Vascular cognitive impairment (VCI) is a vast disease with chronic cerebral hypoperfusion (CCH) being the major driver. In our previous study, we have verified that Da ChuanXiong (DCX) formula could alleviate VCI, and obviously upregulate the decreased cerebral blood flow, indicating its potential benefits on vascular remodeling. This study aims to explore the pro-angiogenic and vasodilatory effects of DCX formula after CCH injuries, and clarify its pharmacodynamic substances and potential mechanisms. The rat model of CCH was established by bilateral common carotid artery occlusion. CD31+ immunofluorescent staining and quail chorioallantoic membrane (qCAM) experiments were conducted to assess the angiogenic effect of DCX. Isolated vascular ring experiments and NO assay in qCAM were performed to evaluate the vasodilatory effect of DCX. Then we analyzed the chemical components absorbed in the blood of DCX under CCH conditions by UPLC-Q-TOF/MS, and further combined network pharmacology, molecular docking to predict the potential mechanisms and effective components. Western blot was performed in CCH rats to verify the possible mechanisms and in vitro pharmacodynamic verification experiment was conducted for innovative active ingredients in DCX. Our results revealed that DCX obviously increased the number of CD31+ cells in the hippocampus and brain cortex of CCH rats, and significantly increased the vascular areas in qCAM. DCX significantly dilated the pre-constricted vascular ring and increased NO contents in qCAM. Besides, Senkyunolide A, Butylphthalide, Ferulic acid, Senkyunolide I, Levistilide A, Ligustilide, Senkyunolide H and Gastrodin were identified as the 8 major blood-absorbed compounds of DCX under CCH condition. Next, network pharmacology and molecular docking revealed NOS3 and PI3K/Akt signaling as the critical targets and pathway of DCX improving vascular remodeling against CCH injuries, which was further validated by western blot. Moreover, we firstly discovered that Senkyunolide A and Senkyunolide H could obviously promote cell migration and enhance the tube formation ability of HUVECs in vitro. Taken together, DCX formula effectively enhanced the angiogenesis and vasodilation via activating PI3K/Akt/eNOS signaling, which might be the important pathways for DCX promoting vascular remodeling against CCH injuries. Notably, Senkyunolide A and Senkyunolide H were firstly identified as innovative components responsible for the pro-angiogenic effects of DCX.

Chaihu Guizhi Ganjiang Decoction (CGGD) is a clinically proven prescription effective against metabolic dysfunction-associated steatohepatitis (MASH). However, its chemical constituents with the lipid-lowering and anti-inflammatory bioactivities and underlying mechanisms remain unclear. This study aimed to characterize the chemical profile of CGGD while elucidating their lipolytic and anti-inflammatory activities and mechanisms. Using UHPLC-Q-TOF-MS/MS, 209 compounds were identified in CGGD, categorized into 52 saponins, 103 flavonoids, 17 gingerols, 21 organic acids, and 16 others, with 13 putative unvalidated new compounds. Nineteen prototype components were detected in hepatic tissues, with saikosaponin A, baicalin, wogonoside, skullcapflavone II, and glycyrrhizin exhibiting strong binding affinities toward PPARα and TLR4 proteins. These components significantly reduced triglyceride (TG) accumulation in FFA-induced HepG2 steatosis cells, confirmed by oil red O staining and TG quantification. Mechanistically, saikosaponin A and baicalin upregulated PPARα and its downstream genes (CPT1A, FABP1, ACOX1) to promote fatty acid transport and oxidation. Meanwhile, saikosaponin A, baicalin, and wogonoside significantly suppressed mRNA expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in LPS-stimulated RAW264.7 macrophages, and saikosaponin A and wogonoside further inhibited the expression of TLR4, MyD88, and phosphorylated NFκB, thereby attenuating inflammation. In conclusion, this study established a comprehensive methodology for profiling the chemical constituents of CGGD and identified saikosaponin A, baicalin, wogonoside, skullcapflavone II, and glycyrrhizin as its key anti-MASH components. Their lipid-lowering and anti-inflammatory activities were mediated through the PPARα-regulated fatty acid metabolism pathway and the TLR4/MyD88/NFκB signaling pathway, respectively, offering a scientific basis for the clinical application of CGGD in MASH.

