Secreted Signaling Molecules at the Neuromuscular Junction in Physiology and Pathology

Signal transduction at the neuromuscular junction (NMJ) is affected in many human diseases, including congenital myasthenic syndromes (CMS), myasthenia gravis, Lambert–Eaton myasthenic syndrome, Isaacs’ syndrome, Schwartz–Jampel syndrome, Fukuyama-type congenital muscular dystrophy, amyotrophic lateral sclerosis, and sarcopenia. The NMJ is a prototypic cholinergic synapse between the motor neuron and the skeletal muscle. Synaptogenesis of the NMJ has been extensively studied, which has also been extrapolated to further understand synapse formation in the central nervous system. Studies of genetically engineered mice have disclosed crucial roles of secreted molecules in the development and maintenance of the NMJ. In this review, we focus on the secreted signaling molecules which regulate the clustering of acetylcholine receptors (AChRs) at the NMJ. We first discuss the signaling pathway comprised of neural agrin and its receptors, low-density lipoprotein receptor-related protein 4 (Lrp4) and muscle-specific receptor tyrosine kinase (MuSK). This pathway drives the clustering of acetylcholine receptors (AChRs) to ensure efficient signal transduction at the NMJ. We also discuss three secreted molecules (Rspo2, Fgf18, and connective tissue growth factor (Ctgf)) that we recently identified in the Wnt/β-catenin and fibroblast growth factors (FGF) signaling pathways. The three secreted molecules facilitate the clustering of AChRs by enhancing the agrin-Lrp4-MuSK signaling pathway.     

The Multifaceted Roles of USP15 in Signal Transduction

Ubiquitination and deubiquitination are protein post-translational modification processes that have been recognized as crucial mediators of many complex cellular networks, including maintaining ubiquitin homeostasis, controlling protein stability, and regulating several signaling pathways. Therefore, some of the enzymes involved in ubiquitination and deubiquitination, particularly E3 ligases and deubiquitinases, have attracted attention for drug discovery. Here, we review recent findings on USP15, one of the deubiquitinases, which regulates diverse signaling pathways by deubiquitinating vital target proteins. Even though several basic previous studies have uncovered the versatile roles of USP15 in different signaling networks, those have not yet been systematically and specifically reviewed, which can provide important information about possible disease markers and clinical applications. This review will provide a comprehensive overview of our current understanding of the regulatory mechanisms of USP15 on different signaling pathways for which dynamic reverse ubiquitination is a key regulator.     

Eupatilin Suppresses OVA-Induced Asthma by Inhibiting NF-κB and MAPK and Activating Nrf2 Signaling Pathways in Mice

To investigate the effect of eupatilin in asthma treatment, we evaluated its therapeutic effect and related signal transduction in OVA-induced asthmatic mice and LPS-stimulated RAW264.7 cells. The BALF was tested for changes in lung inflammatory cells. Th2 cytokines in the BALF and OVA-IgE in the serum were measured by ELISA. H&E and PAS staining were used to evaluate histopathological changes in mouse lungs. The key proteins NF-κB, MAPK, and Nrf2 in lung tissues were quantitatively analyzed by Western blotting. Finally, we evaluated the effect of eupatilin on cytokines and related protein expression in LPS-stimulated RAW 264.7 cells in vitro. In OVA-induced asthmatic mice, eupatilin reduced the numbers of inflammatory cells, especially neutrophils and eosinophils. Eupatilin also decreased the levels of IL-5, IL-13 in the BALF and OVA-IgE in the serum. Furthermore, eupatilin inhibited the activation of NF-κB and MAPK pathways and increased the expression of Nrf2 in OVA-induced asthmatic mice. In vitro, eupatilin significantly reduced LPS-stimulated NO, IL-6, and ROS production. Additionally, the NF-κB, MAPK, and Nrf2 protein expression in LPS-stimulated RAW264.7 cells was consistent with that in OVA-induced asthmatic lung tissues. In summary, eupatilin attenuated OVA-induced asthma by regulating NF-κB, MAPK, and Nrf2 signaling pathways. These results suggest the utility of eupatilin as an anti-inflammatory drug for asthma treatment.     

