Nintedanib Regulates GRK2 and CXCR2 to Reduce Neutrophil Recruitment in Endotoxin-Induced Lung Injury

The role of nintedanib, a multiple tyrosine kinase inhibitor, in the treatment of sepsis-induced acute lung injury (ALI) remains unclear. Lipopolysaccharide (LPS), also known as endotoxin, has been used to induce ALI. The goal of this study was to assess the effect of nintedanib in attenuating the histopathological changes of LPS-induced ALI. Nintedanib was administered via oral gavage to male C57BL/6 mice 24 h and 10 min before intratracheal endotoxin instillation. Lung histopathological characteristics, adhesion molecule expression, and the regulatory signaling pathways of neutrophil chemotaxis were analyzed after 24 h. We found that nintedanib significantly reduced histopathological changes and neutrophil recruitment in LPS-induced ALI. The number of neutrophils in bronchoalveolar lavage fluid (BALF) was reduced in nintedanib-treated relative to untreated mice with ALI. Nintedanib mediated the downregulation of the chemotactic response to LPS by reducing the expression of adhesion molecules and the phosphorylated p38:total p38 mitogen-activated protein kinase (MAPK) ratio in the lungs of mice with ALI. Nintedanib also reduced the expression of lymphocyte antigen 6 complex locus G6D (Ly6G) and very late antigen 4 (VLA-4) in BALF neutrophils and mediated the downregulation of chemokine (C-X-C motif) receptor 2 (CXCR2) and upregulation of G protein-coupled receptor kinase 2 (GRK2) activity in peripheral blood neutrophils in mice with LPS-induced ALI. Nintedanib improved the histopathological changes of LPS-induced ALI by reducing neutrophil chemotaxis. These effects were mediated by the inhibition of adhesion molecules via the activation of GRK2 and the inhibition of p38 MAPK and CXCR2.     

Immunomodulation via MyD88-NFκB Signaling Pathway from Human Umbilical Cord-Derived Mesenchymal Stem Cells in Acute Lung Injury

Excess inflammatory processes play a key detrimental role in the pathophysiology of acute lung injury (ALI). Mesenchymal stem cells (MSCs) were reported to be beneficial to ALI, but the underlying mechanisms have not been completely understood. The present study aimed to examine the involvement of MyD88–NFκB signaling in the immunomodulation of MSCs in mice with lipopolysaccharides (LPS)-induced ALI. We found that serum concentrations of IL-6, TNF-α, MCP-1, IL-1β, and IL-8 were significantly decreased at 6 h after LPS-induced ALI in the MSC group (p < 0.05). For each of the five cytokines, the serum concentration of each individual mouse in either group declined to a similar level at 48 h. The intensity of lung injury lessened in the MSC group, as shown by histopathology and lung injury scores (p < 0.001). The expressions of MyD88 and phospho-NFκB in the lung tissue were significantly decreased in mice receiving MSCs as measured by Western blotting and immunohistochemistry. Our data demonstrated that human umbilical cord-derived MSCs could effectively alleviate the cytokine storm in mice after LPS-induced ALI and attenuated lung injury. Firstly, we documented the correlation between the down-regulation of MyD88–NFκB signaling and immunomodulatory effects of MSCs in the situation of ALI.     

The Fab Fragment of a Humanized Anti-Toll Like Receptor 4 (TLR4) Monoclonal Antibody Reduces the Lipopolysaccharide Response via TLR4 in Mouse Macrophages

Lipopolysaccharides (LPS) can induce acute inflammation, sepsis, or chronic inflammatory disorders through the Toll receptor 4 (TLR4) signaling pathway. The TLR4/MD2 (myeloid differentiation protein 2) complex plays a major role in the immune response to LPS. However, there is not a good method to suppress the immune response induced by LPS via this complex in macrophages. In this article, we aimed to evaluate the effects of humanized anti-TLR4 monoclonal antibodies on LPS-induced responses in mouse macrophages. The peritoneal macrophages of mice were incubated with anti-TLR4 monoclonal antibodies and stimulated with LPS. The expression levels of cytokines were analyzed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assays. Additionally, activation of various signaling pathways was evaluated by Western blotting. The results showed that the humanized anti-TLR4 monoclonal antibody blocked the inflammatory cytokines expression at both the mRNA and protein level. We also found that the Fab fragment significantly inhibited the nuclear factor kappaB signaling pathway by reducing the phosphorylation of the inhibitor of kappaBalpha and decreasing the translocation of p65, resulting in the suppression of p38, extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase 1/2, and IFN-β regulatory factor 3 phosphorylation. Therefore, our study showed that this humanized anti-TLR4 monoclonal antibody could effectively protect against LPS-induced responses by blocking the TLR4 signaling pathway in mouse peritoneal macrophages.     

