Siponimod ameliorates metabolic oligodendrocyte injury via the sphingosine-1 phosphate receptor 5.

Multiple sclerosis (MS), an autoimmune-driven, inflammatory demyelinating disease of the central nervous system (CNS), causes irreversible accumulation of neurological deficits to a variable extent. Although there are potent disease-modifying agents for its initial relapsing-remitting phase, immunosuppressive therapies show limited efficacy in secondary progressive MS (SPMS). Although modulation of sphingosine-1 phosphate receptors has proven beneficial during SPMS, the underlying mechanisms are poorly understood. In this project, we followed the hypothesis that siponimod, a sphingosine-1 phosphate receptor modulator, exerts protective effects by direct modulation of glia cell function (i.e., either astrocytes, microglia, or oligodendrocytes). To this end, we used the toxin-mediated, nonautoimmune MS animal model of cuprizone (Cup) intoxication. On the histological level, siponimod ameliorated cuprizone-induced oligodendrocyte degeneration, demyelination, and axonal injury. Protective effects were evident as well using GE180 translocator protein 18-kDa (TSPO) imaging with positron emission tomography (PET)/computed tomography (CT) imaging or next generation sequencing (NGS). Siponimod also ameliorated the cuprizone-induced pathologies in Rag1-deficient mice, demonstrating that the protection is independent of T and B cell modulation. Proinflammatory responses in primary mixed astrocytes/microglia cell cultures were not modulated by siponimod, suggesting that other cell types than microglia and astrocytes are targeted. Of note, siponimod completely lost its protective effects in S1pr5-deficient mice, suggesting direct protection of degenerating oligodendrocytes. Our study demonstrates that siponimod exerts protective effects in the brain in a S1PR5-dependent manner. This finding is not just relevant in the context of MS but in other neuropathologies as well, characterized by a degeneration of the axon-myelin unit.

Siponimod ameliorates experimental autoimmune neuritis.

BACKGROUND: Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are human autoimmune peripheral neuropathy. Besides humoral immunity, cellular immunity is also believed to contribute to these pathologies, especially CIDP. Sphingosine-1-phosphate receptor 1 (S1PR1) regulates the maturation, migration, and trafficking of lymphocytes. As of date, the therapeutic effect of sphingosine-1-phosphate receptor (S1PR) agonists on patients with GBS or CIDP remains unclear. METHODS: To evaluate the effect of siponimod, an agonist of S1PR1 and S1PR5, on experimental autoimmune neuritis (EAN), an animal model of autoimmune peripheral neuropathy, was used. Lewis rats were immunized with 125 µg of synthetic peptide from bovine P2 protein. Rats in the siponimod group were orally administered 1.0 mg/kg siponimod and those in the EAN group were administrated the vehicle on days 5-27 post-immunization (p.i.) daily. The symptom severity was recorded daily. The changes in the expression of cytokines and transcription factors in the lymph nodes and cauda equina (CE) which correlate with the pathogenesis of EAN and recovery of injured nerve were measured using reverse transcription quantitative PCR. Histological study of CE was also performed. RESULTS: Flaccid paralysis developed on day 11 p.i. in both groups. Siponimod relieved the symptom severity and decreased the expression of interferon-gamma and IL-10 mRNAs in lymph nodes and CE compared with that in the EAN group. The expression of Jun proto-oncogene (c-Jun) mRNA increased from the peak to the recovery phase and that of Sonic hedgehog signaling molecule (Shh) and Glial cell line-derived neurotrophic factor (Gdnf) increased prior to increase in c-Jun with no difference observed between the two groups. Histologically, siponimod also reduced demyelinating lesions and inflammatory cell invasion in CE. CONCLUSIONS: Siponimod has a potential to ameliorate EAN. Shh and Gdnf, as well as C-Jun played a significant role during the recovery of injured nerves.

[Platycladi Semen oil ameliorates Aß_(25-35)-induced brain injury in mice based on network pharmacology and gut microbiota].

