The classical D1 dopamine receptor antagonist SCH23390 is a functional sigma-1 receptor allosteric modulator.

SCH23390 is a widely used D1 dopamine receptor (D1R) antagonist that also elicits some D1R-independent effects. We previously found that the benzazepine, SKF83959, an analog of SCH23390, produces positive allosteric modulation of the Sigma-1 receptor (Sig1R). SCH23390 does not bind to the orthodoxic site of Sig1R but enhances the binding of 3H (+)-pentazocine to Sig1R. In this study, we investigated whether SCH23390 functions as an allosteric modulator of Sig1R. We detected increased Sig1R dissociation from binding immunoglobulin protein (BiP) and translocation of Sig1R to the plasma membrane in response to SCH23390 in transfected HEK293T and SH-SY5Y cells, respectively. Activation of Sig1R by SCH23390 was further confirmed by inhibition of GSK3ß activity in a time- and dose-dependent manner; this effect was blocked by pretreatment with the Sig1R antagonist, BD1047, and by knockdown of Sig1R. SCH23390 also inhibited GSK3ß in wild-type mice but not in Sig1R knockout mice. Finally, we showed that SCH23390 allosterically modulated the effect of the Sig1R agonist SKF10047 on inhibition of GSK3ß. This positive allosteric effect of SCH23390 was further confirmed via promotion of neuronal protection afforded by SKF10047 in primary cortical neurons challenged with MPP+. These results provide the first evidence that SCH23390 elicits functional allosteric modulation of Sig1R. Our findings not only reveal novel pharmacological effects of SCH23390 but also indicate a potential mechanism for SCH23390-mediated D1R-independent effects. Therefore, attention should be paid to these Sig1R-mediated effects when explaining pharmacological responses to SCH23390.

Fraxetin alleviates BLM-induced idiopathic pulmonary fibrosis by inhibiting NCOA4-mediated epithelial cell ferroptosis.

INTRODUCTION: Idiopathic pulmonary fibrosis (IPF) is a debilitating lung condition with few available treatments. The early driver of wound repair that contributes to IPF has been extensively identified as repetitive alveolar epithelial damage. According to recent reports, IPF is linked to ferroptosis, a unique type of cell death characterized by a fatal buildup of iron and lipid peroxidation. OBJECTIVE AND METHOD: There is little information on epithelial cells that induce pulmonary fibrosis by going through ferroptosis. In this study, we used bleomycin (BLM) to examine the impact of ferroptosis on IPF in mouse lung epithelial cells (MLE-12). RESULTS: We discovered that BLM increases ferroptosis in MLE-12. Additionally, we found that NCOA4 is overexpressed and plays a key role in the ferroptosis of epithelial cells throughout the IPF process. Using Molecular docking, we found that Fraxetin, a natural component extracted from Fraxinus rhynchophylla, formed a stable binding to NCOA4. In vitro investigations showed that Fraxetin administration greatly decreased ferroptosis and NCOA4 expression, which in turn lowered the release of inflammatory cytokines. CONCLUSION: Fraxetin treatment significantly alleviated BLM-induced lung inflammation and fibrosis. Our findings imply that fraxetin possesses inhibitory roles in ferroptosis and can be a potential drug against IPF.

Sigma-1 receptor-regulated efferocytosis by infiltrating circulating macrophages/microglial cells protects against neuronal impairments and promotes functional recovery in cerebral ischemic stroke.

Background: Efferocytosis of apoptotic neurons by macrophages is essential for the resolution of inflammation and for neuronal protection from secondary damage. It is known that alteration of the Sigma-1 receptor (Sig-1R) is involved in the pathological development of some neurological diseases, including ischemic stroke. The present study aimed to investigate whether and how Sig-1R regulates the phagocytic activity of macrophages/microglia and its significance in neuroprotection and neurological function in stroke. Methods: The roles of Sig-1R in the efferocytosis activity of microglia/macrophages using bone marrow-derived macrophages (BMDMs) or using Sig-1R knockout mice subjected to transient middle artery occlusion (tMCAO)-induced stroke were investigated. The molecular mechanism of Sig-1R in the regulation of efferocytosis was also explored. Adoptive transfer of Sig-1R intact macrophages to recipient Sig-1R knockout mice with tMCAO was developed to observe its effect on apoptotic neuron clearance and stroke outcomes. Results: Depletion of Sig-1R greatly impaired the phagocytic activity of macrophages/microglia, accordingly with worsened brain damage and neurological defects in Sig-1R knockout mice subjected to tMCAO. Adoptive transfer of Sig-1R intact bone marrow-derived macrophages (BMDMs) to Sig-1R knockout mice restored the clearance activity of dead/dying neurons, reduced infarct area and neuroinflammation, and improved long-term functional recovery after cerebral ischemia. Mechanistically, Sig-1R-mediated efferocytosis was dependent on Rac1 activation in macrophages, and a few key sites of Rac1 in its binding pocket responsible for the interaction with Sig-1R were identified. Conclusion: Our data provide the first evidence of the pivotal role of Sig-1R in macrophage/microglia-mediated efferocytosis and elucidate a novel mechanism for the neuroprotection of Sig-1R in ischemic stroke.

