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Weixu Luo,1,* Yuanshan Han,2,* Pan Meng,1 Qin Yang,3 Hongqing Zhao,1 Jia Ling,1 Yuhong Wang1 1Institute of Innovation and Applied Research in Chinese Medicine, Hunan University of Chinese Medicine, Changsha, Hunan, People’s Republic of China; 2Medical Experimental Innovation Center, The First Affiliated Hospital, Hunan University of Chinese Medicine, Changsha, Hunan, People’s Republic of China; 3Department of Pharmacy, Yueyang Hospital of Traditional Chinese Medicine, Yueyang, Hunan, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yuhong WangInstitute of Innovation and Applied Research in Chinese Medicine, Hunan University of Chinese Medicine, Changsha, Hunan 410208, People’s Republic of ChinaEmail wyhwyh1007@126.comBackground: Breast cancer is one of the most common cancer with high risk in females all over the world. It is usually complicated with depression, which can further accelerate the development and progression of breast tumors. We aim to identify a new drug and identify its functional mechanism in the regulation of hippocampal microglia (MG) in breast cancer complicated with depression (BCCD).Methods: The activation model of MG was established by treatments from corticosterone (CORT) or lipopolysaccharides (LPS). The inhibitory effects of resatorvid on MG were investigated by CCK-8, ROS, immunofluorescence, TUNEL, scratch test, ELISA, RT-qPCR and Western blot. BCCD animal model was established using 4T1 inflammatory breast cancer cells and CORT treatment in vitro. Open field experiment (OFE), tail suspension test (TST), ELISA, RT-qPCR and Western blot experiments were utilized to examine the effects of resatorvid on the animal model in vivo.Results: The cell viability and migration ability of the BCCD model group were suppressed. The expressions of inflammatory factors, ROS, and the apoptotic rate of the BCCD model group were up-regulated, in contrast to the control group. The expressions related to the TLR4/NF-κB/NLRP3 signaling in the BCCD model group were also elevated. Resatorvid reversed the above changes, which showed good therapeutic effects in depression-related behavioral changes, tumor treatment, and blood–brain barrier function.Conclusion: In summary, resatorvid inhibited the activation of hippocampal MG in BCCD by regulating TLR4/NF-κB/NLRP3 signaling pathway.Keywords: resatorvid, breast cancer complicated with depression(BCCD), microglia(MG), TLR4/NF-κB/NLRP3 signaling pathway

To explore the potential role of HMGB1 on TDI-induced NLRP3 inflammasome activation, HBE cells were treated with TDI-HSA conjugate to observe the changes of HMGB1, TLR4, NF-κB, Nrf2 and NLRP3 inflammasome related proteins expressions, ROS release and MMP. NAC, TPCA-1 and Resatorvid pre-treatments were applied to explore the effects of ROS, NF-κB and TLR4 on TDI-induced NLRP3 inflammasome activation. The CRISPR/Cas9 system was used to construct HMGB1 gene knockout HBE cell line and then to explore the role of HMGB1 on TDI-HSA induced NLRP3 inflammasome activation. GL pre-treatment was applied to further confirm the role of HMGB1. Results showed that TDI increased HMGB1, TLR4, P-p65, Nrf2 proteins expressions and ROS release, decreased MMP level and activated NLRP3 inflammasome in HBE cells in a dose dependent manner. NAC, TPCA-1 and Resatorvid pre-treatments decreased the expression of P-p65 and inhibited NLRP3 inflammasome activation. Inhibition of HMGB1 decreased Nrf2 expression and ROS release, improved MMP level and reduced NLRP3 inflammasome activation. GL ameliorated NLRP3 inflammasome activation via inhibiting HMGB1 regulated ROS/NF-κB pathway. These results indicated that HMGB1 was involved in TDI-induced NLRP3 inflammasome activation as a positive regulatory mechanism. The study provided a potential target for early prevention and treatment of TDI-OA.

Background In recent years, more and more attention has been paid to repetitive mild TBI (rmTBI), which can increase the incidence of chronic neurodegenerative disorders. Microglia are the main mediators of the innate immune response in the central nervous system (CNS), and their polarization phenotype plays a dual role in exerting beneficial and detrimental effects on neuroinflammation. This study investigated the mechanism and effect of Resatorvid (TAK242), a specific inhibitor of TLR4, on learning and memory in mice with rmTBI. Methods A controlled cortical impact (CCI) method was used to establish the mild TBI model in this study. Mice in the rmTBI model underwent four head impacts with a 24-hour interval between each impact. Results TAK242 treatment significantly reduced the expression levels of APP and p-Tau, promoted neurological recovery, and improved learning and memory after rmTBI. Furthermore, TAK242 promoted the polarization of microglia from the M1 to M2 phenotype, accompanied by the upregulation of anti-inflammatory factors and downregulation of pro-inflammatory factors. The inhibition of the TLR4/MyD88/NF-κB signalling pathway might be involved in the protective effect of TAK242 mentioned above. Conclusions TAK242 significantly inhibits the neuroinflammatory response by regulating microglial M1/M2 polarization, thereby improving cognitive function after rmTBI.

