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.

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.

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 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.

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.

Matrine ameliorates experimental autoimmune encephalomyelitis by modulating chemokines and their receptors.

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are autoimmune diseases characterized by immune-mediated neuroinflammation, demyelination and neurodegeneration of the central nervous system (CNS). While matrine (MAT), a monomer that is used in traditional Chinese medicine as an anti-inflammatory treatment, delayed onset and ameliorated severity of EAE, the underlying mechanisms have not been fully elucidated. In this study, we investigated the relationship between the clinical effect of MAT and the levels of certain important chemokines/chemokine receptors. Our results showed that attenuated severity of EAE resulting from MAT treatment was positively correlated with the reduction of CCL2 and CXCL10 levels in the periphery and the CNS; both of these chemokines play a crucial role in the recruitment and accumulation of inflammatory cells, especially monocytes/macrophages and T cells, into the CNS. The levels of their corresponding receptors, CCR2 and CXCR3, were also significantly reduced after MAT treatment. Taken together, our data indicate that MAT may be an effective immunomodulatory therapeutic approach for MS/EAE by countering the immune cell recruitment mechanisms.