Development and Evaluation of Novel Metformin Derivative Metformin Threonate for Brain Ischemia Treatment.

Epidemiologic data reveal that diabetes patients taking metformin exhibit lower incidence of stroke and better functional outcomes during post-stroke neurologic recovery. We previously demonstrated that chronic post-ischemic administration of metformin improved functional recovery in experimental cerebral ischemia. However, few beneficial effects of metformin on the acute phase of cerebral ischemia were reported either in experimental animals or in stroke patients, which limits the application of metformin in stroke. We hypothesized that slow cellular uptake of metformin hydrochloride may contribute to the lack of efficacy in acute stroke. We recently developed and patented a novel metformin derivative, metformin threonate (SHY-01). Pharmacokinetic profile in vivo and in cultured cells revealed that metformin is more rapidly uptaken and accumulated from SHY-01 than metformin hydrochloride. Accordingly, SHY-01 treatment exhibited more potent and rapid activation of AMP-activated protein kinase (AMPK). Furthermore, SHY-01 elicited a stronger inhibition of microglia activation and more potent neuroprotection when compared to metformin hydrochloride. SHY-01 administration also had superior beneficial effects on neurologic functional recovery in experimental stroke and offered strong protection against acute cerebral ischemia with reduced infarct volume and mortality, as well as the improved sensorimotor and cognitive functions in rats. Collectively, these results indicated that SHY-01 had an improved pharmacokinetic and pharmacological profile and produced more potent protective effects on acute stroke and long-term neurological damage. We propose that SHY-01 is a very promising therapeutic candidate for cerebral ischemic stroke.

Combined administration of SHP2 inhibitor SHP099 and the α7nAChR agonist PNU282987 protect mice against DSS­induced colitis.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition with complex pathogenesis that currently has no cure. α7 nicotinic acetylcholine receptor (α7nAChR) is known to regulate multiple aspects of immune function. The present study aimed to evaluate the protective effects of PNU282987 and SHP099, which are a selective agonist of α7nAChR and an SHP2 inhibitor, respectively, in dextran sulfate sodium (DSS)­induced colitis in mice. Acute colitis was induced in mice using 3% DSS, and weight loss, colonic histology and cytokine production from colonic lamina propria were analyzed to evaluate disease severity. Bone marrow­derived macrophages were treated with lipopolysaccharide (LPS) to induce an inflammatory response. Cytokine expression and reactive oxygen species (ROS) levels were quantified. The α7nAChR agonist, PNU282987, and the SHP2 inhibitor, SHP099, were administered alone or in combination to LPS­induced macrophages or to colitic model mice to evaluate the inflammatory response and protective efficacy in colitis. α7nAChR protein levels were found to be markedly increased in the colon of DSS­induced colitic mice, and were found to co­localize with macrophages. Consistently, α7nAChR mRNA and protein levels were upregulated with colitis progression in DSS­induced colitic mice. Colonic inflammation was attenuated by PNU282987 treatment in DSS­induced mice, as evidenced by reduced weight loss and alleviated colonic epithelial cell disruption. These effects of PNU282987 on colitis were enhanced when it was combined with SHP099. Cytokine production and ROS levels induced by LPS in macrophages were decreased by a combination treatment of PNU282987 and SHP099. These findings identified α7nAChR as an essential element in the role of intestinal macrophages in colonic repair and demonstrated a synergistic effect of PNU282987 and SHP099, suggesting a new potential therapy for IBD.