Neuroprotective Effect of Bcl-2 on Lipopolysaccharide-Induced Neuroinflammation in Cortical Neural Stem Cells

Neuroinflammation is involved in the pathogenesis of neurodegenerative diseases due to increased levels of pro-inflammatory cytokines in the central nervous system (CNS). Chronic neuroinflammation induced by neurotoxic molecules accelerates neuronal damage. B-cell lymphoma 2 (Bcl-2) is generally accepted to be an important anti-apoptotic factor. However, the role of Bcl-2 in neuroprotection against neuroinflammation remains to be determined. The purpose of this study was to investigate the neuroprotective effect of Bcl-2 on lipopolysaccharide (LPS)-induced neuroinflammation in cortical neural stem cells (NSCs). LPS decreased mRNA and protein levels of Tuj-1, a neuron marker, and also suppressed neurite outgrowth, indicating that LPS results in inhibition of neuronal differentiation of NSCs. Furthermore, LPS treatment inhibited Bcl-2 expression during neuronal differentiation; inhibition of neuronal differentiation by LPS was rescued by Bcl-2 overexpression. LPS-induced pro-inflammatory cytokines, including interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α), were decreased by Bcl-2 overexpression. Conversely, Bcl-2 siRNA increased the LPS-induced levels of IL-6 and TNF-α, and decreased neuronal differentiation of NSCs, raising the possibility that Bcl-2 mediates neuronal differentiation by inhibiting the LPS-induced inflammatory response in NSC. These results suggest that Bcl-2 has a neuroprotective effect by inhibiting the LPS-induced inflammatory response in NSCs.     

Dexmedetomidine Promotes Lipopolysaccharide-Induced Differentiation of Cardiac Fibroblasts and Collagen I/III Synthesis through α2A Adrenoreceptor-Mediated Activation of the PKC-p38-Smad2/3 Signaling Pathway in Mice

Dexmedetomidine (DEX), a selective α₂ adrenergic receptor (AR) agonist, is commonly used as a sedative drug during critical illness. In the present study, we explored a novel accelerative effect of DEX on cardiac fibroblast (CF) differentiation mediated by LPS and clarified its potential mechanism. LPS apparently increased the expression of α-SMA and collagen I/III and the phosphorylation of p38 and Smad-3 in the CFs of mice. These effects were significantly enhanced by DEX through increasing α_2A-AR expression in CFs after LPS stimulation. The CFs from α_2A-AR knockout mice were markedly less sensitive to DEX treatment than those of wild-type mice. Inhibition of protein kinase C (PKC) abolished the enhanced effects of DEX on LPS-induced differentiation of CFs. We also found that the α-SMA level in the second-passage CFs was much higher than that in the nonpassage and first-passage CFs. However, after LPS stimulation, the TNF-α released from the nonpassage CFs was much higher than that in the first- and second-passage CFs. DEX had no effect on LPS-induced release of TNF-α and IL-6 from CFs. Further investigation indicated that DEX promoted cardiac fibrosis and collagen I/III synthesis in mice exposed to LPS for four weeks. Our results demonstrated that DEX effectively accelerated LPS-induced differentiation of CFs to myofibroblasts through the PKC-p38-Smad2/3 signaling pathway by activating α_2A-AR.     