Wnt-Signaling Inhibitor Wnt-C59 Suppresses the Cytokine Upregulation in Multiple Organs of Lipopolysaccharide-Induced Endotoxemic Mice via Reducing the Interaction between β-Catenin and NF-κB

Sepsis is characterized by multiple-organ dysfunction caused by the dysregulated host response to infection. Until now, however, the role of the Wnt signaling has not been fully characterized in multiple organs during sepsis. This study assessed the suppressive effect of a Wnt signaling inhibitor, Wnt-C59, in the kidney, lung, and liver of lipopolysaccharide-induced endotoxemic mice, serving as an animal model of sepsis. We found that Wnt-C59 elevated the survival rate of these mice and decreased their plasma levels of proinflammatory cytokines and organ-damage biomarkers, such as BUN, ALT, and AST. The Wnt/β-catenin and NF-κB pathways were stimulated and proinflammatory cytokines were upregulated in the kidney, lung, and liver of endotoxemic mice. Wnt-C59, as a Wnt signaling inhibitor, inhibited the Wnt/β-catenin pathway, and its interaction with the NF-κB pathway, which resulted in the inhibition of NF-κB activity and proinflammatory cytokine expression. In multiple organs of endotoxemic mice, Wnt-C59 significantly reduced the β-catenin level and interaction with NF-κB. Our findings suggest that the anti-endotoxemic effect of Wnt-C59 is mediated via reducing the interaction between β-catenin and NF-κB, consequently suppressing the associated cytokine upregulation in multiple organs. Thus, Wnt-C59 may be useful for the suppression of the multiple-organ dysfunction during sepsis.     

α-Bisabolol Attenuates Doxorubicin Induced Renal Toxicity by Modulating NF-κB/MAPK Signaling and Caspase-Dependent Apoptosis in Rats

Doxorubicin (DOX) is a well-known and effective antineoplastic agent of the anthracycline family. But, multiple organ toxicities compromise its invaluable therapeutic usage. Among many toxicity types, nephrotoxicity is one of the major concerns. In recent years many approaches, including bioactive agents of natural origin, have been explored to provide protective effects against chemotherapy-related complications. α-Bisabolol is a naturally occurring monocyclic sesquiterpene alcohol identified in the essential oils of various aromatic plants and possesses a wide range of pharmacological properties such as antioxidant, anti-inflammatory, analgesic, cardioprotective, antibiotic, anti-irritant, and anticancer activities. The present study aimed to evaluate the effects of α-Bisabolol on DOX-induced nephrotoxicity in Wistar male albino rats. Nephrotoxicity was induced in rats by injecting a single dose of DOX (12.5 mg/kg, i.p.), and the test compound, α-Bisabolol (25 mg/kg) was administered intraperitoneally along with DOX as a co-treatment daily for 5 days. DOX-injected rats showed reduction in body weight along with a concomitant fall in antioxidants and increased lipid peroxidation in the kidney. DOX-injection also increased levels/expressions of proinflammatory cytokines namely tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) and inflammatory mediators like inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and activated nuclear factor kappa-B (NF-κB)/mitogen-activated protein kinases (MAPK) signaling in the kidney tissues. DOX also triggered apoptotic cell death, evidenced by the increased expression of pro-apoptotic markers like BCL2-Associated X Protein (Bax), cleaved caspase-3, caspase- 9, and cytochrome-C) and a decrease in the expressions of anti-apoptotic markers namely B-cell lymphoma 2 (Bcl2) and B-cell lymphoma-extra large (Bcl-xL) in the kidney. These biochemical alterations were additionally supported by light microscopic findings, which revealed structural alterations in the kidney. However, treatment with α-Bisabolol prevented body weight loss, restored antioxidants, mitigated lipid peroxidation, and inhibited the rise in proinflammatory cytokines, as well as favorably modulated the expressions of NF-κB/MAPK signaling and apoptosis markers in DOX-induced nephrotoxicity. Based on the results observed, it can be concluded that α-Bisabolol has potential to attenuate DOX-induced nephrotoxicity by inhibiting oxidative stress and inflammation mediated activation of NF-κB/MAPK signaling alongwith intrinsic pathway of apoptosis in rats. The study findings are suggestive of protective potential of α-Bisabolol in DOX associated nephrotoxicity and this could be potentially useful in minimizing the adverse effects of DOX and may be a potential agent or adjuvant for renal protection.     

