Study on the mechanism of Orthosiphon aristatus (Blume) Miq. in the treatment of hyperuricemia by microbiome combined with metabonomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Growing evidence indicates that hyperuricemia is closely associated with gut microbiota dysbiosis. Orthosiphon aristatus (Blume) Miq. (O. aristatus), as a traditional Chinese medicine, has been widely used to treat hyperuricemia in China. However, the mechanism by which O. aristatus treats hyperuricemia has not been clarified. AIM OF THE STUDY: In this study, we investigated whether the molecular mechanism underlying the anti-hyperuricemia effect of O. aristatus is related to the regulation of gut microbiota by 16S rDNA gene sequencing combined with widely targeted metabolomics. MATERIALS AND METHODS: Hyperuricemia was induced in rats by administration of 10% fructose and 20% yeast, and the uricosuric effect was assessed by measuring the uric acid (UA) levels in serum and cecal contents. Intestinal morphology was observed by hematoxylin and eosin (HE) staining. To explore the effects of O. aristatus on the gut microbiota and its metabolites, we utilized 16S rDNA gene sequencing combined with widely targeted metabolomics. Furthermore, metabolic pathway enrichment analysis was performed on the screened differential metabolites. The real time quantitative polymerase chain reaction (RT-PCR) and western blotting (WB) were used to detect the expression of relevant proteins in the key pathway. RESULTS: Our results indicated that O. aristatus intervention decreased serum UA levels and increased the UA levels in cecal contents in hyperuricemic rats. Additionally, O. aristatus improved intestinal morphology and altered the composition of the gut microbiota and its metabolites. Specifically, 16S rDNA revealed that O. aristatus treatment significantly reduced the abundance of unidentified-Ruminococcaceae and Lachnospiraceae-NK4A136-group. Meanwhile, widely targeted metabolomics showed that 17 metabolites, including lactose, 4-oxopentanoate and butyrate, were elevated, while 55 metabolites, such as flavin adenine dinucleotide and xanthine, were reduced. Metabolic pathway enrichment analysis found that O. aristatus was mainly involved in purine metabolism. Moreover, RT-PCR and WB suggested that O. aristatus could significantly up-regulate the expression of UA excretion transporter ATP-binding cassette subfamily G member 2 (ABCG2) in the intestine. CONCLUSION: O. aristatus exerts UA-lowering effect by regulating the gut microbiota and ABCG2 expression, indicating that this herb holds great promise in the treatment of hyperuricemia.

Exploring the effect of the Uyghur medicine Munziq Balgam on a collagen-induced arthritis rat model by UPLC-MS/MS-based metabolomics approach.

ETHNOPHARMACOLOGICAL RELEVANCE: Munziq Balgam (MBm) is a classic preparation of a traditional Uyghur medicine used for many years to treat abnormal body fluid diseases. The formula, as an in-hospital preparation, has already been used in the Hospital of Xinjiang Traditional Uyghur Medicine to treat rheumatoid arthritis (RA) with significant clinical effects. AIM OF THE STUDY: This study intends to reveal the intervention effect of MBm on collagen-induced arthritis (CIA) rats, discover the potential biomarkers with efficacy, and explore the mechanisms of metabolic regulation by using metabolomics method. MATERIAL AND METHODS: Sprague Dawley (SD) rats were randomly divided into five groups: blank group, CIA model group, Munziq Balgam nomal-dosage, Munziq Balgam high-dosage group and control group. Body weight, paw swelling, arthritis index, immune indices and histopathological experiments were carried out. Plasma from rats were detected by UPLC-MS/MS. Metabolomics of plasma was performed to analyze metabolic profiles, potential biomarkers, and metabolic pathways of MBm for CIA rats. The main metabolic result of Uyghur medicine MBm was compared with that of Zhuang medicine Longzuantongbi granules (LZTBG) to explore the characteristics of two ethnic medicines from different regions for RA. RESULTS: MBm could significantly alleviate symptoms of CIA rats by relieving arthritis symptoms on paw redness and swelling, inflammatory cell infiltration, synovial hyperplasia, pannus, cartilage and bone tissue destruction, as well as inhibiting the expression of IL-1ß, IL-6, TNF-α, UA and ALP. Linoleic acid, alpha-linolenic acid, pantothenate and CoA biosynthesis, achidonic acid, gycerophospholipid, sphingolipid metabolism, primary bile acid biosynthesis, porphyrin and chlorophyll metabolism and fatty acid degradation served as the main nine pathways of the interventional effect of MBm on CIA rats. Twenty-three different metabolites were screened out and strongly associated with the indicator makes of RA. Eight potential efficacy-related biomarkers were finally discovered in metabolic pathway network (phosphatidylcholine, bilirubin, sphinganine 1-phosphate, phytosphingosine, SM (d18:1/16:0), pantothenic acid, l-palmitoylcarnitine, chenodeoxycholate). Three metabolites (chenodeoxycholate, hyodeoxycholic acid and O-palmitoleoylcarnitine) were changed in both the metabolic study of MBm and LZTBG intervention effects on CIA rats. Additionally, MBm and LZTBG shared the same 6 metabolic pathways including linoleic acid, alpha-linolenic acid, pantothenate and CoA biosynthesis, achidonic acid, gycerophospholipid, and primary bile acid biosynthesis. CONCLUSION: The study suggested that MBm may effectively alleviate RA by regulating inflammation, immunity-related pathways and multiple targets. Metabolomics analysis showed that MBm (Xinjiang, the north of China) and LZTBG (Guangxi, the south of China), two ethnic medicines from different regions in China, share common metabolites and pathways but also have distinct differences in their interventions for RA.