Uterine Micro-Environment and Estrogen-Dependent Regulation of Osteopontin Expression in Mouse Blastocyst

Embryo implantation is a highly synchronized bioprocess between an activated blastocyst and a receptive uterus. In mice, successful implantation relies on the dynamic interplay of estrogen and progesterone; however, the key mediators downstream of these hormones that act on blastocyst competency and endometrium receptivity acquisition are largely unknown. In this study, we showed that the expression of osteopontin (OPN) in mouse blastocysts is regulated by ovarian estrogen and uterine micro-environment. OPN mRNA is up-regulated in mouse blastocyst on day 4 of pregnancy, which is associated with ovarian estrogen secretion peak. Hormone treatment in vivo demonstrated that OPN expression in a blastocyst is regulated by estrogen through an estrogen receptor (ER). Our results of the delayed and activated implantation model showed that OPN expression is induced after estrogen injection. While estrogen treatment during embryo culture in vitro showed less effect on OPN expression, the tubal ligation model on day 3 of pregnancy confirmed that the regulation of estrogen on OPN expression in blastocyst might, through some specific cytokines, have existed in a uterine micro-environment. Collectively, our study presents that estrogen regulates OPN expression and it may play an important role during embryo implantation by activating blastocyst competence and facilitating the endometrium acceptable for active blastocyst.     

A Novel 1,8-Naphthyridine-2-Carboxamide Derivative Attenuates Inflammatory Responses and Cell Migration in LPS-Treated BV2 Cells via the Suppression of ROS Generation and TLR4/Myd88/NF-κB Signaling Pathway

Novel 1,8-naphthyridine-2-carboxamide derivatives with various substituents (HSR2101-HSR2113) were synthesized and evaluated for their effects on the production of pro-inflammatory mediators and cell migration in lipopolysaccharide (LPS)-treated BV2 microglial cells. Among the tested compounds, HSR2104 exhibited the most potent inhibitory effects on the LPS-stimulated production of inflammatory mediators, including nitric oxide (NO), tumor necrosis factor-α, and interleukin-6. Therefore, this compound was chosen for further investigation. We found that HSR2104 attenuated levels of inducible NO synthase and cyclooxygenase 2 in LPS-treated BV2 cells. In addition, it markedly suppressed LPS-induced cell migration as well as the generation of intracellular reactive oxygen species (ROS). Moreover, HSR2104 abated the LPS-triggered nuclear translocation of nuclear factor-κB (NF-κB) through inhibition of inhibitor kappa Bα phosphorylation. Furthermore, it reduced the expressions of Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) in LPS-treated BV2 cells. Similar results were observed with TAK242, a specific inhibitor of TLR4, suggesting that TLR4 is an upstream regulator of NF-κB signaling in BV2 cells. Collectively, our findings demonstrate that HSR2104 exhibits anti-inflammatory and anti-migratory activities in LPS-treated BV2 cells via the suppression of ROS and TLR4/MyD88/NF-κB signaling pathway. Based on our observations, HSR2104 may have a beneficial impact on inflammatory responses and microglial cell migration involved in the pathogenesis of various neurodegenerative disorders.     

Acute Effect of Caffeine on the Synthesis of Pro-Inflammatory Cytokines in the Hypothalamus and Choroid Plexus during Endotoxin-Induced Inflammation in a Female Sheep Model

This study was designed to determine the effect of acute caffeine (CAF) administration, which exerts a broad spectrum of anti-inflammatory activity, on the synthesis of pro-inflammatory cytokines and their receptors in the hypothalamus and choroid plexus (ChP) during acute inflammation caused by the injection of bacterial endotoxin—lipopolysaccharide (LPS). The experiment was performed on 24 female sheep randomly divided into four groups: control; LPS treated (iv.; 400 ng/kg of body mass (bm.)); CAF treated (iv.; 30 mg/kg of bm.); and LPS and CAF treated. The animals were euthanized 3 h after the treatment. It was found that acute administration of CAF suppressed the synthesis of interleukin (IL-1β) and tumor necrosis factor (TNF)α, but did not influence IL-6, in the hypothalamus during LPS-induced inflammation. The injection of CAF reduced the LPS-induced expression of TNF mRNA in the ChP. CAF lowered the gene expression of IL-6 cytokine family signal transducer (IL6ST) and TNF receptor superfamily member 1A (TNFRSF1) in the hypothalamus and IL-1 type II receptor (IL1R2) in the ChP. Our study on the sheep model suggests that CAF may attenuate the inflammatory response at the hypothalamic level and partly influence the inflammatory signal generated by the ChP cells. This suggests the potential of CAF to suppress neuroinflammatory processes induced by peripheral immune/inflammatory challenges.     

