The presence of protozoan viruses can influence host immunological responses triggered by protozoa. However, these effects of G. duodenalis remain unknown. In this study, mice treated with the RAGE inhibitor FPS-ZM1 showed more severe intestinal damage, including increased intestinal permeability and lesions, compared to that of the untreated group. Next, we found that G. duodenalis infection activated RAGE, leading to increased secretion of pro-inflammatory cytokines, including IL-1 β, IL-6, IL-12, TNF-α and IFN-γ in mouse intestinal epithelial cells. Notably, these pro-inflammatory responses were significantly higher in Giardiavirus (GLV)-free Giardia than those of GLV-containing Giardia, except for IFN-γ. Additionally, lactate dehydrogenase (LDH) release, GSDMD-N cleavage, and the morphological observation of pyroptosis were significantly higher in cells induced by GLV-free Giardia than those infected with GLV-carrying Giardia. Differences were also observed in the MAPK (p-JNK, p-38, p-ERK) and NF-κB pathway activation, as well as reactive oxygen species (ROS) levels, with higher activation in cells infected by GLV-free Giardia, and the ROS was involved in the regulation of p38 MAPK and JNK activation. These findings reveal the potential of RAGE as a target for developing vaccines or drugs, suggesting the differences in the regulation of host immune responses induced by GLV-free Giardia or GLV-containing Giardia, providing new insights for the prevention and treatment of giardiasis.

Inhibition of JNK signaling attenuates photoreceptor ferroptosis caused by all-trans-retinal

The disruption of the visual cycle leads to the accumulation of all-trans-retinal (atRAL) in the retina, a hallmark of autosomal recessive Stargardt disease (STGD1) and dry age-related macular degeneration (AMD), both of which cause retinal degeneration. Although our previous studies have shown that atRAL induces ferroptosis and activates c-Jun N-terminal kinase (JNK) signaling in the retina, the relationship between JNK signaling and ferroptosis in atRAL-mediated photoreceptor damage remains unclear. Here, we reported that JNK activation by atRAL drove photoreceptor ferroptosis through ferritinophagy. In photoreceptor cells loaded with atRAL, activated JNK phosphorylated c-Jun, which facilitated its nuclear translocation and promoted the expression of the nuclear receptor coactivator 4 (NCOA4). Elevated NCOA4 induced ferritin degradation via lysosomal processing, a process known as ferritinophagy, thereby releasing a large amount of labile iron. Iron overload led to the generation of reactive oxygen species (ROS) and lipid peroxidation, ultimately culminating in ferroptosis. Treatment with the JNK inhibitor JNK-IN-8, as well as the knockout of Jnk1 and Jnk2 genes, significantly rescued atRAL-loaded photoreceptor cells from ferritinophagy-induced ferroptosis. Abca4-/-Rdh8-/- mice, which exhibit atRAL accumulation in the retina following light exposure, are commonly used to study the pathological processes of STGD1 and dry AMD. In these mice, light exposure activated the JNK/c-Jun/NCOA4 axis, resulting in ferritinophagy in the neural retina. Importantly, intraperitoneal administration of JNK-IN-8 significantly rescued retinal function and photoreceptors from ferritinophagy-induced ferroptosis and effectively mitigated retinal degeneration in light-exposed Abca4-/-Rdh8-/- mice. This study underscores the critical role of the JNK/c-Jun/NCOA4 axis in mediating atRAL-induced ferritinophagy, which drives ferroptosis and retinal atrophy, suggesting that targeting this pathway may offer a potential therapeutic approach for STGD1 and dry AMD.

GPNMB attenuates neuroinflammation and improves ischemic stroke via modulation of PI3K/Akt and p38 MAPK signaling pathways