Selenium-Rich Yeast Peptide Fraction Ameliorates Imiquimod-Induced Psoriasis-like Dermatitis in Mice by Inhibiting Inflammation via MAPK and NF-κB Signaling Pathways

Psoriasis, a chronic and immune-mediated inflammatory disease, adversely affects patients’ lives. We previously prepared selenium-rich yeast peptide fraction (SeP) from selenium-rich yeast protein hydrolysate and found that SeP could effectively alleviate ultraviolet radiation-induced skin damage in mice and inhibited H₂O₂-induced cytotoxicity in cultured human epidermal keratinocyte (HaCaT) cells. This study aimed to investigate whether SeP had a protective effect on imiquimod (IMQ)-induced psoriasis-like dermatitis in mice and the underlying mechanisms. Results showed that SeP significantly ameliorated the severity of skin lesion in IMQ-induced psoriasis-like mouse model. Moreover, SeP treatment significantly attenuated the expression of key inflammatory cytokines, including interleukin (IL)-23, IL-17A, and IL-17F, in the dorsal skin of mice. Mechanistically, SeP application not only inhibited the activation of JNK and p38 MAPK, but also the translocation of NF-κB into the nucleus in the dorsal skin. Furthermore, SeP treatment inhibited the levels of inflammatory cytokines and the activation of MAPK and NF-κB signaling induced by lipopolysaccharide in HaCaT cells and macrophage cell line RAW264.7. Overall, our findings showed that SeP alleviated psoriasis-like skin inflammation by inhibiting MAPK and NF-κB signaling pathways, which suggested that SeP would have a potential therapeutic effect against psoriasis.     

Pantoprazole Attenuates MAPK (ERK1/2, JNK,p38)–NF-κB and Apoptosis Signaling Pathways after Renal Ischemia/Reperfusion Injury in Rats

Ischemia/reperfusion injury (IRI) in the kidney is the most common cause of acute renal dysfunction through different cell damage mechanisms. This study aimed to investigate, on molecular basics for the first time, the effect of pantoprazole on renal IRI in rats. Different biochemical parameters and oxidative stress markers were assessed. ELISA was used to estimate proinflammatory cytokines. qRT-PCR and western blot were used to investigate the gene and protein expression. Renal histopathological examination was also performed. IRI resulted in tissue damage, elevation of serum levels of creatinine, urea nitrogen, malondialdehyde, TNF-α, IL-6, IL-1β, up-regulation of NF-κB, JNK1/2, ERK1/2, p38, and cleaved caspase-3 proteins. Furthermore, it up-regulated the expression of the Bax gene and down-regulated the expression of the Bcl-2 gene. Treatment of the injured rats with pantoprazole, either single dose or multiple doses, significantly alleviated IRI-induced biochemical and histopathological changes, attenuated the levels of proinflammatory cytokines, down-regulated the expression of NF-κB, JNK1/2, ERK1/2, p38, and cleaved caspase-3 proteins, and the Bax gene, and up-regulated Bcl-2 gene expression. Moreover, treatment with pantoprazole multiple doses has an ameliorative effect that is greater than pantoprazole single-dose. In conclusion, pantoprazole diminished renal IRI via suppression of apoptosis, attenuation of the pro-inflammatory cytokines’ levels, and inhibition of the intracellular signaling pathway MAPK (ERK1/2, JNK, p38)–NF-κB.     

The Interaction between microRNAs and the Wnt/β-Catenin Signaling Pathway in Osteoarthritis

Osteoarthritis (OA) is a chronic disease affecting the whole joint, which still lacks a disease-modifying treatment. This suggests an incomplete understanding of underlying molecular mechanisms. The Wnt/β-catenin pathway is involved in different pathophysiological processes of OA. Interestingly, both excessive stimulation and suppression of this pathway can contribute to the pathogenesis of OA. microRNAs have been shown to regulate different cellular processes in different diseases, including the metabolic activity of chondrocytes and osteocytes. To bridge these findings, here we attempt to give a conclusive overview of microRNA regulation of the Wnt/β-catenin pathway in bone and cartilage, which may provide insights to advance the development of miRNA-based therapeutics for OA treatment.     