ETHNOPHARMACOLOGICAL RELEVANCE:Neuroinflammation in the trigeminal nucleus caudalis (TNC) plays an important role in the pathological process of chronic migraine (CM). The Paeoniae Radix Alba (Baishao, BS)-Chuanxiong Rhizoma (Chuanxiong, CX) herb pair (BSCX) is widely used in the treatment of migraine, but its mechanism of action and representative candidate constituents remain unclear. AIM OF THE STUDY:This study aimed to evaluate the effects of BSCX on TNC neuroinflammation and to explore its representative candidate constituents and potential mechanisms associated with microglial inflammatory phenotype-related changes and P2Y12R/PPARγ-related NF-κB signaling. MATERIALS AND METHODS:A nitroglycerin-induced rat model of CM was used to evaluate the anti-migraine effects of BSCX through behavioral testing and molecular analyses. ELISA, RT-qPCR, immunofluorescence staining, and Western blot were used to evaluate changes in inflammatory factors, pro-/anti-inflammatory phenotype-related markers, and pathway-related proteins in blood samples and the TNC. Potential candidate constituents in the TNC were identified using ultrahigh-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS). The associations between the candidate constituents and their putative targets were further investigated by integrating molecular docking, molecular dynamics simulation, drug affinity responsive target stability (DARTS) assays, and cellular thermal shift assays (CETSA). A lipopolysaccharide (LPS)-induced BV2 cell model was established, with additional interventions using the P2Y12R agonist 2-MeS-ADP and the PPARγ inhibitor GW9662. Flow cytometry was used to assess CD86/CD206-positive cells and phagocytic activity, while immunofluorescence staining and Western blot were used to evaluate related protein expression and signaling changes. RESULTS:BSCX alleviated NTG-induced migraine-like behaviors and pain sensitization. Compared with the model group, the medium- and high-dose BSCX groups showed reduced spontaneous head-scratching episodes, increased periorbital mechanical withdrawal threshold, and prolonged thermal withdrawal latency. BSCX reduced the expression of CGRP and c-Fos in the TNC by 16.2%-21.6% and 19.6%-55.1%, respectively. BSCX also reduced serum TNF-α and IL-1β levels while increasing IL-10 and TGF-β levels. Immunofluorescence analysis of the TNC further showed that BSCX decreased the proportion of iNOS+/Iba1+ cells by 49.8%-86.2% and increased the proportion of Arg-1+/Iba1+ cells by 222%-271%. These changes were accompanied by reduced expression of P2Y12R/RhoA/ROCK2/NF-κB-related proteins and increased PPARγ expression. Among the constituents detected in the TNC, benzoylpaeoniflorin (Ben) and senkyunolide I (SENI) reduced LPS-induced TNF-α release by 13.7% and 21.6%, respectively, at 2.5 μM, and increased IL-10 release by 18.1% and 21.0%, respectively, at 5 μM. Molecular docking showed favorable binding energies for the P2Y12R-Ben and PPARγ-SENI complexes. Molecular dynamics simulations further showed that their binding free energies were -23.31 and -22.06 kcal/mol, respectively, which were more favorable than those of the corresponding cross-combinations. In DARTS and CETSA assays, Ben enhanced the protease resistance and thermal stability of P2Y12R, while SENI enhanced the protease resistance and thermal stability of PPARγ. In reversal experiments, 2-MeS-ADP increased the phagocytic activity and P2Y12R expression relative to the Ben group by 40.6% and 76.8%, respectively; GW9662 increased phagocytic activity by 41.7% and decreased PPARγ expression by 26.8% relative to the SENI group. CONCLUSIONS:BSCX alleviates TNC neuroinflammation in CM, accompanied by regulation of pro-/anti-inflammatory phenotype-related markers and changes in P2Y12R/RhoA/ROCK2/NF-κB signaling and PPARγ-related signaling. Ben and SENI may represent candidate constituents associated with the P2Y12R- and PPARγ-related signaling branches, respectively, and may partly contribute to the pharmacological effects of the BSCX herb pair against CM.

