LLDT-8 ameliorates experimental autoimmune encephalomyelitis by mediating macrophage functions in the priming stage.

Multiple sclerosis (MS) is an inflammatory demyelinating disease in the central nervous system caused by T cell activation mediated by peripheral macrophages, resulting in severe neurological deficits and disability. Due to the currently limited and expensive treatments for MS, we here introduce an economic Chinese medicine extract, (5R)-5-Hydroxytriptolide (LLDT-8), which shows low toxicity and high immunosuppressive activity. We used the widely accepted mouse model of MS, experimental autoimmune encephalomyelitis (EAE), to examine the immunosuppressive effect of LLDT-8 in vivo. Through the RNA-sequence analysis of peripheral macrophages in EAE mice, we discovered that LLDT-8 alleviates the symptoms of EAE by inhibiting the proinflammatory effect of macrophages, thereby blocking the activation and proliferation of T cells. In all, we found that LLDT-8 could be a potential treatment for MS.

Infiltrating myeloid cell-derived properdin markedly promotes microglia-mediated neuroinflammation after ischemic stroke.

BACKGROUND: Emerging evidence has shown that myeloid cells that infiltrate into the peri-infarct region may influence the progression of ischemic stroke by interacting with microglia. Properdin, which is typically secreted by immune cells such as neutrophils, monocytes, and T cells, has been found to possess damage-associated molecular patterns (DAMPs) properties and can perform functions unrelated to the complement pathway. However, the role of properdin in modulating microglia-mediated post-stroke neuroinflammation remains unclear. METHODS: Global and conditional (myeloid-specific) properdin-knockout mice were subjected to transient middle cerebral artery occlusion (tMCAO). Histopathological and behavioral tests were performed to assess ischemic brain injury in mice. Single-cell RNA sequencing and immunofluorescence staining were applied to explore the source and the expression level of properdin. The transcriptomic profile of properdin-activated primary microglia was depicted by transcriptome sequencing. Lentivirus was used for macrophage-inducible C-type lectin (Mincle) silencing in microglia. Conditioned medium from primary microglia was administered to primary cortex neurons to determine the neurotoxicity of microglia. A series of cellular and molecular biological techniques were used to evaluate the proinflammatory response, neuronal death, protein-protein interactions, and related signaling pathways, etc. RESULTS: The level of properdin was significantly increased, and brain-infiltrating neutrophils and macrophages were the main sources of properdin in the ischemic brain. Global and conditional myeloid knockout of properdin attenuated microglial overactivation and inflammatory responses at the acute stage of tMCAO in mice. Accordingly, treatment with recombinant properdin enhanced the production of proinflammatory cytokines and augmented microglia-potentiated neuronal death in primary culture. Mechanistically, recombinant properdin served as a novel ligand that activated Mincle receptors on microglia and downstream pathways to drive primary microglia-induced inflammatory responses. Intriguingly, properdin can directly bind to the microglial Mincle receptor to exert the above effects, while Mincle knockdown limits properdin-mediated microglial inflammation. CONCLUSION: Properdin is a new medium by which infiltrating peripheral myeloid cells communicate with microglia, further activate microglia, and exacerbate brain injury in the ischemic brain, suggesting that targeted disruption of the interaction between properdin and Mincle on microglia or inhibition of their downstream signaling may improve the prognosis of ischemic stroke.

Modulation of adipose tissue metabolism by microbial-derived metabolites.

Obesity and its complications, including type 2 diabetes, cardiovascular disease, and certain cancers, have posed a significant burden on health and healthcare systems over the years due to their high prevalence and incidence. Gut microbial derivatives are necessary for the regulation of energy metabolism and host immunity, as well as for maintaining homeostasis of the intestinal environment. Gut flora metabolites may be a link between gut microbes and diseases, such as obesity, and help understand why alterations in the microbiota can influence the pathophysiology of human disease. This is supported by emerging evidence that microbial-derived metabolites, such as short-chain fatty acids, bile acids, tryptophan, trimethylamine-N-oxide, and lipopolysaccharides, can be beneficial or detrimental to the host by affecting organs outside the gut, including adipose tissue. Adipose tissue is the largest lipid storage organ in the body and an essential endocrine organ that plays an indispensable role in the regulation of lipid storage, metabolism, and energy balance. Adipose tissue metabolism includes adipocyte metabolism (lipogenesis and lipolysis), thermogenesis, and adipose tissue metabolic maladaptation. Adipose tissue dysfunction causes the development of metabolic diseases, such as obesity. Here, we review the current understanding of how these microbial metabolites are produced and discuss both established mechanisms and the most recent effects of microbial products on host adipose tissue metabolism. We aimed to identify novel therapeutic targets or strategies for the prevention and treatment of obesity and its complications.

