Establishment of a multi-strategy platform for quality control and quality markers screen of Mailuoshutong pill.

Thromboangiitis obliterans (TAO) is a non-atherosclerotic segmental inflammatory occlusive disease with a high recurrence rate, high disability rate, difficulty to cure, and poor prognosis. It has been clinically proven that Mailuoshutong pill (MLSTP) is an effective traditional Chinese medicine for treating TAO. As MLSTP contains hundreds of chemical components, the quality control of which is a challenge in the development of reliable quality evaluation metrics. This study aimed to evaluate the quality uniformity of MLSTP by establishing a multi-strategy platform. In the present study, the key targets and signaling pathways of MLSTP treating TAO were predicted by network pharmacology. It was further shown by in vivo validation experiments that MLSTP exerted therapeutic effects on TAO by modulating the PI3K-AKT signaling pathway, VEGF signaling pathway, and HIF-1 signaling pathway. In addition, UPLC fingerprints of MLSTP were established and screened for potential Q-markers of MLSTP in combination with network pharmacology results. Six components, including chlorogenic acid, liquiritin, paeoniflorin, calycosin-7-glucoside, berberine, and formononetin, were selected as potential quality markers (Q-markers) in MLSTP. Finally, the quantitative analysis of multi-components by single marker (QAMS) method was established to quantitatively analyze the six potential Q-markers, and the results were consistent with those obtained by the external standard method (ESM). Taken together, the multi-strategy platform established in this study would be conducive to the Q-markers screening and quality control of MLSTP, improving the quality standard of MLSTP and providing favorable assurance for the clinical management of TAO.

Matrine Mediated Immune Protection in MS by Regulating Gut Microbiota and Production of SCFAs.

There is clearly an unmet need for more effective and safer treatments for multiple sclerosis (MS). Our previous studies showed a significant therapeutic effect of matrine, a monomer of traditional herbal medicine, on experimental autoimmune encephalomyelitis (EAE) mice. To explore the mechanism of matrine action, we used 16S rRNA sequencing technology to determine the gut microbes in matrine-treated EAE mice and controls. The concentrations of short-chain fatty acids (SCFAs) were then tested by metabonomics. Finally, we established pseudo-sterile mice and transplanted into them fecal microbiota, which had been obtained from the high-dose matrine-treated EAE mice to test the effects of matrine. The results showed that matrine could restore the diversity of gut microbiota and promote the production of SCFAs in EAE mice. Transplantation of fecal microbiota from matrine-treated mice significantly alleviated EAE severity, reduced CNS inflammatory infiltration and demyelination, and decreased the level of IL-17 but increased IL-10 in sera of mice. In conclusion, matrine treatment can regulate gut microbiota and metabolites and halt the progression of MS.

Integrated pharmacokinetics and pharmacometabolomics to reveal the synergistic mechanism of a multicomponent Chinese patent medicine, Mailuo Shutong pills against thromboangiitis obliterans.

BACKGROUND: Mailuo Shutong Pills (MLST) have displayed pharmacological activity against thromboangiitis obliterans (TAO). However, the active ingredients and therapeutic mechanism of MLST against TAO remained to be further clarified. PURPOSE: The aim of this study was to explore the active components of MLST and their synergistic mechanism against TAO by integrating pharmacokinetics (PK) and pharmacometabolomics (PM). METHODS: TAO model rats were established by sodium laurate solution. Firstly, the efficacy of MLST was evaluated by gangrene score, blood flow velocity, and hematoxylin-eosin (H&E) staining. Secondly, PK research was conducted on bioavailable components to characterize their dynamic behaviors under TAO. Thirdly, multiple plasma and urine metabolic biomarkers for sodium laurate-induced TAO rats were found by untargeted metabolomics, and then variations in TAO-altered metabolites following MLST treatment were analyzed utilizing multivariate and bioinformatic analysis. Additionally, metabolic pathway analysis was performed using MetaboAnalyst. Finally, the dynamic link between absorbed MLST-compounds and TAO-associated endogenous metabolites was established by correlation analysis. RESULTS: MLST significantly alleviated gangrene symptoms by improving the infiltration of inflammatory cells and blood supply in TAO rats. Significant differences in metabolic profiles were found in 17 differential metabolites in plasma and 24 in urine between Sham and TAO rats. The 10 bioavailable MLST-compounds, such as chlorogenic acid and paeoniflorin, showed positive or negative correlations with various TAO-altered metabolites related to glutamate metabolism, histidine metabolism, arachidonic acid metabolism and so on. CONCLUSION: This study originally investigated the dynamic interaction between MLST and the biosystem, providing unique insight for disclosing the active components of MLST and their synergistic mechanisms against TAO, which also shed light on new therapeutic targets for TAO and treatment.