Study of Novel Furocoumarin Derivatives on Anti-Vitiligo Activity,Molecular Docking and Mechanism of Action

Vitiligo is a common chronic dermatological abnormality that afflicts tens of millions of people. Furocoumarins isolated from Uygur traditional medicinal material Psoralen corylifolia L. have been proven to be highly effective for the treatment of vitiligo. Although many furocoumarin derivatives with anti-vitiligo activity have been synthesized, their targets with respect to the disease are still ambiguous. Fortunately, the JAKs were identified as potential targets for the disease and its inhibitors have been proved to be effective in the treatment of vitiligo in many clinical trials. Thus, sixty-five benzene sulfonate and benzoate derivatives of furocoumarins (7a–7ad, 8a–8ag) with superior anti-vitiligo activity targeting JAKs were designed and synthesized based on preliminary research. The SAR was characterized after the anti-vitiligo-activity evaluation in B16 cells. Twenty-two derivatives showed more potent effects on melanin synthesis in B16 cells than the positive control (8-MOP). Among them, compounds 7y and 8 not only could increase melanin content, but they also improved the catecholase activity of tyrosinase in a concentration-dependent manner. The docking studies indicated that they were able to interact with amino acid residues in JAK1 and JAK2 via hydrogen bonds. Furthermore, candidate 8 showed a moderate inhibition of CXCL−10, which plays an important role in JAK–STAT signaling. The RT-PCR and Western blotting analyses illustrated that compounds 7y and 8 promoted melanogenesis by activating the p38 MAPK and Akt/GSK-3β/β-catenin pathways, as well as increasing the expressions of the MITF and tyrosinase-family genes. Finally, furocoumarin derivative 8 was recognized as a promising candidate for the fight against the disease and worthy of further research in vivo.     

Prunetinoside Inhibits Lipopolysaccharide-Provoked Inflammatory Response via Suppressing NF-κB and Activating the JNK-Mediated Signaling Pathway in RAW264.7 Macrophage Cells

Inflammation is a multifaceted response of the immune system at the site of injury or infection caused by pathogens or stress via immune cells. Due to the adverse effects of chemical drugs, plant-based compounds are gaining interest in current research. Prunetinoside or prunetin-5-O-glucoside (PUG) is a plant-based active compound, which possesses anti-inflammatory effects on immune cells. In this study, we investigate the effect of PUG on mouse macrophage RAW264.7 cells with or without stimulation of lipopolysaccharide (LPS). Cytotoxicity results showed that PUG is non-cytotoxic to the cells and it reversed the cytotoxicity in LPS-stimulated cells. The levels of nitric oxide (NO) and interleukin-6 (IL-6) were determined using a NO detection kit and IL-6 ELISA kit, respectively, and showed a significant decrease in NO and IL-6 in PUG-treated cells. Western blot and qRT-PCR were performed for the expression of two important pro-inflammatory cytokines, COX2 and iNOS, and found that their expression was downregulated in a dose-dependent manner. Other pro-inflammatory cytokines, such as IL-1β, IL-6, and TNFα, had reduced mRNA expression after PUG treatment. Furthermore, a Western blot was performed to calculate the expression of NF-κB and MAPK pathway proteins. The results show that PUG administration dramatically reduced the phosphorylation of p-Iκbα, p-NF-κB 65, and p-JNK. Remarkably, after PUG treatment, p-P38 and p-ERK remain unchanged. Furthermore, docking studies revealed that PUG is covalently linked to NF-κB and suppresses inflammation. In conclusion, PUG exerted the anti-inflammatory mechanism by barring the NF-κB pathway and activating JNK. Thus, prunetinoside could be adopted as a therapeutic compound for inflammatory-related conditions.     