Microglial lnc-U90926 facilitates neutrophil infiltration in ischemic stroke via MDH2/CXCL2 axis.

Dysregulated long non-coding RNAs (lncRNAs) have been shown to contribute to the pathogenesis of ischemic stroke. However, the potential role of lncRNAs in post-stroke microglial activation remains largely unknown. Here, we uncovered that lncRNA-U90926 was significantly increased in microglia exposed to ischemia/reperfusion both in vivo and in vitro. In addition, adenovirus-associated virus (AAV)-mediated microglial U90926 silencing alleviated neurological deficits and reduced infarct volume in experimental stroke mice. Microglial U90926 knockdown could reduce the infiltration of neutrophils into ischemic lesion site, which might be attributed to the downregulation of C-X-C motif ligand 2 (CXCL2). Mechanistically, U90926 directly bound to malate dehydrogenase 2 (MDH2) and competitively inhibited the binding of MDH2 to the CXCL2 3' untranslated region (UTR), thus protecting against MDH2-mediated decay of CXCL2 mRNA. Taken together, our study demonstrated that microglial U90926 aggravated ischemic brain injury via facilitating neutrophil infiltration, suggesting that U90926 might be a potential biomarker and therapeutic target for ischemic stroke.

Role of the gut microbiota in type 2 diabetes and related diseases.

Type 2 diabetes is the fastest-growing metabolic disease in the world. Many clinical studies have found that type 2 diabetes patients have metabolic disorders and chronic inflammatory states accompanied by disturbances in the gut microbiota. The gut microbiota plays an important role in body metabolism and immune regulation, and disturbances in the gut microbiota in conjunction with destruction of the intestinal barrier in type 2 diabetes patients causes damage to multiple organs. Therefore, the gut microbiota may be a new therapeutic target for treating type 2 diabetes and related diseases. In this review, we introduce the characteristics of the gut microbiota in type 2 diabetes and related diseases, as well as highlight the potential molecular mechanisms of their effects on intestinal barrier disruption, metabolic disorders, and chronic inflammation. Finally, we summarize an intestinal microecological therapeutic strategy, with a focus on shaping the intestinal bacteria, to improve the malignant progress of type 2 diabetes and related diseases. AUTHOR SUMMARY: Type 2 diabetes (T2D) is the fastest-growing metabolic disease in the world. Many clinical studies have found that T2D patients have metabolic disorders and chronic inflammatory states, accompanied by disturbances of the gut microbiota and increased intestinal permeability. The number of human gut microbiota is more than 10 times of human cells, and they play an important role in the body's metabolism and immune regulation. The abnormal intestinal metabolites and intestinal barrier disruption caused by the gut microbiota dysbiosis in the T2D facilitate intestinal bacteria and their harmful metabolites entering the circulatory system. The abnormal entering will cause the damage to multiple organs through disturbing insulin sensitivity, glucose metabolism, and immune homeostasis. Therefore, the gut microbiota may be a new therapeutic target for improving T2D and its related diseases. In this review, we introduce the compositional characteristics of the gut microbiota in T2D, and highlight some new molecular mechanisms of their effects on intestinal barrier disruption, metabolic disorders and chronic inflammation in T2D and its related diseases. Finally, we summarize an intestinal microecological therapeutic strategy, with a focus on shaping the intestinal bacteria, to improve the malignant progress of T2D and related diseases.

The role of short-chain fatty acids in intestinal barrier function, inflammation, oxidative stress, and colonic carcinogenesis.

Short-chain fatty acids (SCFAs), mainly including acetate, propionate, and butyrate, are metabolites produced during the bacterial fermentation of dietary fiber in the intestinal tract. They are believed to be essential factors affecting host health. Most in vitro and ex vivo studies have shown that SCFAs affect the regulation of inflammation, carcinogenesis, intestinal barrier function, and oxidative stress, but convincing evidence in humans is still lacking. Two major SCFA signaling mechanisms have been identified: promotion of histone acetylation and activation of G-protein-coupled receptors. In this review, we introduce the production and metabolic characteristics of SCFAs, summarize the potential effects of SCFAs on the four aspects mentioned above and the possible mechanisms. SCFAs have been reported to exert a wide spectrum of positive effects and have a high potential for therapeutic use in human-related diseases.

Poncirin suppresses lipopolysaccharide (LPS)-induced microglial inflammation and ameliorates brain ischemic injury in experimental stroke in mice.