Ferroptosis as a mechanism of oligodendrocyte loss and demyelination in experimental autoimmune encephalomyelitis.

Ferroptosis, distinct from necrosis, autophagy and apoptosis, is a unique form of regulated cell death，and is a potential pathogenic mechanism of neuronal loss and defunction in many neurodegenerative disorders. Recent studies have shown a presence of iron deposition in the central nervous system (CNS) of patients with multiple sclerosis (MS). However, whether ferroptosis is involved in the pathogenesis of MS remains unclear. In the present study, we tested certain classical biomarkers of ferroptosis in the spinal cord of mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS, to substantiate the relationship between ferroptosis and oligodendrocyte (OL) loss and demyelination. Our results revealed decreased levels of key molecules in glutathione antioxidant mechanisms, including system xC (xCT) and glutathione peroxidase 4 (GPX4) in spinal cord of EAE mice, with evident lipid peroxidation in OLs. Moreover, transferrin receptor and ferritinophagy further catalyzed the generation of lipid reactive oxygen species through the fenton reaction, which induced OL death and demyelination at disease peak of EAE. This phenomenon was largely reversed by administering Fer-1, an inhibitor of ferritin phagocytosis, further validating the key role of ferritin phagocytosis in EAE. Taken together, these findings demonstrate that OL loss and demyelination may be induced in EAE through, at least in part, a mechanism of ferroptosis.

Rapid and comprehensive identification of chemical constituents in Mai-Luo-Shu-Tong pill by UHPLC-Q-Orbitrap HRMS combined with a data mining strategy.

Mai-Luo-Shu-Tong pill is an effective traditional Chinese medicine formula for the treatment of superficial thrombophlebitis, but it was insufficiently chemically scrutinized. In this study, the mass spectral data of Mai-Luo-Shu-Tong pill were acquired by ultra-high performance liquid chromatography coupled with Q Exactive hybrid Quadrupole-Orbitrap high resolution mass spectrometry. Then, a data mining strategy combining multiple data processing methods was used to identify chemical constituents in Mai-Luo-Shu-Tong pill by constructing a database of precursor ions and summarizing the mass spectral fragmentation behaviors. As a result, a total of 211 compounds including 70 flavonoids, 56 terpenoids, 37 phenolic acids and 48 others were identified in positive and negative ion modes. Among them, 66 compounds have passed comparison verification with reference standards, 145 compounds were identified based on the data mining strategy combining the characteristic cleavage behaviour of homologous compounds and fragment ions and 4 compounds were potentially new compounds. This study provides a database for quality evaluation and further study of Mai-Luo-Shu-Tong pill in vivo. Moreover, it provides a reference for the characterization of the chemical constituents of other traditional Chinese medicine formulae.

Identifying quality markers of Mailuoshutong pill against thromboangiitis obliterans based on chinmedomics strategy.