The occurrence of isomeric dihydroflavone and chalcone, two important subfamilies of flavonoid class, extensively happens in herbal medicines. However, identical MS/MS spectra make the identity confirmation a tough job, the complexity will be further boosted in biological samples. Inspired by that isomers possess distinct inherent physicochemical parameters, optimal collision energy (OCE), which is positively correlated with the bond dissociation energies (BDEs), was evaluated towards differentiating isomeric dihydroflavone and chalcone. It was achieved by plotting their relative response-collision energy curves (RRCECs) and comparing the unique OCEs obtained. As a result, difference were observed for either OCE of bond dissociations involving Retro Diels-Alder (RDA) reaction as well as glycosidic cleavage when comparing two pairs of isomers, which were liquiritin vs. isoliquiritin and liquiritigenin vs. isoliquiritigenin, because the energies required for bond dissociation to produce identical fragment ions varies among isomers. Furthermore, OCEs consistently ranked dihydroflavone higher than chalcone, aligning with their BDEs determined through quantum calculation. These results suggested great potential for OCEs in distinguishing between dihydroflavone and chalcone. By applying the discriminating criterion, successful identity recognition was achieved for the two pairs of isomers among the many signals sharing identical MRM transitions in honey-fried licorice treated plasma. Thereafter, the confidence-enhanced OCE-LC-MS/MS method was applied to characterize and determine these 4 isomers along with 3 components of honey-fried licorice in rat plasma, the definite determination as well as the pharmacokinetic courses were subsequently accomplished and discussed. Overall, this study boosted the application of OCE-LC-MS/MS in isomers discrimination of isomeric dihydroflavone and chalcone.

ETHNOPHARMACOLOGICAL RELEVANCE:Slow transit constipation (STC) is a common gastrointestinal disease. Zhutong Decoction (ZTD), a traditional empirical prescription, is composed of Zhizhu Decoction and Prunus persica (L.) Batsch. ZTD can strengthen the spleen by advancing Qi and promote defecation by moistening the intestine. ZTD is commonly employed clinically for the treatment of constipation caused by insufficient gastrointestinal peristalsis. However, the mechanisms of ZTD in treating constipation are still unknown. AIM OF THE STUDY:This study aimed to evaluate the therapeutic efficacy of ZTD in treating STC, and explore its pharmacodynamic substances and underlying mechanisms. MATERIALS AND METHODS:Therapeutic effect of ZTD in STC was evaluated using loperamide-induced STC rat model. Immunofluorescence, western blotting, and RT-qPCR were used to detect the expression of proteins and genes associated with the intestinal motility. Chromatography and mass spectrometry were utilized to identify the potential active components present in ZTD that are absorbed in the rat plasma and colon. Ex vivo intestine motility experiments were conducted to assess the pharmacodynamic substances and elucidate their mechanisms of action. RESULTS:ZTD significantly ameliorated constipation symptoms, including reduced fecal number, shorter stool length, and decreased gastrointestinal transit ratio. ZTD restored balance in the SIP syncytium, which is composed of smooth muscle cells (SMC), interstitial cells of Cajal (ICC), and platelet-derived growth factor receptor α positive cells (PDGFRα+ cells), by upregulating the expression of C-KIT/ANO1 and downregulating the expression of PDGFRα/SK3. Furthermore, ZTD increased SMC contractions in the SIP syncytium through the CAM/MLCK/CPI-17 pathway. In the duodenum, atractylenolide II and atractylenolide III induced contractions through ANO1 and SK3, whereas hesperidin, neohesperidin, and naringin induced contractions only through ANO1. In the colon, atractylenolide II, atractylenolide III, and nobiletin promoted contractions via both ANO1 and SK3. CONCLUSIONS:ZTD ameliorated loperamide-induced STC in rats by promoting intestinal peristalsis, and the mechanisms related to enhanced SMC contractility involved restoring the balance between C-KIT/ANO1 and PDGFRα/SK3 in the SIP syncytium.