Serum amyloid A3 confers protection against acute lung injury in Pseudomonas aeruginosa-infected mice.

Pseudomonas aeruginosa is a gram-negative bacterium associated with serious illnesses, including ventilator-associated pneumonia and various sepsis syndromes in humans. Understanding the host immune mechanisms against P. aeruginosa is, therefore, of clinical importance. The present study identified serum amyloid A3 (SAA3) as being highly inducible in mouse bronchial epithelium following P. aeruginosa infection. Genetic deletion of Saa3 rendered mice more susceptible to P. aeruginosa infection with decreased neutrophil superoxide anion production, and ex vivo treatment of mouse neutrophils with recombinant SAA3 restored the ability of neutrophils to produce superoxide anions. The SAA3-deficient mice showed exacerbated inflammatory responses, which was characterized by pronounced neutrophil infiltration, elevated expression of TNF-α, KC/CXCL1, and MIP-2/CXCL2 in bronchoalveolar lavage fluid (BALF), and increased lung microvascular permeability compared with their wild-type littermates. BALF neutrophils from Saa3 knockout mice exhibited reduced superoxide anion production compared with neutrophils from wild-type mice. Adoptive transfer of SAA3-treated neutrophils to Saa3 knockout mice ameliorated P. aeruginosa-induced acute lung injury. These findings demonstrate that SAA3 not only serves as a biomarker for infection and inflammation, but also plays a protective role against P. aeruginosa infection-induced lung injury in part through augmentation of neutrophil bactericidal functions.

Elevated Expression of Serum Amyloid A 3 Protects Colon Epithelium Against Acute Injury Through TLR2-Dependent Induction of Neutrophil IL-22 Expression in a Mouse Model of Colitis.

Induced expression of serum amyloid A (SAA) is a hallmark of many inflammatory diseases, but whether SAA exacerbates inflammation or protects tissues against injury remains unclear. In dextran sulfate sodium (DSS)-induced colitis, SAA3 is the predominant isoform of inducible SAA proteins that also include SAA1 and SAA2, and mice with genetic deletion of Saa3 exhibits increased production of proinflammatory cytokines, decreased expression of IL-22 along with aggravated epithelium disruption, and reduced colon length compared with wild-type littermates. Colonic neutrophils have been identified as a major source of IL-22 in these mice. Administration of exogenous SAA3 as recombinant protein to Saa3-/- mice improves neutrophil IL-22 production, colonic epithelial integrity, and secretion of the antimicrobial peptides Reg3ß and Reg3γ. Stimulation of mouse bone marrow neutrophils with mouse SAA3 or human SAA1 leads to expansion of IL-22-producing neutrophils. Unlike previously reported IL-22 induction through IL-23, the SAA3-induced neutrophil IL-22 expression utilizes a TLR2-dependent mechanism that does not depend on IL-23. Adoptive transfer of the SAA3-treated neutrophils to Saa3-/- mice ameliorates DSS-induced colitis and improves colonic epithelial integrity. These findings suggest that in the DSS-induced mouse colitis model, SAA isoforms are expressed to different extent in colon and deletion of Saa3 renders these mice more susceptible to DSS-induced injury. The presence of SAA3 in the inflamed colon mucosal serves to protect epithelial barrier in part through expansion of IL-22-producing neutrophils. It is speculated that SAA3 stimulation of autologous neutrophils may have therapeutic potential for inflammatory bowel disease.

4'-Hydroxywogonin suppresses lipopolysaccharide-induced inflammatory responses in RAW 264.7 macrophages and acute lung injury mice.