Ischemic stroke is a leading cause of disability and mortality worldwide, with limited effective treatments. Neuroinflammation plays a crucial role in the progression of ischemic brain injury. Glycoprotein nonmetastatic melanoma protein B (GPNMB) has emerged as a potential regulator of inflammation, but its role and underlying mechanisms in ischemic stroke remain largely unknown. We investigated the expression profile, functional significance, and molecular pathways of GPNMB in ischemic stroke using a mouse model of middle cerebral artery occlusion (MCAO), transcriptome sequencing, and human serum samples. The effects of GPNMB knockdown on stroke outcomes, neuroinflammation, and neuronal damage were assessed in vivo. Bioinformatic analyses and experimental validation were performed to identify the downstream signaling pathways of GPNMB. GPNMB was highly upregulated in the ischemic brain, with its expression peaking at 3-7days post-MCAO. Serum GPNMB levels were elevated in ischemic stroke patients and correlated with stroke severity. GPNMB knockdown exacerbated stroke outcomes, neuroinflammation, and neuronal damage. Mechanistically, GPNMB positively modulated the PI3K/Akt/GSK3β pathway while negatively regulating p38 MAPK, JNK, and ERK activation. GPNMB knockdown enhanced the expression of NF-κB, a master transcriptional regulator of inflammation. GPNMB is highly upregulated in the ischemic brain and confers neuroprotection against ischemic injury by modulating neuroinflammation via the PI3K/Akt and p38 MAPK signaling pathways.

Exploration of the Combined Mechanism of Direct and Indirect Effects of Paeoniflorin in the Treatment of Cholestasis.

Cholestasis is a multifactorial hepatobiliary disorder, characterized by obstruction of bile flow and accumulation of bile, which in turn causes damage to liver cells and other tissues. In severe cases, it can result in the development of life-threatening conditions, including cirrhosis and liver cancer. Paeoniflorin (PF) has been demonstrated to possess favourable therapeutic potential for the treatment of cholestasis. The objective of this research was to examine the molecular mechanism of PF in the treatment of ANIT-induced cholestasis and to propose novel avenues for further research on the pharmacological effects of PF. In vivo and in vitro models of cholestasis were developed. The histopathological changes in the bile ducts and liver were evaluated through the use of hematoxylin and eosin (HE) staining. The extent of apoptosis was evaluated through the use of immunofluorescence (IF), immunoblotting (WB), and electron microscopy. The JNK signalling pathway was identified as the direct mechanism of action of PF through the utilisation of HuProt™ 20K chips and other technologies. The present study demonstrated that PF markedly alleviated liver injury in an ANIT-induced cholestasis model. Specifically, PF was observed to attenuate cholestasis-induced liver injury by reducing the abnormal elevation of liver function indices and suppressing the expression of inflammatory mediators. Furthermore, PF exhibited anti-apoptotic properties in both in vivo and in vitro experiments, thereby mitigating cholestasis-induced hepatocyte apoptosis. These protective effects are attributable to the fact that PF exerts its action through direct interaction with the JNK pathway. It has been demonstrated that PF is capable of binding directly to MAPK8 (JNK1) and MAPK9 (JNK2), thereby inhibiting JNK activation and reducing apoptosis. With regard to the protection of bile ducts, PF may indirectly inhibit hepatocyte apoptosis by maintaining the structural integrity and tight junctions of bile duct cells. PF improved cholestasis by inhibiting hepatocyte apoptosis directly by targeting the JNK signaling pathway and indirectly inhibited hepatocyte apoptosis by improving the tight junctions of bile duct cells to regulate the bile duct microenvironment.

Licochalcone A Induces Uterine Leiomyoma Cell Apoptosis via the ROS-Mediated JNK Activation of the GRP78/NRF2 Pathway In Vitro and In Vivo

Licochalcone A (LicoA) possesses anti-tumor properties. However, the potential therapeutic effect of LicoA on uterine leiomyomas (ULs) remains unknown. In this study, the effects of LicoA on the proliferation of ULs and its underlying mechanism were explored. LicoA treatment significantly decreased the viability of uterine smooth muscle cells (UtSMCs) and ELT3 cells in a dose-dependent manner. The induction of ELT3 cell apoptosis by LicoA was accompanied by the increased generation of reactive oxygen species (ROS), elevated endoplasmic reticulum (ER) stress (GRP78/IRE1α/ATF6/CHOP), and the increased expression of proapoptotic proteins (c-caspase-3, c-caspase-9, and c-PARP). The ability of Z-VAD-FMK (a caspase inhibitor) and n-acetylcysteine (NAC; a cell membrane permeable antioxidant) to reverse LicoA-induced ROS-mediated ER stress pathways also observed. Furthermore, GRP78 or JNK knockdown was involved in LicoA-induced ROS-mediated ER stress and apoptosis in ELT3 cells. In immunodeficient mice, LicoA significantly suppressed the growth of ELT3 tumor cells, without toxicity. This study is the first to show that LicoA exerts anti-leiomyoma effects via the modulation of ROS-mediated ER stress-induced apoptosis through the JNK/GRP78/NRF2 signaling pathway.