Functional and binding studies of gallic acid showing platelet aggregation inhibitory effect as a thrombin inhibitor.

Objective: This study was devoted to identifying natural thrombin inhibitors from traditional Chinese medicine (TCM) and evaluating its biological activity in vitro and binding characteristics. Methods: A combination strategy containing molecular docking, thrombin inhibition assay, surface plasmon resonance (SPR) and molecular dynamics simulation were applied to verify the study result. Results: Gallic acid was confirmed as a direct thrombin inhibitor with IC50 of 9.07 µmol/L and showed a significant inhibitory effect on thrombin induced platelet aggregation. SPR-based binding studies demonstrated that gallic acid interacted with thrombin with a KD value of 8.29 µmol/L. Molecular dynamics and binding free energy analysis revealed that thrombin-gallic acid system attained equilibrium rapidly with very low fluctuations, the calculated binding free energies was -14.61 kcal/mol. Ala230, Glu232, Ser235, Gly258 and Gly260 were the main amino acid residues responsible for thrombin inhibition by gallic acid, providing a mechanistic basis for further optimization. Conclusion: This study proved that gallic acid is a direct thrombin inhibitor with platelet aggregation inhibitory effect, which could provide a basis for the follow-up research and development for novel thrombin inhibitors.

The therapeutic mechanism of PuRenDan for the treatment of diabetic nephropathy: Network pharmacology and experimental verification.

ETHNOPHARMACOLOGICAL RELEVANCE: Purendan (PRD), as a Chinese medicinal formula, behaves remarkable therapeutic effects on diabetes and complications in clinical and experimental research. However, the underlying pharmacological mechanism in the treatment of diabetic nephropathy (DN) is still unclear. AIMS: To investigate the therapeutical effects of PRD on DN and to explore its pharmacological mechanisms using network pharmacology and experimental verification. MATERIALS AND METHODS: The active compounds and putative targets in PRD, and disease-related targets of DN were extracted from public databases. The key targets were identified through the protein-protein interaction (PPI) network and module analysis. The GO and KEGG enrichment analysis were performed to discover potential pharmacological mechanisms. The expression of the key targets was detected in kidney tissue in Gene Expression Omnibus (GEO) dataset. The affinity between key proteins and corresponding compounds was evaluated by molecular docking and validated by the surface plasmon resonance (SPR) assay. The indicators on major pathways and hub genes were verified by in vivo experiments. RESULTS: In network pharmacology, 137 common targets in PRD for DN treatment were screened. The key targets and main signaling pathways including AGE-RAGE and lipid pathways were identified. The statistical difference in the expression of the key targets was verified in GSE96804 database, confirming the association with DN. The docking scores obtained from molecular docking illustrated good binding force between hub proteins and active compounds. And the good component-protein affinities were validated by SPR assay. Furthermore, the results of animal experiment indicated that PRD could ameliorate the level of serum glucose and renal function in rat model. It could regulate the expression of hub targets (AKT1, MAPK3, and STAT3) and improve indicators related with oxidative stress and lipid metabolism. CONCLUSION: The key targets and major signaling pathways in the treatment of PRD on DN were identified. The mechanism might relate to regulation of oxidative stress and lipid metabolism.

Purendan alleviates non-alcoholic fatty liver disease in aged type 2 diabetic rats via regulating mTOR/S6K1/SREBP-1c signaling pathway.

Older people are more likely to develop insulin resistance and lipid metabolism disorders. Purendan (PRD) is a clinically verified traditional Chinese medicine compound, which plays an obvious role in regulating lipid metabolism disorder and improving insulin sensitivity. Our study aimed to investigate the efficacy and mechanism of PRD on aged type 2 diabetes mellitus (T2DM) complicated with non-alcoholic fatty liver disease (NAFLD) rats. Sprague-Dawley rats (13 months) were fed with high-fat diet (HFD) and injected with low-dose STZ to replicate T2DM model. PRD was treated at three concentrations with metformin as a positive control. After administration, blood and liver tissue samples were collected to measure glucose metabolism indexes such as serum glucose and insulin, as well as lipid metabolism indexes such as TC, TG, LDL, HDL and FFA. Liver fat accumulation was observed by HE staining and oil red O staining. And protein expression levels of mTOR, p-mTOR, S6K1, p-S6K1 and SREBP-1c were detected by western blot. After PRD treatment, not only the insulin sensitivity and insulin resistance were significantly improved, but also the TC, TG, LDL, FFA, AST and ALT in serum and the lipid accumulation in liver tissue were significantly decreased. Moreover, PRD significantly down-regulated the expression of p-mTOR, p-S6K1 and SREBP-1c in liver tissues. In conclusion, PRD can alleviate NAFLD in aged T2DM rats by inhibiting the mTOR /S6K1/ SREBP-1c pathway.