BACKGROUND: Mailuoshutong pill (MLSTP) is a traditional Chinese medicine (TCM) for the treatment of Thromboangiitis obliterans (TAO, Buerger's disease) which is a segmental non-atherosclerotic inflammatory occlusive disorder. However, the mechanism and quality standards of MLSTP have not been sufficiently studied. PURPOSE: This work aims to investigate the potential mechanisms and quality markers (Q-markers) of MLSTP treating TAO based on the chinmedomics strategy. METHODS: The therapeutical effect of MLSTP on TAO rats was evaluated by changes in body weight and clinical score, regional blood flow velocity and perfused blood vessel distribution, hematoxylin-eosin (H&E) staining, serum metabolic profile. Moreover, both endogenous metabolites and exogenous components were simultaneously detected in serum based on ultra-high performance liquid chromatography coupled with a Q Exactive hybrid quadrupole-orbitrap high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS), and multivariate analysis was applied to identify the biomarkers, as well as the dynamic changes of metabolites were observed to explore the mechanism of action of MLSTP. In addition, the pharmacodynamic material basis were identified by correlation analysis between biomarkers and absorbed constituents. Finally, the Q-markers of MLSTP were determined according to the screening principles of Q-marker and validated the measurability. RESULTS: MLSTP treatment alleviated disease severity of TAO, reduced inflammatory infiltration, and ameliorated vascular function. 26 potential biomarkers associated with glutamate metabolism, linoleic acid metabolism, arachidonic acid metabolism and so on were identified. Besides, 27 prototypical components were identified in serum, 16 of which were highly correlated with efficacy and could serve as the pharmacodynamic material basis of MLSTP against TAO. In addition, 7 compounds, namely, sweroside, chlorogenic acid, calycosin-7-glucoside, formononetin, paeoniflorin, liquiritigenin and 3-butylidenephthalide, were considered as potential Q-markers of MLSTP. Ultimately, the measurability of the seven Q-markers was validated by rapid identifcation and quantifcation. CONCLUSION: This study successfully clarified the therapeutic effect and Q-markers of MLSTP by chinmedomics strategy, which is of great significance for the establishment of quality standards. Furthermore, it provides a certain reference for the screening of Q-markers in TCM prescriptions.

Matrine inhibits the Wnt3a/ß-catenin/TCF7L2 signaling pathway in experimental autoimmune encephalomyelitis.

Oligodendrocyte (OL) death and remyelination failure lead to progressive neurological deficits in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Matrine (MAT), a quinolizidine alkaloid component derived from the root of Sophora flavescens, has the capacity to effectively inhibit central nervous system (CNS) inflammation and to promote neuroregeneration. In the present study we explored its regulatory mechanism on the Wnt/ß-catenin/TCF7L2 pathway, a negative modulator for myelination, in MOG35--55 peptide-induced EAE. Our results clearly indicate that MAT treatment reduced the activation of Wnt3a and ß-catenin in the CNS of EAE mice, accompanied by the activation of GSK3ß and decreased expression of cyclin D1 and Axin2, two target genes of the Wnt3a/ß-catenin pathway. In addition, MAT increased OL maturation and myelination, as evidenced by the decreased number of NG2+Olig2+ cells and the increased numbers of MBP+ and CC1+Olig2+ cells. Taken together, these findings indicate that MAT treatment promoted the maturation of OLs and myelin repair, which is closely related to the modulation of the Wnt/ß-catenin/TCF7L2 signaling pathway.

Matrix compatibility of typical sol-gel solid-phase microextraction coatings in undiluted plasma and whole blood for the analysis of phthalic acid esters.

Sol-gel materials have been widely used for solid-phase microextraction (SPME) coatings due to their outstanding performance; in contrast, sol-gel SPME coatings have seldom been used for in vivo sampling. The main reason is that their matrix compatibility is unclear. In order to promote the application of this type of coating and accelerate the development of in vivo SPME, in this study, the matrix compatibility of several typical sol-gel coatings was assessed in plasma and whole blood using phthalic acid esters as analytes. The service life of five kinds of sol-gel coatings was among 20-35 times in undiluted plasma, while it was 27 times for a homemade commercial polydimethylsiloxane coating, which indicates good matrix compatibility of sol-gel coatings in untreated plasma. The sol-gel hydroxy-terminated silicone oil/methacrylic acid fiber achieved the highest extraction ability among all of the fibers, and it was tested in pig whole blood. It could be continuously used for at least 22 times, demonstrating good potential for in vivo sampling. Subsequently, a direct-immersion SPME/gas chromatography-flame ionization detection method was established for the determination of 5 phthalic acid esters in blood. Compared with other methods reported in the literature, this method is rapid, simple, sensitive, and accurate, and does not need expensive instruments or tedious procedures. A simulation system of animal blood circulation was constructed to verify the practicability of sol-gel SPME coatings in animal vein sampling. The result illustrated the feasibility of that coating for in vivo blood sampling, but a more accurate quantification calibration approach needs to be explored.