4'-Hydroxywogonin (4'-HW), a flavonoid, has been isolated from various plants and shown to inhibit NO production in macrophages. However, the molecular mechanisms and its in vivo activity have not been determined. Our study aimed to investigate the mechanisms underlying the anti-inflammatory effects of 4'-HW in vitro and in vivo. We showed that 4'-HW potently reduced the expression levels of COX-2 and iNOS as well as their products, prostaglandin E2 (PGE2) and nitric oxide (NO) respectively, in LPS-stimulated RAW 264.7 macrophages. 4'-HW also suppressed LPS-induced pro-inflammatory cytokines at mRNA and protein levels. Moreover, 4'-HW blocked the interaction of TAK1 and TAB1 in LPS-stimulated RAW 264.7 macrophages, resulting in an inhibition of the TAK1/IKK/NF-κB signaling pathway. Furthermore, 4'-HW also reduced the phosphorylation of MAPKs and PI3/Akt signaling pathways in LPS-stimulated RAW 264.7 macrophages. 4'-HW was also significantly decreased the intracellular reactive oxygen species (ROS) level. The effect of 4'-HW was confirmed in vivo. 4'-HW exhibited potent protective effect against LPS-induced ALI in mice. These findings indicate that 4'-HW suppresses the LPS-induced response in vitro and in vivo. It is likely that the inhibition of the TAK1/IKK/NF-κB, MAPKs and PI3/AKT signaling pathways contribute to the anti-inflammatory effects of 4'-HW. Our study suggests that 4'-HW may be an important functional constituent in the plants and has the potential value to be developed as a novel anti-inflammatory agent.

Suppression of Lipopolysaccharide-Induced Inflammatory Response by Fragments from Serum Amyloid A.

Serum amyloid A (SAA) is known as an acute-phase protein and a biomarker for inflammatory diseases. Published studies have shown that SAA possesses proinflammatory cytokine-like activity and is chemotactic for phagocytes, but the structural basis for these activities remains unidentified. In this article, we report that truncated SAA1 proteins lacking N- and C-terminal sequences exhibit reduced proinflammatory activity and strongly suppress LPS-induced expression of IL-1ß, IL-6, and TNF-α in macrophages. A truncated SAA1 containing aa 11-58 was examined further and found to facilitate p38 MAPK phosphorylation while reducing LPS-stimulated phosphorylation of ERK and JNK. In LPS-challenged mice, aa 11-58 reduced the severity of acute lung injury, with significantly less neutrophil infiltration in the lungs and attenuated pulmonary expression of IL-1ß, IL-6, and TNF-α. Coadministration of aa 11-58 markedly improved mouse survival in response to a lethal dose of LPS. A potent induction of IL-10 was observed in a TLR2-dependent, but TLR4-independent, manner in macrophages stimulated with aa 11-58. However, the aa 11-58 fragment of SAA1 was unable to induce chemotaxis or calcium flux through formyl peptide receptor 2. These results indicate that the N- and C-terminal sequences contain structural determinants for the proinflammatory and chemotactic activities of SAA1, and their removal switches SAA1 to an anti-inflammatory role. Given that proteolytic processing of SAA is associated with the pathological changes in several diseases, including secondary amyloidosis, our findings may shed light on the structure-function relationship of SAA1 with respect to its role in inflammation.

Identification of P-Rex1 as an anti-inflammatory and anti-fibrogenic target for pulmonary fibrosis.

Pulmonary fibrosis (PF) leads to progressive and often irreversible loss of lung functions, posing a health threat with no effective cure. We examined P-Rex1, a PI3K- and G protein ßγ-regulated guanine nucleotide exchange factor (GEF) of the Rac small GTPase, for its potential involvement in PF. In a bleomycin-induced PF model, mice deficient in p-rex1 had well-preserved alveolar structure and survived significantly better than their wild type (WT) littermates. The p-rex1(-/-) mice expressed significantly less proinflammatory cytokines and chemokines and had reduced leukocyte infiltration in the lung tissue than their WT littermates. P-Rex1 was detected in lung fibroblasts of WT mice, and its genetic deletion attenuated TGFß-1-stimulated lung fibroblast migration, Rac1 activation and p38 MAPK phosphorylation. The p-rex1(-/-) mice showed significantly reduced pathological changes including the expression of α-smooth muscle actin, fibronectin and TGFß-1 compared with their WT controls. Expression of a GEF-deficient P-Rex1 mutant effectively blocked Smads-dependent transcriptional activation, suggesting that P-Rex1 is a downstream mediator of TGFß-1 signaling. These findings identify P-Rex1 as a novel player of PF, suggesting that targeting P-Rex1 may simultaneously block the inflammatory and fibrogenic processes of PF.

The protective action of ketanserin against lipopolysaccharide-induced shock in mice is mediated by inhibiting inducible NO synthase expression via the MEK/ERK pathway.