Suppression of LPS-Induced Inflammation and Cell Migration by Azelastine through Inhibition of JNK/NF-κB Pathway in BV2 Microglial Cells

The c-Jun N-terminal kinases (JNKs) are implicated in many neuropathological conditions, including neurodegenerative diseases. To explore potential JNK3 inhibitors from the U.S. Food and Drug Administration-approved drug library, we performed structure-based virtual screening and identified azelastine (Aze) as one of the candidates. NMR spectroscopy indicated its direct binding to the ATP-binding site of JNK3, validating our observations. Although the antihistamine effect of Aze is well documented, the involvement of the JNK pathway in its action remains to be elucidated. This study investigated the effects of Aze on lipopolysaccharide (LPS)-induced JNK phosphorylation, pro-inflammatory mediators, and cell migration in BV2 microglial cells. Aze was found to inhibit the LPS-induced phosphorylation of JNK and c-Jun. It also inhibited the LPS-induced production of pro-inflammatory mediators, including interleukin-6, tumor necrosis factor-α, and nitric oxide. Wound healing and transwell migration assays indicated that Aze attenuated LPS-induced BV2 cell migration. Furthermore, Aze inhibited LPS-induced IκB phosphorylation, thereby suppressing nuclear translocation of NF-κB. Collectively, our data demonstrate that Aze exerts anti-inflammatory and anti-migratory effects through inhibition of the JNK/NF-κB pathway in BV2 cells. Based on our findings, Aze may be a potential candidate for drug repurposing to mitigate neuroinflammation in various neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases.     

Effect of Placenta-Derived Mesenchymal Stromal Cells Conditioned Media on an LPS-Induced Mouse Model of Preeclampsia

We tested the pro-angiogenic and anti-inflammatory effects of human placenta-derived mesenchymal stromal cells (hPDMSCs)-derived conditioned media (CM) on a mouse model of preeclampsia (PE), a severe human pregnancy-related syndrome characterized by maternal hypertension, proteinuria, endothelial damage, inflammation, often associated with fetal growth restriction (FGR). At d11 of pregnancy, PE was induced in pregnant C57BL/6N mice by bacterial lipopolysaccharide (LPS) intravenous injection. At d12, 300 μL of unconditioned media (control group) or 300 μL PDMSCs-CM (CM group) were injected. Maternal systolic blood pressure was measured from 9 to 18 days of pregnancy. Urine protein content were analyzed at days 12, 13, and 17 of pregnancy. At d19, mice were sacrificed. Number of fetuses, FGR, fetal reabsorption, and placental weight were evaluated. Placentae were analyzed for sFlt-1, IL-6, and TNF-α gene and protein expressions. No FGR and/or reabsorbed fetuses were delivered by PDMSCs-CM-treated PE mice, while five FGR fetuses were found in the control group accompanied by a lower placental weight. PDMSCs-CM injection significantly decreased maternal systolic blood pressure, proteinuria, sFlt-1, IL-6, and TNF-α levels in PE mice. Our data indicate that hPDMSCs-CM can reverse PE-like features during pregnancy, suggesting a therapeutic role for hPDMSCs for the treatment of preeclampsia.     