Muscle Cell Palmitate-Induced Insulin Resistance, JNK, IKK/NF-κB and STAT3 Activation are Attenuated by Carnosic and Rosmarinic Acid.

The worldwide epidemic of obesity has drastically worsened with the increase in more sedentary lifestyles and increased consumption of fatty foods. Increased blood free fatty acids (FFAs), often observed in obesity, leads to impaired insulin action, and promotes the development of insulin resistance and Type 2 diabetes mellitus (T2DM). JNK, IKK-NF-κB, and STAT3 are known to be involved in skeletal muscle insulin resistance. We reported previously that carnosic acid (CA) and rosmarinic acid (RA), attenuated the palmitate-induced skeletal muscle insulin resistance, an effect that was associated with increased AMPK activation and reduced mTOR-p70S6K signaling. In the present study, we examined the effects of CA and RA on JNK, IKK-NF-κB, and STAT3. Exposure of cells to palmitate increased the phosphorylation/activation of JNK, IKKα/β, IκBα, NF-κBp65, and STAT3. Importantly, CA and RA attenuated the deleterious effects of palmitate. Our data indicate that CA and RA have the potential to counteract the palmitate-induced skeletal muscle cell insulin resistance by modulating JNK, IKK-NF-κB, and STAT3 signaling.

PIM1 enhances insulin secretion and inhibits ferroptosis of high glucose-induced pancreatic β-cells through strengthening PINK1/Parkin-mediated mitophagy via inactivating JNK/p38 signaling pathway.

Diabetes mellitus (DM), a chronic metabolic disease, is characterized by long-term hyperglycemia resulting from the defect of insulin production and insulin resistance. The damage and dysfunction of pancreatic β-cells is a main link in DM development. In this work, pancreatic β-cell line INS-1E cells were exposed to 30 mM glucose for 48 h to construct an in vitro DM model. For gain-of-function experiments, HG-treated INS-1E cells were transfected with Oe-PIM1 to thoroughly discuss the biological role of PIM1 in HG-injured pancreatic β-cells. Furthermore, to probe into whether JNK/p38 signaling involved in the protective role of PIM1 in HG-injured pancreatic β-cells, HG-treated INS-1E cells were pre-treated with a JNK activator anisomycin (0.01 μM) for 1 h for rescue experiments. It was verified that HG treatment obviously downregulated PIM1 expression in INS-1E cells. PIM1 overexpression enhanced insulin secretion, inhibited ferroptosis and strengthened PINK1/Parkin-mediated mitophagy of HG-treated INS-1E cells. PIM1 overexpression inactivated JNK/p38 signaling pathway in HG-treated INS-1E cells. Activation of JNK/p38 signaling pathway partially abolished the strengthening effects of PIM1 overexpression on PINK1/Parkin-mediated mitophagy in HG-treated INS-1E cells. Upregulation of PIM1 strengthened PINK1/Parkin-mediated mitophagy in HG-injured pancreatic β-cells via inactivating JNK/p38 signaling pathway. Besides, activation of JNK/p38 signaling pathway partially abolished the enhancing effects of PIM1 overexpression on insulin secretion and the suppressive effects of PIM1 overexpression on ferroptosis in HG-treated INS-1E cells. Upregulation of PIM1 enhanced insulin secretion and inhibited ferroptosis in HG-injured pancreatic β-cells via inactivating JNK/p38 signaling pathway. In a word, upregulation of PIM1 may alleviate HG-induced pancreatic β-cell injury through strengthening PINK1/Parkin-mediated mitophagy via inactivating JNK/p38 signaling pathway.

GS-4997 halts the progression of tubulointerstitial injury inlupus nephritis.