The present study aimed to investigate the protective effect and underlying mechanism of Platycladi Semen oil(SP) on Aß_(25-35)-induced brain injury in mice to provide a theoretical basis for the clinical treatment of Alzheimer's disease(AD). Male Kunming(KM) mice were randomly divided into a control group, a model group(brain injection of Aß_(25-35), 200 µmol·L~(-1), 0.15 µL·g~(-1)), a positive drug group(donepezil, 10 mg·kg~(-1)), and low-and high-dose SP groups(0.5 and 1 mL·kg~(-1)). Learning and memory ability, neuronal damage, levels of Aß_(1-42)/Aß_(1-40), p-Tau, related indicators of apoptosis and oxidative stress, and immune cells, and protein and mRNA expression related to the sphingosine kinase 1(SPHK1)/sphingosine-1-phosphate(S1P)/sphingosine-1-phosphate receptor 5(S1PR5) signaling pathway of mice in each group were determined. In addition, compounds in SP were analyzed by gas chromatography-mass spectrometry(GC-MS). The mechanism of SP against AD was investigated by network pharmacology, 16S rDNA gene sequencing for gut microbiota(GM), and molecular docking techniques. The results showed that SP could improve the learning and memory function of Aß_(25-35)-induced mice, reduce hippocampal neuronal damage, decrease the levels of Aß_(1-42)/Aß_(1-40), p-Tau, and indicators related to apoptosis and oxidative stress in the brain, and maintain the homeostasis of immune cells and GM. Network pharmacology and sequencing analysis for GM showed that the therapeutic effect of SP on AD was associated with the sphingolipid signaling pathway. Meanwhile,(Z,Z,Z)-9,12,15-octadecatrienoic acid and(Z,Z)-9,12-octadecadienoic acid, the components with the highest content in SP, showed good binding activity to SPHK1 and S1PR5. Therefore, it is inferred that SP exerts anti-apoptosis and antioxidant effects by regulating GM and inhibiting SPHK1/S1P/S1PR5 pathway, thereby improving brain injury induced by Aß_(25-35) in mice. Moreover,(Z,Z,Z)-9,12,15-octadecatrienoic acid and(Z,Z)-9,12-octadecadienoic acid may be the material basis for the anti-AD effect of SP.

Sphingosine 1-Phosphate Receptor 5 (S1P5) Knockout Ameliorates Adenine-Induced Nephropathy.

S1P and its receptors have been reported to play important roles in the development of renal fibrosis. Although S1P5 has barely been investigated so far, there are indications that it can influence inflammatory and fibrotic processes. Here, we report the role of S1P5 in renal inflammation and fibrosis. Male S1P5 knockout mice and wild-type mice on a C57BL/6J background were fed with an adenine-rich diet for 7 days or 14 days to induce tubulointerstitial fibrosis. The kidneys of untreated mice served as respective controls. Kidney damage, fibrosis, and inflammation in kidney tissues were analyzed by real-time PCR, Western blot, and histological staining. Renal function was assessed by plasma creatinine ELISA. The S1P5 knockout mice had better renal function and showed less kidney damage, less proinflammatory cytokine release, and less fibrosis after 7 days and 14 days of an adenine-rich diet compared to wild-type mice. S1P5 knockout ameliorates tubular damage and tubulointerstitial fibrosis in a model of adenine-induced nephropathy in mice. Thus, targeting S1P5 might be a promising goal for the pharmacological treatment of kidney diseases.

Bone marrow-derived mesenchymal stem cells suppress NK cell recruitment and activation in PolyI:C-induced liver injury.

Mesenchymal stem cells (MSCs) have been shown to have an immunomodulatory capability and clinical potential in immune diseases. However, it is unknown how MSCs may affect immunity in liver injury. This study was designed to explore the effect of bone marrow-derived MSCs (BM-MSCs) on hepatic natural killer (NK) cells in polyinosinic-polycytidylic acid (PolyI:C)-induced liver injury. Unlike in controls, adoptive transfer of BM-MSCs in mice ameliorated PolyI:C-induced liver injury, as shown by lower alanine aminotransferase levels and decreased lymphocyte infiltration in the liver. Importantly, BM-MSCs suppress NK cell accumulation and activation in the liver, which plays an important role in PolyI:C-induced liver injury. Furthermore, NK cells co-cultured with BM-MSCs reduced expression of sphingosine-1-phosphate receptor type 5 (S1PR5), an important receptor required for NK cell trafficking in vivo. BM-MSC administration suppressed the elevation of expression of S1PR5 in the liver induced by PolyI:C injection. Accordingly, BM-MSCs inhibited the chemotactic activity of NK cells induced by sphingosine-1-phosphate (S1P, the ligand of S1PR5). Our results provide an additional mechanism for the immunosuppressive effect of BM-MSCs on NK cells, which further supports the therapeutic potential of BM-MSCs in immune-mediated disorders, including those in which NK cells play a major role.

S1PR-1/5 modulator RP-101074 shows beneficial effects in a model of central nervous system degeneration.

Introduction: In multiple sclerosis (MS), chronic disability primarily stems from axonal and neuronal degeneration, a condition resistant to conventional immunosuppressive or immunomodulatory treatments. Recent research has indicated that selective sphingosine-1-phosphate receptor S1PR-1 and -5 modulators yield positive effects in progressive MS and mechanistic models of inflammation-driven neurodegeneration and demyelination. Methods: In this study, the S1PR-1/-5 modulator RP-101074 was evaluated as a surrogate for ozanimod in the non-inflammatory, primary degenerative animal model of light-induced photoreceptor loss (LI-PRL) in CX3CR1-GFP mice to assess potential neuroprotective effects, independent of its immunomodulatory mechanism of action. Results: Prophylactic administration of RP-101074 demonstrated protective effects in the preclinical, non-inflammatory LI-PRL animal model, following a bell-shaped dose-response curve. RP-101074 treatment also revealed activity-modulating effects on myeloid cells, specifically, CX3CR1+ cells, significantly reducing the marked infiltration occurring one week post-irradiation. Treatment with RP-101074 produced beneficial outcomes on both retinal layer thickness and visual function as evidenced by optical coherence tomography (OCT) and optomotor response (OMR) measurements, respectively. Additionally, the myelination status and the quantity of neural stem cells in the optic nerve suggest that RP-101074 may play a role in the activation and/or recruitment of neural stem cells and oligodendrocyte progenitor cells, respectively. Conclusion/Discussion: The data from our study suggest that RP-101074 may have a broader role in MS treatment beyond immunomodulation, potentially offering a novel approach to mitigate neurodegeneration, a core contributor to chronic disability in MS.