Background Breast cancer is one of the most common cancer with high risk in females all over the world. It is usually complicated with depression, which can further accelerate the development and progression of breast tumors. We aim to identify a new drug and identify its functional mechanism in the regulation of hippocampal microglia (MG) in breast cancer complicated with depression (BCCD). Methods The activation model of MG was established by treatments from corticosterone (CORT) or lipopolysaccharides (LPS). The inhibitory effects of resatorvid on MG were investigated by CCK-8, ROS, immunofluorescence, TUNEL, scratch test, ELISA, RT-qPCR and Western blot. BCCD animal model was established using 4T1 inflammatory breast cancer cells and CORT treatment in vitro. Open field experiment (OFE), tail suspension test (TST), ELISA, RT-qPCR and Western blot experiments were utilized to examine the effects of resatorvid on the animal model in vivo. Results The cell viability and migration ability of the BCCD model group were suppressed. The expressions of inflammatory factors, ROS, and the apoptotic rate of the BCCD model group were up-regulated, in contrast to the control group. The expressions related to the TLR4/NF-κB/NLRP3 signaling in the BCCD model group were also elevated. Resatorvid reversed the above changes, which showed good therapeutic effects in depression-related behavioral changes, tumor treatment, and blood-brain barrier function. Conclusion In summary, resatorvid inhibited the activation of hippocampal MG in BCCD by regulating TLR4/NF-κB/NLRP3 signaling pathway.

Secondary brain damage caused by hyperactivation of autophagy and inflammatory responses in neurons plays an important role in hypoxic-ischemic brain damage (HIBD). Although previous studies have implicated Toll-like receptor 4 (TLR4) and nuclear factor kappa-B (NF-κB) in the neuroinflammatory response elicited by brain injury, the role and mechanisms of the TLR4-mediated autophagy signaling pathway in neonatal HIBD are still unclear. We hypothesized that this pathway can regulate brain damage by modulating neuron autophagy and neuroinflammation in neonatal rats with HIBD. Hence, we established a neonatal HIBD rat model using the Rice-Vannucci method, and injected 0.75, 1.5, or 3 mg/kg of the TLR4 inhibitor resatorvid (TAK-242) 30 minutes after hypoxic ischemia. Our results indicate that administering TAK-242 to neonatal rats after HIBD could significantly reduce the infarct volume and the extent of cerebral edema, alleviate neuronal damage and neurobehavioral impairment, and decrease the expression levels of TLR4, phospho-NF-κB p65, Beclin-1, microtubule-associated protein l light chain 3, tumor necrosis factor-α, and interleukin-1β in the hippocampus. Thus, TAK-242 appears to exert a neuroprotective effect after HIBD by inhibiting activation of autophagy and the release of inflammatory cytokines via inhibition of the TLR4/NF-κB signaling pathway. This study was approved by the Laboratory Animal Ethics Committee of Affiliated Hospital of Yangzhou University, China (approval No. 20180114-15) on January 14, 2018.

Systemic sclerosis (SSc) is a poorly understood heterogeneous condition with progressive multi-organ fibrosis. Recent genetic and genomic evidence suggest a pathogenic role for dysregulated innate immunity and toll-like receptor (TLR) activity in SSc. Levels of both TLR4, as well as certain endogenous TLR ligands, are elevated in skin and lung tissues from patients with SSc and correlate with clinical disease parameters. Conversely, genetic targeting of TLR4 or its endogenous “damage-associated” ligands ameliorates progressive tissue fibrosis. Targeting TLR4 signaling therefore represents a pharmacological strategy to prevent intractable fibrosis. We examined the effect of TAK242, a small molecule TLR4 inhibitor, in preclinical fibrosis models and in SSc fibroblasts. TAK242 treatment prevented, promoted regression of, bleomycin-induced dermal and pulmonary fibrosis, and reduced the expression of several pro-fibrotic mediators. Furthermore, TAK242 ameliorated peritoneal fibrosis and reduced spontaneous hypodermal thickness in TSK/+ mice. Importantly, TAK242 abrogated collagen synthesis and myofibroblasts differentiation in explanted constitutively active SSc fibroblast. Altogether, these findings identify TAK242 as an anti-fibrotic agent in preclinical models of organ fibrosis. TAK242 might potentially represent a novel strategy for the treatment of SSc and other fibrotic diseases.