Tubulointerstitial injury has been increasingly recognized as an important component in lupus nephritis (LN) pathology over the last decades. However, current clinical treatment options for this process remain limited. In this study, we aimed to investigate the potential benefits of GS-4997, a selective inhibitor of ASK1, in tubulointerstitial injury of LN. Female MRL/lpr mice were used as a classical lupus-prone murine model. Development of nephritis was assessed by monitoring of proteinuria, renal function, and histologic analysis. GS-4997 (50 mg/kg) or vehicle were treated orally. Invitro study, human kidney-2 (HK-2) cells were stimulated with 1 μg/mL lipopolysaccharide (LPS) to mimic the response of renal tubular epithelial cells undergoing inflammatory responses during LN. GS-4997 could inhibit the activation of the ASK1 in renal tubulointerstitium in MRL/lpr mice and LPS-induced HK-2 cells. GS-4997 treatment improved renal function, proteinuria, and attenuated tubular injury, renal interstitial fibrosis, and inflammation both invivo and invitro. Additionally, we found that in MRL/lpr mice, GS-4997 reduced deposition of IgG and C3 in the kidneys, antibody levels in the serum, splenic enlargement, and inflammatory cell infiltration in the spleen. Mechanistically, GS-4997 inhibited the activation of downstream signaling molecules, p38 and JNK, in the ASK1 signaling pathway. Pharmacological inhibition of ASK1 may prevent the progression of tubulointerstitial injury via inhibiting the ASK1/MAPK pathway in LN. Therefore, our findings demonstrate the potential use of GS-4997 for LN treatment.

Celecoxib paradoxically induces COX-2 expression and astrocyte activation through the ERK/JNK/AP-1 signaling pathway in the cerebral cortex of rats.

Previous studies have shown that celecoxib or NSAID may paradoxically induce cyclooxygenase-2 (COX-2) expression and trigger inflammation-like responses in airway smooth muscle cells and renal mesangial cells. Despite the extensive research on celecoxib, its atypical biological effect on the induction of COX-2 in astroglial cells within the central nervous system (CNS) remains unexplored. In the present study, we investigated the impact of celecoxib on COX-2 and Glial Fibrillary Acidic Protein (GFAP) expression and explored the mechanisms underlying celecoxib-regulated COX-2 expression in cortical astrocytes of rats. Cortical astrocytes were treated with celecoxib (20μM) for 24h, resulting in a significant increase in COX-2 expression and up-regulation of GFAP, a marker of astrocyte activation, and the COX-2 induced by celecoxib is functionally active in prostaglandin E2 (PGE2) synthesis. Celecoxib also enhanced LPS-induced COX-2 expression, but its ability to inhibit PGE2 synthesis decreased at higher concentrations. Celecoxib induced phosphorylation of Extracellular signal-regulated Kinase (ERK) and c-Jun N-terminal Kinase (JNK) but not p38 Mitogen-Activated Protein Kinase (p38 MAPK), and inhibition of activity of ERK and JNK by U0126 and SP600125 effectively blocked COX-2 and GFAP induction by celecoxib. Celecoxib increased the accumulation of transcription factor AP-1 (composed of phosphorylated c-Jun and c-fos) in the nucleus. Inhibition of AP-1 activity with SR11302 significantly prevented celecoxib-induced COX-2 and GFAP expression. Additionally, the inhibiting activity of ERK and JNK can effectively suppress AP-1 expression and activity induced by celecoxib. These findings demonstrated that celecoxib induces COX-2 expression and astrocyte activation through the ERK/JNK/AP-1 signaling pathway, highlighting its potential effect in modulating inflammatory responses in the central nervous system.

Mdivi-1 alleviates nicotine-induced human periodontal ligament cells injury by inhibiting mitochondrial fission and dysfunction through the JNK/Drp1 pathway

Nicotine, a major component of tobacco, is implicated in the pathogenesis of periodontitis. However, the exact mechanisms through which nicotine exerts its harmful effects remain incompletely understood. This study investigates the impact of nicotine-induced mitochondrial fission on human periodontal ligament cells (hPDLCs). A range of assays, including MTT, immunofluorescence staining, flow cytometry, and western blotting, were utilized to evaluate hPDLC viability, apoptosis, mitochondrial fission, and function. Nicotine decreases hPDLC viability in a dose-dependent manner, leading to apoptosis, an elevated BAX/BCL-2 ratio, and cellular injury. Furthermore, nicotine induces phosphorylation of Drp1 at Ser616, which facilitates mitochondrial fission, elevates mitochondrial ROS production, reduces mitochondrial membrane potential, and lowers ATP generation, resulting in mitochondrial dysfunction. Inhibition of Drp1 phosphorylation by Mdivi-1 significantly alleviates mitochondrial fission and dysfunction, reduces nicotine-induced apoptosis, and promotes osteogenic differentiation. Nicotine activates c-Jun N-terminal kinase (JNK), and the inhibition of JNK activity with SP600125 effectively prevents nicotine-induced mitochondrial fission, enhances cell viability, and inhibits Drp1 phosphorylation.