Matrine treatment induced an A2 astrocyte phenotype and protected the blood-brain barrier in CNS autoimmunity.

Type 1 astrocytes (A1), which are highly proinflammatory and neurotoxic, are prevalent in multiple sclerosis (MS). In addition, in MS and its animal model, experimental autoimmune encephalomyelitis (EAE), immune cells must cross the blood-brain barrier (BBB) and infiltrate into the parenchyma of the central nervous system (CNS) in order to induce neurological deficits. We have previously reported that treatment of EAE with matrine (MAT), a quinazine alkaloid derived from Sophorae Flavescens, effectively inhibited CNS inflammation and promoted neuroregeneration. However, the impact of MAT treatment on astrocyte phenotype is not known. In the present study, we showed that MAT treatment inhibited the generation of neurotoxic A1 astrocytes and promoted neuroprotective A2 astrocytes in the CNS of EAE, most likely by inhibiting production of the A1-inducing cytokine cocktail. MAT also downregulated the expression of vascular endothelial growth factor-A (VEGF-A) and upregulated tight junction proteins Claudin 5 and Occludin, thus protecting the BBB from CNS inflammation-induced damage. Moreover, MAT treatment promotes the formation of astrocyte tight junctions at glia limitans, thereby limiting parenchymal invasion of the CNS by immune cells. Taken together, the inhibition of A1 astrogliogenesis, and the dual effects on the BBB and astrocytic glia limitans, may be the mechanisms whereby MAT significantly improves EAE clinical scores and neuroprotection.

Matrine treatment reduces retinal ganglion cell apoptosis in experimental optic neuritis.

Inflammatory demyelination and axonal injury of the optic nerve are hallmarks of optic neuritis (ON), which often occurs in multiple sclerosis and is a major cause of visual disturbance in young adults. Although a high dose of corticosteroids can promote visual recovery, it cannot prevent permanent neuronal damage. Novel and effective therapies are thus required. Given the recently defined capacity of matrine (MAT), a quinolizidine alkaloid derived from the herb Radix Sophorae flavescens, in immunomodulation and neuroprotection, we tested in this study the effect of matrine on rats with experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. MAT administration, started at disease onset, significantly suppressed optic nerve infiltration and demyelination, with reduced numbers of Iba1+ macrophages/microglia and CD4+ T cells, compared to those from vehicle-treated rats. Increased expression of neurofilaments, an axon marker, reduced numbers of apoptosis in retinal ganglion cells (RGCs). Moreover, MAT treatment promoted Akt phosphorylation and shifted the Bcl-2/Bax ratio back towards an antiapoptotic one, which could be a mechanism for its therapeutic effect in the ON model. Taken as a whole, our results demonstrate that MAT attenuated inflammation, demyelination and axonal loss in the optic nerve, and protected RGCs from inflammation-induced cell death. MAT may therefore have potential as a novel treatment for this disease that may result in blindness.

Modulation of the HMGB1/TLR4/NF-κB signaling pathway in the CNS by matrine in experimental autoimmune encephalomyelitis.