肺炎链球菌dnaJ基因缺陷对小鼠肺炎感染模型天然免疫应答的影响

目的探讨肺炎链球菌(Streptococcus pneumonia,S.pn)dnaJ基因缺陷对肺炎感染模型小鼠天然免疫应答的影响。方法将BALB/c小鼠随机分为空白对照组、D39感染组和△dnaJ感染组,分别经鼻腔滴注30μl无菌PBS、30μl含2×107cfu的S.pn D39和△dnaJ(dnaJ基因缺陷株)菌液,复制小鼠肺炎感染模型。于感染后6、12、24、36和48 h处死小鼠,取肺组织,匀浆后分离上清,ELISA检测促炎因子TNF-α、IL-1β、IL-6和IFNγ的表达水平,HE染色观察感染12 h的小鼠肺组织炎症反应变化;将小鼠巨噬细胞株RAW264.7体外感染S.pn D39和△dnaJ,细菌与细胞的比例为100∶1,感染1、2、3 h后分离细胞培养上清液,ELISA检测TNF-α和IL-6的表达水平。实时定量PCR和Western blot分别检测感染12 h的小鼠肺组织中模式识别受体TLRs基因mRNA的转录水平和炎症相关信号p38MAPK的磷酸化水平。结果与D39感染组相比,△dnaJ感染组小鼠肺组织炎症反应强度下降,TNF-α、IL-6和IL-1β等促炎因子水平达峰时间延迟,且峰值降低,RAW264.7细胞感染3 h分泌TNF-α和IL-6显著减少(P<0.01或P<0.05);小鼠肺组织Tlr2和Tlr13基因mRNA转录水平显著降低(P<0.05),p38MAPK的磷酸化水平也明显下降。结论肺炎链球菌dnaJ基因缺陷可下调肺炎感染模型小鼠的炎症反应、促炎因子分泌及胞内信号分子的激活,也可影响小鼠感染后肺组织中Trl2和Tlr13基因mRNA的表达,为进一步研究机体对肺炎链球菌DnaJ蛋白的天然免疫应答分子机制奠定了基础。     

Angiotensin-(1-7) Prevents Lipopolysaccharide-Induced Autophagy via the Mas Receptor in Skeletal Muscle

Skeletal muscle atrophy, which occurs in lipopolysaccharide (LPS)-induced sepsis, causes a severe muscle function reduction. The increased autophagy contributes to sepsis-induced skeletal muscle atrophy in a model of LPS injection, increasing LC3II/LC3I ratio, autophagy flux, and autophagosomes. Angiotensin-(1-7) (Ang-(1-7)) has anti-atrophic effects via the Mas receptor in skeletal muscle. However, the impact of Ang-(1-7) on LPS-induced autophagy is unknown. In this study, we determined the effect of Ang-(1-7) on sepsis-induced muscle autophagy. C57BL6 wild-type (WT) mice and mice lacking the Mas receptor (KO Mas) were injected with LPS together with the systemic administration of Ang-(1-7) to determine autophagy in skeletal muscle. We also evaluated autophagy and p38 and c-Jun N-terminal kinase (JNK)activation. Our results show that Ang-(1-7) prevents LPS-induced autophagy in the diaphragm, tibialis anterior, and gastrocnemius of WT mice, which is demonstrated by a decrease in the LC3II/LC3I ratio and mRNA levels of lc3b and ctsl. This effect was lost in KO Mas mice, suggesting the role of the Mas receptor. The results in C2C12 cells show that Ang-(1-7) reduces several LPS-dependent effects, such as autophagy (LC3II/LC3I ratio, autophagic flux, and autophagosomes), activation of p38 and JNK, B-cell lymphoma-2 (BCL2) phosphorylation, and disassembly of the Beclin1/BCL2 complex. In conclusion, Ang-(1-7)/Mas receptor reduces LPS-induced autophagy in skeletal muscle. In vitro assays indicate that Ang-(1-7) prevents LPS-induced autophagy and modifies the MAPK signaling and the disassembly of a complex involved at the beginning of autophagy.     

Norepinephrine Inhibits Lipopolysaccharide-Stimulated TNF-α but Not Oxylipin Induction in n-3/n-6 PUFA-Enriched Cultures of Circumventricular Organs

Sensory circumventricular organs (sCVOs) are pivotal brain structures involved in immune-to-brain communication with a leaky blood–brain barrier that detect circulating mediators such as lipopolysaccharide (LPS). Here, we aimed to investigate the potential of sCVOs to produce n-3 and n-6 oxylipins after LPS-stimulation. Moreover, we investigated if norepinephrine (NE) co-treatment can alter cytokine- and oxylipin-release. Thus, we stimulated rat primary neuroglial sCVO cultures under n-3- or n-6-enriched conditions with LPS or saline combined with NE or vehicle. Supernatants were assessed for cytokines by bioassays and oxylipins by HPLC-MS/MS. Expression of signaling pathways and enzymes were analyzed by RT-PCR. Tumor necrosis factor (TNF)α bioactivity and signaling, IL-10 expression, and cyclooxygenase (COX)2 were increased, epoxide hydroxylase (Ephx)2 was reduced, and lipoxygenase 15-(LOX) was not changed by LPS stimulation. Moreover, LPS induced increased levels of several n-6-derived oxylipins, including the COX-2 metabolite 15d-prostaglandin-J2 or the Ephx2 metabolite 14,15-DHET. For n-3-derived oxylipins, some were down- and some were upregulated, including 15-LOX-derived neuroprotectin D1 and 18-HEPE, known for their anti-inflammatory potential. While the LPS-induced increase in TNFα levels was significantly reduced by NE, oxylipins were not significantly altered by NE or changes in TNFα levels. In conclusion, LPS-induced oxylipins may play an important functional role in sCVOs for immune-to-brain communication.     