Herbacetin ameliorates lipopolysaccharide-elicited inflammatory response by suppressing NLRP-3/AIM-2 inflammasome activation, PI3K/Akt/MAPKs/NF-κB redox inflammatory signalling, modulating autophagy and macrophage polarization imbalance.

Herbacetin, a flavonol abundant in traditional medicines, is documented as an anti-inflammatory agent. However, information regarding its attributes on lipopolysaccharide (LPS)-induced inflammatory immunopathies has not been delineated yet. The present study aimed to comprehend herbacetin effects on LPS-induced aspects of unwarranted, non-resolving inflammation, particularly via targeting the vicious circle of oxi-inflammatory stress, autophagy-apoptosis, macrophages polarization, impaired inflammasome activation, and inflammatory cascades. In-vitro model of LPS-stimulated RAW 264.7 macrophage was recapitulated to investigate different inflammatory anomalies using enzyme-linked immunosorbent assay, qRT-PCR (Real-Time Quantitative Reverse Transcription PCR), immunoblotting. Concanavalin A challenged splenocytes and in silico studies were performed to measure Tregs population and binding affinity, respectively. Herbacetin administration caused remarkable reduction in nitric oxide, reactive oxygen species, mitochondrial membrane potential hyperpolarization, tumor necrosis factor-α, interferon-γ, interleukin-6, inducible nitric oxide synthase and ratio of M1/M2 markers (inducible nitric oxide synthase/arginase-1/macrophage scavenger receptor-1/mannose receptor C type-1) in in vitro model of persistent inflammation. Suppression of interleukins-5,17 and matrix metalloproteinases-2,3,9,13 and proliferating cell nuclear antigen, signifies its anti-inflammatory attributes. Noticeable decline in monodansylcadaverine-Lysotracker staining, caspase-6, and enhanced p62, B-cell lymphoma-2 expression indicates apoptosis-autophagosome accumulation inhibition and lysosomal destabilization. These were accompanied by reduced NLRP3 activation, caspase-1, AIM-2 expression, and interleukin-1β release. Subsequently, up-regulated activation of TLR-4, NF-κB, PI3K, Akt, ERK1/2, and JNK was decisively thwarted by herbacetin. In silico investigation signified the interaction of herbacetin with these targets. Decreased cytokines and enhanced Tregs conferred its role in extenuating inflammation facilitated by T-cells depletion. Collectively, these findings comprehend attributes of herbacetin as an alternative therapeutic strategy in relieving LPS-associated chronic inflammatory disorders.

Resveratrol and Its Derivatives Diminish Lipid Accumulation in Adipocytes In Vitro-Mechanism of Action and Structure-Activity Relationship.

Expansion of white adipose tissue causes systemic inflammation and increased risk of metabolic diseases due to its endocrine function. Resveratrol was suggested to be able to prevent obesity-related disorders by mimicking caloric restriction; however, its structure-activity relationships and molecular targets are still unknown. We aimed to compare the effects of resveratrol and its analogues on adipocyte metabolism and lipid accumulation in vitro. Mouse embryonic fibroblasts were differentiated to adipocytes in the absence or presence of resveratrol or its derivatives (oxyresveratrol, monomethylated resveratrol, or trimethylated resveratrol). Intracellular lipid content was assessed by Oil Red O staining. Glucose uptake and its response to insulin were estimated by 2-NBDG, and mitochondrial activity was assayed via resazurin reduction. Involvement of potential molecular pathways was investigated by concurrent treatment with their inhibitors. Although lipid accumulation was significantly reduced by all analogues without altering protein content, oxyresveratrol was the most potent (IC50 = 4.2 μM), while the lowest potency was observed with trimethylated resveratrol (IC50 = 27.4 μM). Increased insulin-stimulated glucose uptake was restored by each analogue with comparable efficiency. The enhanced mitochondrial activity was normalized by resveratrol and its methylated derivatives, while oxyresveratrol had a minor impact on it. Among the examined pathways, inhibition of SIRT1, PGC-1α, and JNK diminished the lipid-reducing effect of the compounds. Autophagy appeared to play a key role in the effect of all compounds but oxyresveratrol. Resveratrol and its analogues can mimic caloric restriction with complex mechanisms, including activation of SIRT1, PGC-1α, and JNK, making them possible drug candidates to treat obesity-related diseases.