The inflammatory mediator high-mobility group box 1 (HMGB1)-induced signaling pathway has been shown to play an important role in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Matrine (MAT), a quinolizidine alkaloid component derived from the root of Sophorae flavescens, has the capacity to effectively suppress EAE. However, the impact of MAT treatment on HMGB1-induced signaling is not known. In the present study, we show that MAT treatment alleviated disease severity of ongoing EAE, reduced inflammatory infiltration and demyelination, and reduced the production of inflammatory factors including TNF-α, IL-6, and IL-1ß in the CNS. Moreover, MAT administration significantly reduced the protein and RNA expression of HMGB1 and TLR4 in the spinal cord, particularly in astrocytes and microglia/infiltrating macrophages. The expression of MyD88 and TRAF6, and the phosphorylation of NF-κB p65, was also down-regulated after MAT treatment. In contrast, the level of IκB-α, an inhibitory molecule for NF-κB activation, was significantly increased. Furthermore, the direct inhibitory effect of MAT on HMGB1/TLR4/NF-κB signaling in macrophages was further confirmed in vitro. Taken together, these findings demonstrate that MAT treatment alleviated CNS inflammatory demyelination and activation of astrocytes and microglia/macrophages in EAE rats, and that the mechanism underlying these effects may be closely related to modulation of HMGB1/TLR4/NF-κB signaling pathway.

Matrine Inhibits CNS Autoimmunity Through an IFN-ß-Dependent Mechanism.

Matrine (MAT), a quinolizidine alkaloid component derived from the root of Sophora flavescens, suppresses experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS), by inducing the production of immunomodulatory molecules, e.g., IL-10. In an effort to find the upstream pathway(s) of the mechanism underlying these effects, we have tested certain upregulated immunomodulatory molecules. Among them, we found increased levels of IL-27 and IFN-ß, one of the first-line MS therapies. Indeed, while low levels of IFN-ß production in sera and type I interferon receptor (IFNAR1) expression in spinal cord of saline-treated control EAE mice were detected, they were significantly increased after MAT treatment. Increased numbers of CD11b+IFN-ß+ microglia/infiltrating macrophages were observed in the CNS of MAT-treated mice. The key role of IFN-ß induction in the suppressive effect of MAT on EAE was further verified by administration of anti-IFN-ß neutralizing antibody, which largely reversed the therapeutic effect of MAT. Further, we found that, while MAT treatment induced production of IL-27 and IL-10 by CNS microglia/macrophages, this effect was significantly reduced by IFN-ß neutralizing antibody. Finally, the role of IFN-ß in MAT-induced IL-27 and IL-10 production was further confirmed in human monocytes in vitro. Together, our study demonstrates that MAT exerts its therapeutic effect in EAE through an IFN-ß/IL-27/IL-10 pathway, and is likely a novel, safe, low-cost, and effective therapy as an alternative to exogenous IFN-ß for MS.

Matrine protects oligodendrocytes by inhibiting their apoptosis and enhancing mitochondrial autophagy.

Stressed oligodendrocytes (OLGs) activate microglia to produce an inflammatory response, and the impairment of mitochondria further aggravates OLG damage, which is the earliest pathological change in multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system. Matrine (MAT), a tetracyclic quinolizine alkaloid derived from the herb radix sophorae flavescentis, has been shown to effectively ameliorate clinical signs of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. However, the mechanisms underlying the effect of MAT treatment need to be further studied. In the present study, we show that MAT effectively suppressed ongoing EAE, and significantly reduced the expression of caspase-3 and alpha B-crystallin in OLGs, therefore lessen OLG apoptosis, microglial activation and inflammatory factors secretion. MAT treatment also reduced the content of cytochrome c and malondialdehyde, an oxidative stress marker, in the central nervous system. In contrast, the levels of autophagy-related proteins Beclin1, microtubule-associated protein l light chain 3 and glutathione peroxidase was upregulated, hence enhancing mitochondrial autophagy and alleviating the imbalance of the oxidation/antioxidation system caused by mitochondrial damage. Our research indicates that MAT is effective in treating EAE, at least in part, by protecting OLGs through inhibiting their apoptosis and enhancing mitochondrial autophagy.

Matrine Treatment Blocks NogoA-Induced Neural Inhibitory Signaling Pathway in Ongoing Experimental Autoimmune Encephalomyelitis.