Comprehensive pan-cancer analysis on CBX3 as a prognostic and immunological biomarker.

BACKGROUND: Increased evidence supports the relationship between chromobox protein homolog 3 (CBX3) and tumorigenesis of some cancers. However, the role of CBX3 in pan-cancers remains poorly defined. In the research, we aimed to investigate the prognostic value and the immunological functions of CBX3. RESULTS: We explored the potential oncogenic roles of CBX3 in mRNA and protein levels based on the diverse databases, including the expression, the correlation with prognosis, tumor microenvironment (TME), DNA methylation, protein phosphorylation and enrichment analysis across all TCGA tumors. The results show that CBX3 is overexpressed in multiple cancers, and significant correlations exist between high expression and adverse prognosis in most tumor patients. We observed an enhanced phosphorylation level in uterine corpus endometrial carcinoma, colon cancer and lung adenocarcinoma. A distinct relationship was also found between CBX3 expression and TME, including immune infiltration of tumor-infiltrating lymphocytes and cancer-associated fibroblasts, immune score or matrix score, immune checkpoints. The correlative transcription factors and miRNAs of CBX3-binding hub genes were analyzed to investigate the molecular mechanism. Moreover, alcoholism and alteration of DNA cellular biology may be involved in the functional mechanisms of CBX3. CONCLUSION: The first pan-cancer study offers a relatively comprehensive cognition on the oncogenic roles of CBX3 as a prognostic and immunological marker in various malignant tumors.

Bioactivity-guided separation of potential α-glycosidase inhibitor from clerodendranthus spicatus based on HSCCC coupled with molecular docking.

Clerodendranthus Spicatus is a traditional Dais medi-edible plant and it has been proven to have good blood glucose-lowering efficacy. However, the material basis of Clerodendranthus Spicatus has not been clarified yet and therefore needs to be determined. In this paper, the effective ingredients of this medicine were purified by high-speed counter-current chromatography. Alongside, their potential hypoglycemic activity was determined by α-glucosidase inhibitory activities in vitro and molecular docking. Finally, five compounds were purified and identified as 2-caffeoyl-L-tartaric acid (1), N-(E)-caffeoyldopamine (2), rosmarinc acid (3), methyl rosmarinate (4), 6,7,8,3',4'-Pentamethoxyflavone (5). Examination of α-glucosidase inhibitory activity in vitro showed that 2-caffeoyl-L-tartaric acid and rosmarinic acid had a higher inhibitory activity than acarbose. Molecular docking indicated that the affinity energy of the identified compounds ranged from - 7.6 to - 8.6 kcal/mol, a more desirable result than acarbose (- 6.6 kcal/mol). Particularly, rosmarinc acid with the lowest affinity energy of - 8.6 kcal/mol was wrapped with 6 hydrogen bonds. Overall, α-glucosidase inhibitory activities and molecular docking suggested that rosmarinc acid was likely to be a promising hypoglycemic drug.

The spectrum-effect relationship-a rational approach to screening effective compounds, reflecting the internal quality of Chinese herbal medicine.

Since the chromatographic fingerprint was introduced, it has been accepted by many countries to assess the quality and authenticity of Chinese herbal medicine (CHM). However, solely using the chromatographic fingerprint to assay numerous chemicals is not suitable for the assessment of the whole internal quality and pharmacodynamics of CHM. Consequently, it is necessary to develop a rational approach to connecting the chromatographic fingerprint with effective components to assess the internal quality of CHM. For this purpose, a spectrum-effect relationship theory was proposed and accepted as a new method for the assessment of CHM because of its potential use to screen effective components from CHM. In this paper, we systematically reviewed the application of the spectrum-effect relationship theory in the research of CHM, including research mentality, different chromatographic analysis techniques, data processing technologies, and structure determination.

[Molecular docking analysis of xanthine oxidase inhibition by constituents of cichory].

Human xanthine oxidase is considered to be a target for therapy of hyperuricemia. Cichorium intybus is a Chinese plant medicine which widely used in Xinjiang against various diseases. In order to screen the inhibitors of xanthine oxidase from C. intybus and to explore main pharmacological actions of cichory a compound collection of C. intybus was built via consulting related references about chemical research on cichory. The three-dimensional crystal structure of xanthine oxidase (PDB code: 1N5X) from Protein Data Bank was downloaded.. Autodock 4.2 was employed to screen the inhibitors of xanthine oxidase from cichory 70 compounds were found to possess quite low binding free energy comparing with TEI (febuxostat). C. intybus contains constituents possessing potential inhibitive activity against xanthine oxidase. It can explain the main pharmacological actions of cichory which can significantly lower the level of serum uric acid.