In Silico Study Active Compounds of Jamu Against Pre-Eclampsia Through Anti-Vasoconstriction and Anti-Inflammation

Preeclampsia is one of serious pregnancy-related condition that marked by elevated blood pressure and proteinuria that typically emerges during the second trimester or above 20 week, this disease become the second common cause of maternal death globally per year. One factors that can initiate preeclampsia are immunologic factor. Preeclampsia is started with abnormal trophoblast development and poor or shallow placental blood vessel development. Several protein that are induce by pre eclampsia are ACE1 (as vasoconstrictor), p38α, and JNK (stress-related protein and induce inflammation). One of the natural medicine in Indonesia is a “Jamu” that refers to traditional herbal medicine derived from natural ingredients such as herbs, roots, and spices.. In this research we are trying to predict several compounds in early-month pregnant jamu to tackle activation of ACE1, JNK, and p38α. Firstly, all of jamu compound was screened using Lipinski and bioavaibility test. Then, all of the jamu compound that pass the test before will be docked with ACE1, JNK, and p38α using Vina wizard in PyRx. The highest affinity in docking result will be visualized using Biovia Discovery Studio 2019, and will advance to molecular dynamics simulation test to determine stability of the complex in cell environment. The result show that kaempferol can stabily bind to p38α protein, with only induce minimum fluctuation in all RMSD and RMSF result. Piperanine can stabily bind JNK protein with also minimum fluctuation in all RMSD and RMSF result. Both kaempferol and piperanine did’t violate any Lipinski rule and Bioavaibility test.

The ribotoxic stress response drives acute inflammation, cell death, and epidermal thickening in UV-irradiated skin in vivo

Solar UVB light causes damage to the outermost layer of skin. This insult induces rapid local responses, such as dermal inflammation, keratinocyte cell death, and epidermal thickening, all of which have traditionally been associated with DNA damage response signaling. Another stress response that is activated by UVB light is the ribotoxic stress response (RSR), which depends on the ribosome-associated mitogen-activated protein 3 kinases (MAP3K) ZAKα and culminates in p38 and JNK activation. Using ZAK knockout mice, we here show that it is the RSR that is responsible for the early manifestation of UVB-induced skin inflammation and keratinocyte death and subsequent proliferation invivo. We also show that the RSR controls both p38-mediated pyroptotic and JNK-mediated apoptotic programmed cell death of human keratinocytes invitro. In sum, our work highlights that skin cells rely on a cytoplasmic and ribosomal stress signal rather than a nuclear and DNA-templated signal for rapid inflammatory responses to UV exposure.

Leptin on the apical surface inhibits casein production and STAT5 phosphorylation in mammary epithelial cells

Leptin is a peptide hormone present in both the blood and milk. A close relationship between leptin and milk production in lactating mammary glands has been previously reported. However, how leptin influences milk production in lactating mammary glands remains unclear. Also, whether leptin in milk or blood influences mammary epithelial cells (MECs) during lactation needs further investigation. This study investigated the effects of leptin on mouse MECs using a culture model in which MECs produced milk components and formed less permeable tight junctions. Our results showed that β-casein production in MEC was inhibited by leptin in a concentration-dependent manner. Leptin also inactivated the signal transducer and activator of transcription 5 (STAT5), a transcription factor that facilitates milk production in MECs. Leptin treatment induced the activation of p38 and c-Jun N-terminal kinase (JNK) in MEC before STAT5 inactivation, and anisomycin, an activator of p38 and JNK, induced the inactivation of STAT5. Furthermore, leptin exposure on the apical surface of MECs inhibited β-casein production and inactivated STAT5. However, leptin exposure on the basolateral surface hardly caused these effects. These findings suggested that milk leptin, but not plasma leptin, inhibited milk production in MECs.

IL-4 Downregulates PD-L1 Level Via SOCS1 Upregulation-Induced JNK Deactivation to Enhance Antitumor Immunity in In Vitro Colorectal Cancer.