BACKGROUND: Based on accumulating evidence, excessive activation of microglia-mediated inflammatory responses plays an essential role in ischemic stroke. Poncirin (Pon) exerts anti-hyperalgesic, anti-osteoporotic and anti-tumor effects on various diseases. However, the roles of Pon in microglial activation and the underlying mechanism have not been elucidated. This study aimed to explore whether Pon inhibits lipopolysaccharide (LPS)-induced microglial neuroinflammation and protects against brain ischemic injury in experimental stroke in mice. METHODS: Primary microglia cells were prepared from the cerebral cortices of 1- to 2-day-old C57BL/6J mice. Murine BV2 cells and primary microglia were stimulated with LPS and the effects of a non-cytotoxic concentration of Pon on LPS-stimulated pro-inflammatory factors were measured using real-time PCR and enzyme-linked immunosorbent assays (ELISAs). Western blot analyses were used for mechanistic studies. In an in vivo study, 8-week-old male C57BL/6J mice were subjected to focal cerebral ischemia through middle cerebral artery occlusion (MCAO). Pon (30 mg/kg, i.p.) or the same volume of saline was administered after the MCAO model was established, and the infarct volume was evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) staining. We also evaluated animal behaviours, the expression of pro-inflammatory cytokines and microglial activation in the ischemic hemisphere. RESULTS: Pon prevented the release of nitric oxide (NO), prostaglandin E2 (PGE2), interleukin (IL)-1ß, IL-6 and tumor necrosis factor-alpha (TNF-α) in both BV2 cells and primary microglia stimulated with LPS. The inhibitory effects of Pon were associated with the regulation of the ERK1/2, JNK and nuclear factor kappa B (NF-κB) signaling pathways. In mice that underwent MCAO, Pon administration decreased the lesion size and improved neurological deficits. Furthermore, Pon attenuated the production of inflammatory cytokines mainly by restraining microglial activation after ischemic stroke. CONCLUSIONS: Based on the findings from the present study, Pon provides neuroprotection through its anti-inflammatory effects on microglia and it may be a useful treatment for ischemic stroke.

FasL-PDPK1 Pathway Promotes the Cytotoxicity of CD8+ T Cells During Ischemic Stroke.

CD8+ T cells are recognized as key players in exacerbation of ischemic stroke; however, the underlying mechanism in modulating the function of CD8+ T cells has not been completely elucidated. Here, we uncovered that FasL enhanced the cytotoxicity of CD8+ T cells to neurons after ischemic stroke. Inactivation of FasL specific on CD8+ T cells protected against brain damage and neuron loss. Proteomic analysis identified that PDPK1 functioned downstream of FasL signaling and inhibition of PDPK1 effectively reduced cytotoxicity of CD8+ T cells and improved ischemic neurological deficits. Taken together, these results highlight an intrinsic FasL-PDPK1 pathway regulating the cytotoxicity of CD8+ T cells in ischemic stroke.

A SIRPα-Fc fusion protein enhances the antitumor effect of oncolytic adenovirus against ovarian cancer.

Oncolytic viruses armed with therapeutic transgenes of interest show great potential in cancer immunotherapy. Here, a novel oncolytic adenovirus carrying a signal regulatory protein-α (SIRPα)-IgG1 Fc fusion gene (termed SG635-SF) was constructed, which could block the CD47 'don't eat me' signal of cancer cells. A strong promoter sequence (CCAU) was chosen to control the expression of the SF fusion protein, and a 5/35 chimeric fiber was utilized to enhance the efficiency of infection. As a result, SG635-SF was found to specifically proliferate in hTERT-positive cancer cells and largely increased the abundance of the SF gene. The SF fusion protein was effectively detected, and CD47 was successfully blocked in SK-OV3 and HO8910 ovarian cancer cells expressing high levels of CD47. Although the ability to induce cell cycle arrest and cell death was comparable to that of the control empty SG635 oncolytic adenovirus in vitro, the antitumor effect of SG635-SF was significantly superior to that of SG635 in vivo. Furthermore, CD47 was largely blocked and macrophage infiltration distinctly increased in xenograft tissues of SK-OV3 cells but not in those of CD47-negative HepG2 cells, indicating that the enhanced antitumor effect of SG635-SF was CD47-dependent. Collectively, these findings highlight a potent antitumor effect of SG635-SF in the treatment of CD47-positive cancers.

YAP/TAZ: a promising target for squamous cell carcinoma treatment.

Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are two homologous transcriptional coactivators and the final effectors of the Hippo signaling transduction pathway. The transcriptional activity of YAP/TAZ is dependent on their recruitment to the nucleus, which promotes binding to the transcription factor of TEA domain family members 1-4 (TEAD1-4). In Hippo-signaling pathway, YAP/TAZ is inactivated and its translocation to the nucleus is blocked via a core kinase cascade stimulated by a variety of upstream signals, such as G-protein-coupled receptor signaling, mechanical pressure, and adherens junction signaling. This pathway plays a very important role in regulating organ size, tissue homeostasis, and tumor development. In recent years, many studies have reported upregulation or nuclear localization of YAP/TAZ in a number of human malignancies, such as breast cancer, melanoma, lung cancer, especially squamous cell carcinoma in different organs. A large number of experiments demonstrate that YAP/TAZ activation promotes cancer formation, progression, and metastasis. Therefore, in this review, we summarize the evidence of regulation and function of YAP/TAZ and discuss its role in squamous cell carcinoma. Collectively, this summary strongly suggests that targeting aberrant YAP/TAZ activation is a promising strategy for the suppression of squamous cell carcinoma.

EZH2 inhibitor DZNep modulates microglial activation and protects against ischaemic brain injury after experimental stroke.

Enhancer of zeste homolog-2 (EZH2), a histone methyltransferase, has been recognized to play a pivotal role in regulating the immune response in various diseases. However, its role in the inflammatory response induced by ischaemic stroke remains to be further investigated. The aim of this study was to determine the role of EZH2 in microglia-associated inflammation in ischaemic stroke and to further detect the effects of the EZH2 inhibitor, 3-deazaadenosine A (DZNep), in ischaemic brain injury. Here, we found that both in vivo ischemic/reperfusion (I/R) injury and in vitro oxygen-glucose deprivation (OGD) treatment induced a marked upregulation of EZH2 in microglia. The administration of the EZH2 inhibitor DZNep improved behavioural performance and reduced the infarct volume in mice after experimental stroke. Furthermore, we showed that DZNep blocked pro-inflammatory (CD86+) microglial activation and triggered anti-inflammatory (CD206+) microglial polarization in experimental stroke. Pro-inflammatory cytokines such as IL-1ß, IL-6, TNF-α and CXCL10 were also significantly downregulated by DZNep. In addition, it was found that DZNep blocked the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in microglia, which was increased by I/R injury and OGD. Collectively, we demonstrated that EZH2 is implicated in regulating microglial activation and exacerbates neurological deficits after ischaemic stroke, probably via activating STAT3, and that the EZH2 inhibitor DZNep can exert neuroprotective effects after ischaemic stroke.

11R-P53 and GM-CSF Expressing Oncolytic Adenovirus Target Cancer Stem Cells with Enhanced Synergistic Activity.

Targeting cancer stem cells with oncolytic virus (OV) holds great potential for thorough elimination of cancer cells. Based on our previous studies, we here established 11R-P53 and mGM-CSF carrying oncolytic adenovirus (OAV) SG655-mGMP and investigated its therapeutic effect on hepatocellular carcinoma stem cells Hep3B-C and teratoma stem cells ECCG5. Firstly, the augmenting effect of 11R in our construct was tested and confirmed by examining the expression of EGFP with Fluorescence and FCM assays after transfecting Hep3B-C and ECCG5 cells with OVA SG7605-EGFP and SG7605-11R-EGFP. Secondly, the expressions of 11R-P53 and GM-CSF in Hep3B-C and ECCG5 cells after transfection with OAV SG655-mGMP were detected by Western blot and Elisa assays, respectively. Thirdly, the enhanced growth inhibitory and augmented apoptosis inducing effects of OAV SG655-mGMP on Hep3B-C and ECCG5 cells were tested with FCM assays by comparing with the control, wild type 5 adenovirus, 11R-P53 carrying OVA in vitro. Lastly, the in vivo therapeutic effect of OAV SG655-mGMP toward ECCG5 cell-formed xenografts was studied by measuring tumor volumes post different treatments with PBS, OAV SG655-11R-P53, OAV SG655-mGM-CSF and OAV SG655-mGMP. Treatment with OAV SG655-mGMP induced significant xenograft growth inhibition, inflammation factor AIF1 expression and immune cells infiltration. Therefore, our OAV SG655-mGMP provides a novel platform to arm OVs to target cancer stem cells.

Efficient synthesis of boron-containing α-acyloxyamide analogs via microwave irradiation.

In this report, a Passerini three-component reaction utilizing boron-containing carboxylic acids or aldehydes is discussed. The reaction was carried out in water and facilitated by the use of microwave irradiation. This methodology allowed for the efficient formation of a broad range of boron-containing α-acyloxyamides under mild conditions within a short time. Two series of boron-containing α-acyloxyamides were synthesized and subsequently screened for cytotoxicity using the MTT cell viability assay. Two potential lead compounds were found to have potent activity against the HepG2 cancer cell line, demonstrating the potential of this methodology for use in the development of novel pharmaceuticals.