Nitric oxide (NO) plays an important role in the pathogenesis of endotoxic shock. This work tested the hypothesis that ketanserin could attenuate endotoxic shock by inhibiting the expression of inducible NO synthase (iNOS). The results demonstrated that ketanserin could inhibit iNOS expression in the heart, lungs, liver, and kidneys and nitrate production in the serum upon endotoxic shock in mice. In RAW264.7 cells, ketanserin significantly inhibited the expression of iNOS and decreased the production of NO, TNFα, IL-6, and reactive oxygen species upon lipopolysaccharide (LPS) challenge. Ketanserin also increased the level of ATP and mitochondrial membrane potential in RAW264.7 cells upon LPS exposure. LPS-induced iNOS expression was inhibited by the 5-HT2A receptor antagonist ritanserin and not the α1 receptor antagonist prazosin. Knockdown of 5-HT2A receptor by siRNA abolished the inhibitory effect of ketanserin on the expression of iNOS. These results indicated that the inhibitory effect of ketanserin on the expression of iNOS is mediated by blocking the 5-HT2A receptor. Furthermore, ketanserin significantly inhibited the activation of ERK1/2 and NF-κB signal. Pretreatment with PD184352, a specific inhibitor of ERK1/2, blocked the inhibitory effect of ketanserin on the expression of iNOS and NO production, indicating a critical role for the MEK/ERK1/2 signaling pathway. Collectively, our findings indicate that inhibition of the expression of iNOS via the MEK/ERK pathway mediates the protective effects of ketanserin against LPS-induced shock in mice.

Combined administration of anisodamine and neostigmine produces anti-shock effects: involvement of α7 nicotinic acetylcholine receptors.

AIM: To evaluate the anti-effects of anisodamine and neostigmine in animal models of endotoxic and hemorrhagic shock. METHODS: Kunming mice were injected with lipopolysaccharide (LPS 30 mg/kg, ip) to induce endotoxic shock. Anisodamine (12.5, 25, and 50 mg/kg, ip) and neostigmine (12.5, 25, and 50 µg/kg, ip) were administered immediately after LPS injection. Survival rate was monitored, and the serum levels of TNF-α and IL-1ß were analyzed using ELISA assays. The effects of anisodamine and neostigmine were also examined in α7 nicotinic acetylcholine receptor (α7 nAChR) knockout mice with endotoxic shock and in Beagle dogs with hemorrhagic shock. RESULTS: In mice with experimental endotoxemia, combined administration of anisodamine and neostigmine significantly increased the survival rate and decreased the serum levels of inflammatory cytokines, as compared to those produced by either drug alone. The anti-shock effect of combined anisodamine and neostigmine was abolished in α7 nAChR knockout mice. On the other hand, intravenous injection of the combined anisodamine and neostigmine, or the selective α7 nAChR agonist PNU282987 exerted similar anti-shock effects in dogs with hemorrhagic shock. CONCLUSION: The results demonstrate that combined administration of anisodamine and neostigmine produces significant anti-shock effects, which involves activation of α7 nAChRs.

Effects of ketanserin on endotoxic shock and baroreflex function in rodents.

BACKGROUND: Ketanserin, a 5-hydroxytryptamine receptor antagonist, is clinically used as an antihypertensive agent and could enhance baroreflex function. The present work tested the hypothesis that restoration of baroreflex function is an effective treatment for lipopolysaccharide (LPS)-induced shock. METHODS: Kunming mice were injected with LPS (30 mg/kg; intraperitoneal) to induce endotoxic shock. Ketanserin (0.3, 1, 3, or 10 mg/kg; intraperitoneal) was administered immediately after LPS injection. Survival time was monitored, and serum cytokines were analyzed after the onset of LPS. Effects of ketanserin were also examined in IL-10-deficient mice and mice with sinoaortic denervation. Finally, effects of ketanserin on blood pressure, heart rate, and baroreflex sensitivity were examined in Wistar-Kyoto (WKY) rats with endotoxic shock. RESULTS: Ketanserin significantly increased survival time and decreased serum levels of tumor necrosis factor α and interleukin (IL) 1ß in mice with endotoxic shock. At a dose of 10 mg/kg, ketanserin also significantly increased serum IL-10 concentration. The antishock effect of ketanserin was also apparent in IL-10-knockout mice. In mice with sinoaortic denervation, however, ketanserin had little antishock effects. In WKY rats, ketanserin significantly prevented the baroreflex impairment induced by LPS and prolonged the survival time. CONCLUSIONS: Ketanserin could ameliorate endotoxic shock by restoring baroreflex function.