Interleukin-4 (IL-4) controls cell growth and immune system regulation in tumorigenesis and can inhibit the growth of colon cancer cell lines, but the possible mechanism is unclear. In this study, we investigated the possible mechanism of IL-4 in colorectal cancer (CRC) through in vitro experiments. CRC cells received treatment with IL-4 (50 ng/mL), investigating the suppressor of cytokine signaling 1 (SOCS1)-related mechanism underlying the role of IL-4 in the progression and immunosuppression of CRC. The malignant processes of CRC cells and CD8+T cell-mediated immune response in CRC cells were determined by CCK-8, Transwell, wound healing, and flow cytometry assays. Programmed death ligand 1 (PD-L1), SOCS1 expressions, and c-Jun N-terminal kinase (JNK) activation in CRC cells were analyzed by quantitative reverse transcription polymerase chain reaction and/or Western blot. IL-4 repressed the malignant processes, yet promoted the apoptosis of CRC cells. Besides, IL-4 downregulated PD-L1 level, upregulated SOCS1 level, and restrained JNK activation in CRC cells, while enhancing CRC cell-killing effect of CD8+T cells. IL-4-induced effects on the aforementioned malignant processes of CRC cells and the killing effect of CD8+T cells toward CRC cells were all reversed when SOCS1 was knocked down in the CRC cells. IL-4 downregulates PD-L1 level via SOCS1 upregulation-induced JNK deactivation to enhance antitumor immunity in in vitro CRC. The study provides a theoretical basis for the clinical application of IL-4 in antitumor immunity in CRC.

Mechanism of Marinobufagenin-Induced Hyperpermeability of Human Brain Microvascular Endothelial Cell Monolayer: A Potential Pathogenesis of Seizure in Preeclampsia.

Preeclampsia (preE) is a hypertensive disorder in pregnancies. It is the third leading cause of mortality among pregnant women and fetuses worldwide, and there is much we have yet to learn about its pathophysiology. One complication includes cerebral edema, which causes a breach of the blood-brain barrier (BBB). Urinary marinobufagenin (MBG) is elevated in a preE rat model prior to developing hypertension and proteinuria. We investigated what effect MBG has on the endothelial cell permeability of the BBB. Human brain microvascular endothelial cells (HBMECs) were utilized to examine the permeability caused by MBG. The phosphorylation of ERK1/2, Jnk, p38, and Src was evaluated after the treatment with MBG. Apoptosis was evaluated by examining caspase 3/7. MBG ≥ 1 nM inhibited the proliferation of HBMECs by 46-50%. MBG induced monolayer permeability, causing a decrease in the phosphorylation of ERK1/2 and the activated phosphorylation of Jnk, p38, and Src. MBG increased the caspase 3/7 expression, indicating the activation of apoptosis. Apoptotic signaling or the disruption of endothelia tight junction proteins was not observed when using the p38 inhibitor as a pretreatment in MBG-treated cells. The MBG-induced enhancement of the HBMEC monolayer permeability occurs by the downregulation of ERK1/2, the activation of Jnk, p38, Src, and apoptosis, resulting in the cleavage of tight junction proteins, and are attenuated by p38 inhibition.