Network Pharmacology and Experimental Verification to Explore the Potential Mechanism of Yin-Huo-Tang for Lung Adenocarcinoma Recurrence.

PURPOSE: Yin-Huo-Tang (YHT) is a classic traditional Chinese prescription, used to prevent lung adenocarcinoma (LUAD) relapse by "nourishing yin and clearing heat". In this study, the mechanism of YHT in LUAD recurrence was investigated. METHODS: Firstly, the bioactive compounds and targets of YHT, as well as related targets of LUAD recurrence, were collected from public databases. The protein-protein interaction network, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed to find the pivotal compounds, hub genes, functional annotation and main pathways. Subsequently, RNA sequencing of recurrent tumor tissues from Lewis lung carcinoma mice treated with YHT was used to explore the main pathways. At the same time, pathways screened by network pharmacology and RNA sequencing analysis were considered the most important pathways. Finally, liquid chromatography mass spectrometry was used to validate the pivotal active ingredients. Molecular docking technology was performed to validate the binding association between the hub genes and the pivotal active ingredients. PCR and WB analysis were used to validate the main pathways. RESULTS: There were 128 active compounds and 419 targets interacting with YHT and LUAD recurrence. Network analysis identified 4 pivotal compounds, 28 hub genes and 30 main pathways. Sphingolipid signaling pathway was the common main pathway in network pharmacology and RNA sequencing results. The hub gene related to the sphingolipid signaling pathway was S1PR5. Qualitative phytochemical analysis confirmed the presence of 3 pivotal compounds, namely stigmasterol, nootkatone and ergotamine. The molecular docking verified that the pivotal compounds could good affinity with S1PR5. The PCR and WB analysis verified YHT suppressed Lewis lung cancer cells proliferation and migration by inhibiting the sphingolipid signaling pathway. CONCLUSION: The potential mechanism and therapeutic effect of YHT against the recurrence of LUAD may be ascribed to inhibition of the sphingolipid signaling pathway.

The Role and Mechanism of S1PR5 in Colon Cancer.

PURPOSE: To investigate the role and mechanism of S1PR5 in colon cancer. MATERIALS AND METHODS: Lentiviral infection and drug screening helped to establish colon cancer cell lines with stable overexpression and knockdown of S1PR5. Effects of S1PR5 expression on cell growth, proliferation, migration, and invasion were analyzed using a subcutaneous xenograft model in nude mice. Western blot (WB) was used to detect the effects of S1PR5 expression on p-AKT, STAT3, NF-κB, and p-JNK. The distribution of p65 was evaluated in nuclear and cytoplasmic fractions using WB. CCK-8, Transwell migration, and Transwell invasion assays analyzed cell growth, proliferation, migration, and invasion. qRT-PCR analysis revealed that S1PR5 expression was associated with altered expression levels of NF-κB downstream target genes, such as IL-6, TNF-α, and indoleamine 2, 3-dioxygenase 1 (IDO1). RESULTS: qRT-PCR and WB analysis showed that the S1PR5 level in colon cancer cell lines-SW480, SW620, HCT116, and LoVo-was significantly higher than in NCM460, a healthy colonic epithelial cell line. SW620 and SW480, with high and low expression of S1PR5, respectively, were selected as model cell lines. S1PR5 knockdown in SW620 caused the growth rate, proliferation, migration, invasion, and subcutaneous tumor formation rate to decrease in mice, whereas S1PR5 overexpression in SW480 caused all of these parameters to increase. WB analysis showed an increase in phospho-p65 and its nuclear translocation. S1PR5 knockdown caused a decrease in phospho-p65 levels and its nuclear import, thereby inhibiting its activity. In S1PR5 knockdown and overexpressing cells, p65 was overexpressed and knocked down, respectively. qRT-PCR and WB showed that S1PR5 over-expression up-regulates IDO1, and S1PR5 knockdown inhibits IDO1. CCK-8 and Transwell assays showed that p65 and IDO1 overexpression antagonizes the antitumor effect of S1PR5 knockdown, and that p65 and IDO1 knockdown antagonizes the tumorigenic effect of S1PR5 overexpression. CONCLUSION: S1PR5 overexpression promotes the growth, migration, and invasion of cancer by activating the NF-κB/IDO1 signaling pathway.