Myelin-associated inhibitors, such as NogoA, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp), play a pivotal role in the lack of neuroregeneration in multiple sclerosis, an inflammatory demyelinating disease of the central nervous system (CNS). Matrine (MAT), a monomer that is used in traditional Chinese medicine as an anti-inflammatory agent, has shown beneficial effects in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. However, the underlying mechanisms of MAT-induced EAE amelioration are not fully understood. In the present study, we show that MAT treatment suppressed ongoing EAE, and this effect correlated with an increased expression of growth-associated protein 43, an established marker for axonal regeneration. MAT treatment significantly reduced the levels of NogoA, its receptor complex NgR/p75NTR/LINGO-1, and their downstream RhoA/ROCK signaling pathway in the CNS. In contrast, intracellular cyclic AMP (cAMP) levels and its protein kinase (protein kinase A (PKA)), which can promote axonal regrowth by inactivating the RhoA, were upregulated. Importantly, adding MAT in primary astrocytes in vitro largely induced cAMP/PKA expression, and blockade of cAMP significantly diminished MAT-induced expression of PKA and production of BDNF, a potent neurotrophic factor for neuroregeneration. Taken together, our findings demonstrate that the beneficial effects of MAT on EAE can be attributed not only to its capacity for immunomodulation, but also to its directly promoting regeneration of the injured CNS.

Matrine downregulates IL-33/ST2 expression in the central nervous system of rats with experimental autoimmune encephalomyelitis.

Interleukin (IL)-33 is a recently described member of the IL-1 family and functions as a ligand for ST2, a member of the IL-1 receptor family. The role of IL-33/ST2 axis in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an experimental model of multiple sclerosis (MS), remains controversial. Matrine (MAT), a quinolizidine alkaloid derived from the herb Radix Sophorae Flave, has been recently found to suppress clinical EAE and CNS inflammation. However, the underlying immunoregulatory mechanisms have not been fully elucidated, and whether this effect of MAT is through inhibiting the function of the IL-33/ST2 axis is not known. In this study, we investigated the relationship between the therapeutic effects of MAT and IL-33/ST2 expression. MAT treatment successfully attenuated severe clinical deficit and histopathological changes, compared to untreated controls. While IL-33/ST2 mRNA expression was largely increased in spinal cord of EAE rats compared to naïve rats, this expression was significantly inhibited in rats treated with MAT. These results were further confirmed by their protein levels tested with immunohistochemistry. Together, our study demonstrates that MAT treatment regulates the inflammatory IL-33/ST2 axis, thus being a novel mechanism underlying the effect of MAT.

Protective effects of matrine on experimental autoimmune encephalomyelitis via regulation of ProNGF and NGF signaling.

Inflammation, demyelination, oligodendrocyte (OLG) death, and axonal degeneration are primary characteristics of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). OLGs generate myelin sheaths that surround axons, while damage to OLGs leads to demyelination and neurological functional deficit. Matrine (MAT), a quinolizidine alkaloid derived from the herb Radix Sophorae Flave, has been recently found to effectively ameliorate clinical signs in EAE. Its therapeutic mechanism has, however, not been completely elucidated. In the present study, we found that MAT retarded the disease process, attenuated the clinical severity of EAE rats, ameliorated inflammation and demyelination, and suppressed the apoptosis of OLGs in the central nervous system (CNS) of EAE rats. In addition, MAT markedly blocked increased expression of the proNGF-p75(NTR) death signaling complex, which is known to mediate OLG death in EAE animals. At the same time, MAT also prevented a decrease in the levels of NGF and its receptor TrkA, which together mediate the cell survival pathway and differentiation of OLGs. ProNGF, NGF, and the downstream effector proteins play an important role in the growth, differentiation, and apoptosis of OLGs as well as the reparative response to neuronal damage. These findings thus indicate that MAT improves clinical severity of EAE in part by reducing OLG apoptosis via restoring the ratios of proNGF:NGF and the respective receptors p75(NTR):TrkA in vivo. Taken together, these results suggest that MAT may be a promising agent for MS treatment based on its protective effect on OLGs.