The role of cellular senescence in aortic dissection

Abstract Background Aortic dissection (AD) is a catastrophic disease with high mortality. Survived patients frequently suffer from serious complications due to progressive destruction of the aortic walls. Recently we have reported that cell proliferation precedes the inflammatory response which is important in AD development and progression. Since cell proliferation promotes cellular senescence that can induce inflammatory response through SASP (senescence-associated secretary phenotype), we hypothesized that cellular senescence plays a critical role in AD pathogenesis. Purpose To investigate if cellular senescence contributes to AD pathogenesis in mouse AD model. Methods and Results A mouse AD model was created by the continuous infusion of beta-aminopropionitrile and angiotensin II (BAPN+AngII) for 14days. BAPN+AngII infusion induced senescence markers and senescent cells in mouse AD model. Senescent cells, as demonstrated by the expression of senescence-associated beta-galactosidase, were evident in intimal endothelial cells, medial smooth muscle cells, adventitial macrophages, and fibroblasts. Rapamycin, an inhibitor of mTOR pathway, suppressed the expression of senescent cells on the aortic tissue and the senescent marker protein expression. To examine the role of cellular senescence in AD, we orally administrated ABT263 known as "senolytics" that eliminates senescent cells. ABT263 treatment prevented cellular senescence in mouse AD model. The AD mortality of ABT263 group was 35%, which was lower than that of vehicle group (66.7%, P < 0.05 by log-rank test). The severity of AD, as assessed by the lesion length in vehicle group was 33.2 ± 3.1mm, whereas that in ABT263 group was 24.6 ± 1.8mm (P < 0.05). Transcriptome analysis revealed that ABT263 treatment suppressed the immune and inflammatory response in the aorta before AD onset. Quantitative RT-PCR confirmed that ABT263 treatment prevented the induction of p21Cip1, interleukin-6, several chemokines and M1 macrophage marker CD80 and CD86. It is known that Jnk activation induces SASP and accelerates cellular senescence. Western blotting confirmed that ABT263 reduced the ratio of pJnk to total Jnk, which was induced by BAPN + AngⅡ. Moreover, aortic smooth muscle cells（SMCs）remained contractile phenotype in ABT263 group, whereas BAPN+AngII reduced that phenotype in vehicle group. Conclusions Cell proliferation via mTOR pathway causes cellular senescence in mouse AD model. Jnk activation was confirmed in mouse AD model. Cellular senescence contributes to AD progression and death associated with inflammatory responses including SASP, the differentiation to M1 macrophage, and the phenotype change of SMCs.Cellular senescence represents a potential predictor and a therapeutic target for AD.

The novel role of complement 3 in Wnt5a-mediated vascular smooth muscle cell calcification

Abstract Background Although vascular calcification (VC) is a risk factor for cardiovascular and all-cause mortality, a therapy is not yet available. VC involves the transdifferentiation of vascular smooth muscle cells (VSMC) towards an osteochondrogenic lineage that results in calcium phosphate salts deposition in the arterial wall. We have previously shown an association between plasma WNT5A levels and new onset and progression of coronary artery calcification. Purpose We aimed to study the poorly understood role of Wnt5a in VSMC calcification. Methods Study 1 included 18 patients with coronary artery disease (CAD) undergoing cardiac surgery; VSMC were isolated from their saphenous veins to perform in vitro assays and microarrays. Study 2 included 11 patients with CAD; IMA were treated ex vivo with Wnt5a recombinant protein (rWnt5a) to perform western blot (WB) analyses. Study 3 included 647 patients with CAD; RNA sequencing was performed on samples from their internal mammary arteries (IMA), IMA perivascular adipose (PVAT), and thoracic adipose tissue (ThAT). VSMC and human aortic smooth muscle cells (HASMC) from healthy donors were grown in osteogenic medium in the presence or absence of rWnt5a with or without specific protein inhibitors. Calcium deposition and expression of VC markers (i.e. RUNX2, BMP2, and MSX2) were assessed with a colorimetric assay and by RT-qPCR, respectively. Phosphorylation/activation of non-canonical Wnt pathway effectors was evaluated by WB analyses. Results rWnt5a treatment significantly increased calcium deposition in both VSMC and HASMC at a concentration that activates both JNK and CaMKII, that are effectors of the non-canonical planar cell polarity pathway and the calcium-dependent pathway, respectively. Inhibition of either JNK or CaMKII could not counteract rWnt5a-mediated increase in VSMC calcium deposition; whereas, inhibition of both non-canonical pathway effectors (JNK-i and CaMKII-i) prevented rWnt5a-mediated increase in calcium deposition by VSMC (A) and HASMC. Inhibition of JNK and CaMKII also prevented rWnt5a-induced gene expression of VC markers, such as RUNX2 (B). rWnt5a treatment in IMA increased the activation of JNK and CaMKII. Patients with higher WNT5A levels in IMA had significantly higher RUNX2 levels. By intersecting RNA sequencing and microarray data complement 3 (C3) resulted as the most upregulated gene in rWnt5a-treated VSMC among those genes whose levels positively correlated in IMA with both WNT5A and RUNX2 expression (C). Patients with higher WNT5A levels in IMA, their PVAT, and ThAT had significantly higher C3 levels (D), and patients with higher levels of C3AR1, that is the gene coding for the C3a receptor, had higher VC markers expression. C3 inhibition (C3-i) with a C3AR1 antagonist counteracted Wnt5a-induced VSMC calcium deposition (E). Conclusions Non-canonical Wnt signalling promotes VSMC calcification through C3. Wnt5a may be a therapeutic target in VC.Abstract Figure