Vanillic Acid,a Bioactive Phenolic Compound,Counteracts LPS-Induced Neurotoxicity by Regulating c-Jun N-Terminal Kinase in Mouse Brain

The receptor for advanced glycation end products (RAGE), a pattern recognition receptor signaling event, has been associated with several human illnesses, including neurodegenerative diseases, particularly in Alzheimer’s disease (AD). Vanillic acid (V.A), a flavoring agent, is a benzoic acid derivative having a broad range of biological activities, including antioxidant, anti-inflammatory, and neuroprotective effects. However, the underlying molecular mechanisms of V.A in exerting neuroprotection are not well investigated. The present study aims to explore the neuroprotective effects of V.A against lipopolysaccharides (LPS)-induced neuroinflammation, amyloidogenesis, synaptic/memory dysfunction, and neurodegeneration in mice brain. Behavioral tests and biochemical and immunofluorescence assays were applied. Our results indicated increased expression of RAGE and its downstream phospho-c-Jun n-terminal kinase (p-JNK) in the LPS-alone treated group, which was significantly reduced in the V.A + LPS co-treated group. We also found that systemic administration of LPS-injection induced glial cells (microglia and astrocytes) activation and significantly increased expression level of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB) and secretion of proinflammatory cytokines including tumor necrosis factor alpha (TNF-α), interleukin-1 β (IL1-β), and cyclooxygenase (COX-2). However, V.A + LPS co-treatment significantly inhibited the LPS-induced activation of glial cells and neuroinflammatory mediators. Moreover, we also noted that V.A treatment significantly attenuated LPS-induced increases in the expression of AD markers, such as β-site amyloid precursor protein (APP)–cleaving enzyme 1 (BACE1) and amyloid-β (Aβ). Furthermore, V.A treatment significantly reversed LPS-induced synaptic loss via enhancing the expression level of pre- and post-synaptic markers (PSD-95 and SYP), and improved memory performance in LPS-alone treated group. Taken together; we suggest that neuroprotective effects of V.A against LPS-induced neurotoxicity might be via inhibition of LPS/RAGE mediated JNK signaling pathway; and encourage future studies that V.A would be a potential neuroprotective and neurotherapeutic candidate in various neurological disorders.     

Protective Role of Proton-Sensing TDAG8 in Lipopolysaccharide-Induced Acute Lung Injury

Acute lung injury is characterized by the infiltration of neutrophils into lungs and the subsequent impairment of lung function. Here we explored the role of TDAG8 in lung injury induced by lipopolysaccharide (LPS) administrated intratracheally. In this model, cytokines and chemokines released from resident macrophages are shown to cause neutrophilic inflammation in the lungs. We found that LPS treatment increased TDAG8 expression in the lungs and confirmed its expression in resident macrophages in bronchoalveolar lavage (BAL) fluids. LPS administration remarkably increased neutrophil accumulation without appreciable change in the resident macrophages, which was associated with increased penetration of blood proteins into BAL fluids, interstitial accumulation of inflammatory cells, and damage of the alveolar architecture. The LPS-induced neutrophil accumulation and the associated lung damage were enhanced in TDAG8-deficient mice as compared with those in wild-type mice. LPS also increased several mRNA and protein expressions of inflammatory cytokines and chemokines in the lungs or BAL fluids. Among these inflammatory mediators, mRNA and protein expression of KC (also known as CXCL1), a chemokine of neutrophils, were significantly enhanced by TDAG8 deficiency. We conclude that TDAG8 is a negative regulator for lung neutrophilic inflammation and injury, in part, through the inhibition of chemokine production.