Lactobacillus brevis BGZLS10-17 and Lb. plantarum BGPKM22 Exhibit Anti-Inflammatory Effect by Attenuation of NF-κB and MAPK Signaling in Human Bronchial Epithelial Cells

Bronchial epithelial cells are exposed to environmental influences, microbiota, and pathogens and also serve as a powerful effector that initiate and propagate inflammation by the release of pro-inflammatory mediators. Recent studies suggested that lung microbiota differ between inflammatory lung diseases and healthy lungs implicating their contribution in the modulation of lung immunity. Lactic acid bacteria (LAB) are natural inhabitants of healthy human lungs and also possess immunomodulatory effects, but so far, there are no studies investigating their anti-inflammatory potential in respiratory cells. In this study, we investigated immunomodulatory features of 21 natural LAB strains in lipopolysaccharide (LPS)-stimulated human bronchial epithelial cells (BEAS-2B). Our results show that several LAB strains reduced the expression of pro-inflammatory cytokine and chemokine genes. We also demonstrated that two LAB strains, Lactobacillus brevis BGZLS10-17 and Lb. plantarum BGPKM22, effectively attenuated LPS-induced nuclear factor-κB (NF-κB) nuclear translocation. Moreover, BGZLS10-17 and BGPKM22 reduced the activation of p38, extracellular signal-related kinase (ERK), and c-Jun amino-terminal kinase (JNK) signaling cascade resulting in a reduction of pro-inflammatory mediator expressions in BEAS-2B cells. Collectively, the LAB strains BGZLS10-17 and BGPKM22 exhibited anti-inflammatory effects in BEAS-2B cells and could be employed to balance immune response in lungs and replenish diminished lung microbiota in chronic lung diseases.     

Corilagin Attenuates Aerosol Bleomycin-Induced Experimental Lung Injury

Idiopathic pulmonary fibrosis (IPF) is a progressing lethal disease with few clinically effective therapies. Corilagin is a tannin derivative which shows anti-inflammatory and antifibrotics properties and is potentiated in treating IPF. Here, we investigated the effect of corilagin on lung injury following bleomycin exposure in an animal model of pulmonary fibrosis. Corilagin abrogated bleomycin-induced lung fibrosis as assessed by H&E; Masson’s trichrome staining and lung hydroxyproline content in lung tissue. Corilagin reduced the number of apoptotic lung cells and prevented lung epithelial cells from membrane breakdown, effluence of lamellar bodies and thickening of the respiratory membrane. Bleomycin exposure induced expression of MDA, IKKα, phosphorylated IKKα (p-IKKα), NF-κB P65, TNF-α and IL-1β, and reduced I-κB expression in mice lung tissue or in BALF. These changes were reversed by high-dose corilagin (100 mg/kg i.p) more dramatically than by low dose (10 mg/kg i.p). Last, corilagin inhibits TGF-β1 production and α-SMA expression in lung tissue samples. Taken together, these findings confirmed that corilagin attenuates bleomycin-induced epithelial injury and fibrosis via inactivation of oxidative stress, proinflammatory cytokine release and NF-κB and TGF-β1 signaling. Corilagin may serve as a promising therapeutic agent for pulmonary fibrosis.     

Pharmacological Effects of Polyphenol Phytochemicals on the Intestinal Inflammation via Targeting TLR4/NF-κB Signaling Pathway

TLR4/NF-κB is a key inflammatory signaling transduction pathway, closely involved in cell differentiation, proliferation, apoptosis, and pro-inflammatory response. Toll like receptor 4 (TLR4), the first mammalian TLR to be characterized, is the innate immune receptor that plays a key role in inflammatory signal transductions. Nuclear factor kappa B (NF-κB), the TLR4 downstream, is the key to accounting for the expression of multiple genes involved in inflammatory responses, such as pro-inflammatory cytokines. Inflammatory bowel disease (IBD) in humans is a chronic inflammatory disease with high incidence and prevalence worldwide. Targeting the TLR4/NF-κB signaling pathway might be an effective strategy to alleviate intestinal inflammation. Polyphenol phytochemicals have shown noticeable alleviative effects by acting on the TLR4/NF-κB signaling pathway in intestinal inflammation. This review summarizes the pharmacological effects of more than 20 kinds of polyphenols on intestinal inflammation via targeting the TLR4/NF-κB signaling pathway. We expected that polyphenol phytochemicals targeting the TLR4/NF-κB signaling pathway might be an effective approach to treat IBD in future clinical research applications.