An urgent need exists for the development of more efficacious molecular strategies targeting non-melanoma skin cancer (NMSC), the most common malignancy worldwide. Inflammatory signaling downstream of Toll-like receptor 4 (TLR4) is a causative factor in several forms of tumorigenesis, yet its role in solar UV-induced skin carcinogenesis remains undefined. In our recently published work based upon immunohistochemical (IHC) analysis of NMSC tissue microarrays and proteomic analysis of reverse-phase protein microarrays (RPPA) from banked human tissue, we observed increased TLR4 expression in association with tumorigenic progression from normal skin to actinic keratosis and cutaneous squamous cell carcinoma. In immortalized keratinocytes expressing luciferase reporter constructs, resatorvid potently inhibits UV-induced AP-1 and NF-κB signaling, associated with downregulation of inflammatory mediators (IL-6, IL-8, IL-10) and MAP Kinase phosphorylation. In a subsequent acute exposure model in SKH-1 mice, topical resatorvid efficiently antagonized UV-induced stress signaling while potentiating UV-induced epidermal apoptosis. In the current report, we show for the first time that pharmacological inhibition of TLR4 using the specific antagonist resatorvid (TAK-242) blocks UV-induced tumorigenesis in SKH-1 mice. After assessing photostability, efficient cutaneous delivery, and skin residence time of topical resatorvid, we then tested the feasibility of TLR4-directed inhibition of UV-induced tumorigenesis. To this end, SKH-1 mice were split into three groups (n = 20). Each group was exposed to solar-simulated UV (three times a week; 15 weeks duration), followed by another 10 weeks in the absence of UV exposure (sacrifice at week 25). The control group received topical vehicle (acetone) on their backs 1 hour prior to UV exposure (three times a week until sacrifice). The “Prevention” group received topical resatorvid following an analogous dosing regimen. The “Intervention” group received vehicle 1hr prior to each UV treatment but switched to topical resatorvid three times a week only after UV was terminated. In the Prevention model, topical treatment with resatorvid significantly inhibited both tumor area (66% reduction, p = 0.0052) and tumor multiplicity (32% reduction, p = 0.0329; cross sectional Kruskal-Wallis test). In the Intervention model, a marked inhibition of both measures of tumor yield was observed, yet they did not reach statistical significance. Likewise, suppression of UV-induced TLR4-driven signaling was also confirmed in murine skin harvested at week 14 (prior to tumor onset), as assessed by IHC and RPPA analysis. Taken together, these data generated using resatorvid in a murine photocarcinogenesis model suggest that pharmacological TLR4 antagonism may represent a novel molecular strategy for topical prevention of solar UV-induced NMSC. Citation Format: Sally E. Dickinson, Karen Blohm-Mangone, Nichole B. Burkett, Shekha Tahsin, Paul B. Myrdal, Kelly L. Karlage, Jaroslav Janda, Kathylynn Saboda, Denise J. Roe, Zigang Dong, Ann M. Bode, Emanuel F. Petricoin, Valerie S. Calvert, Clara Curiel-Lewandrowski, David S. Alberts, Georg T. Wondrak. Pharmacological TLR4 antagonism using topical resatorvid blocks solar UV-induced skin tumorigenesis in SKH-1 mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2244. doi:10.1158/1538-7445.AM2017-2244

Currently the treatment mainstay of sepsis is early and appropriate antibiotic therapy, accompanied by aggressive fluid administration, the use of vasopressors when needed and the prompt initiation of measures to support each failing organ. Activated protein C and hydrocortisone, when used accordingly can affect mortality. As the pathophysiologic events that take place during sepsis are being elucidated, new molecules that target each step of those pathways are being tested. However, a lot of those molecules affect various mediators of the sepsis cascade including inflammatory cytokines, cellular receptors, nuclear transcription factors, coagulation activators and apoptosis regulators. Over the last decade, a multitude of clinical trials and animal studies have investigated strategies that aimed to restore immune homeostasis either by reducing inflammation or by stimulating the innate and adaptive immune responses. Antibiotics, statins and other molecules with multipotent immunomodulatory actions have also been studied in the treatment of sepsis. © 2011 Expert Reviews Ltd. 9 11 1013 1033