BACKGROUND:Ischemic stroke remains a major disease leading high disability and mortality worldwide, however, even after timely thrombolytic therapy, the secondary brain injuries induced by cerebral ischemia-reperfusion still cannot be ignored. After cerebral ischemia/reperfusion, abnormal membrane lipids metabolism usually occurred in the brain, which further aggravated the brain injuries and neurological dysfunction. Da ChuanXiong (DCX) Formula, a classic prescription composed of Gastrodia elata Bl. and Ligusticum chuanxiong Hort., exhibits considerable promise in treating ischemic cerebrovascular diseases. This study aims to explore whether DCX could improve cerebral ischemia/reperfusion injury (CIRI) by reshaping membrane lipids homeostasis as well as illustrating the relevant molecular mechanisms. METHODS:Firstly, DCX extract was delivered to middle cerebral artery occlusion-reperfusion (MCAO/R) rats, and neurological function scores, TTC staining and oxidative stress kit assays were conducted to evaluate the neuroprotective effects of DCX. Then, the integrated metabolomics studies of plasma and brain tissue as well as the construction of metabolic network were performed to further detect the alterations of metabolites, unearth the key targets and signaling pathways related to membrane lipid metabolism. Finally, the molecular biological techniques such as real-time PCR and western blot were employed to verify the expression levels of key targets associated with glycerophospholipid metabolism. RESULTS:After CIRI, DCX treatment relieved neurological function deficits, reduced the cerebral infarction areas and elevated the levels of Ach in the brain. Additionally, DCX treatment alleviated the oxidative stress damages via elevating the expressions of GSH and SOD while reducing the level of MDA. Metabolomics studies revealed that DCX was able to reverse 34 differential metabolites in plasma and 40 differential metabolites in brain tissues. Notably, phospholipid-related metabolites were dominant among the differential metabolites, and DCX intervention reversed the abnormality of these differential metabolites. For example, DCX treatment obviously elevated the contents of choline and phosphocholine in the brain tissues, upregulated the contents of PC(16:0/18:2) and PC(16:1(9Z)/18:2(9Z,12Z)), and downregulated the contents of LysoPC, such as LysoPC(14:0), LysoPC(15:0), LysoPC(20:4(5Z,8Z,11Z,14Z)), LysoPC(18:2(9Z,12Z)), LysoPC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)) and LysoPC(P-16:0). KEGG enrichment analyses presented that glycerophospholipid metabolism may be the key regulatory pathway. The constructed metabolic network indicated that PLA2G10, PLA2G2A, PLA2G6, ACHE, CHAT, CHPT1, LPCAT1, LCAT, LPGAT1 and PCYT1B were possible to be ten crucial targets by which DCX improved CIRI via modulating membrane phospholipid metabolisms. Finally, PCR and western blot experiments further testified that DCX could significantly upregulate the expression levels of CHAT and PCYT1B, and obviously downregulate the expression levels of ACHE and PLA2G2A. CONCLUSION:DCX played obvious neuroprotective effects against CIRI. Mechanistically, DCX maintained the balance of lipid metabolites in MCAO/R rats, reshaped cell membrane phospholipids and sustained the stability of cell membrane structure directly or indirectly. And ACHE, CHAT, PCYT1B and PLA2G2A were the crucial metabolic enzyme targets of DCX treating CIRI via regulating membrane phospholipid homeostasis. These findings offered new insights into the potential neuroprotective mechanisms of DCX treating CIRI from the perspective of membrane lipid metabolism for the first time.

Background: Qishen granule (QSG) is a widely prescribed herbal formula for the treatment of chronic heart failure. The mechanisms of action of QSG have been clarified; however, the effective substances remain unclear. This lack of clarity hinders quality control and the consistency of the clinical efficacy of QSG. Methods: In the present study, an integrated strategy for an efficacy- and in vivo exposure-oriented study involving metabolite profiling, molecular docking, in vitro bioassays, and in vivo pharmacokinetics was proposed for investigating the potentially effective components of QSG. Results: In total, 101 prototypes/metabolites were preliminarily identified and characterized by UHPLC-Q TOF-MS/MS. Molecular docking of the absorbed constituents with targeted proteins suggested that 49 potential components were highly related to chronic heart failure (CHF). Then, the effectiveness of these potential compounds was verified by the oxygen glucose deprivation/re-oxygenation (OGD/R)-induced H9c2 cell model. As a result, 14 active components were screened, and their median effective concentration (EC50) was calculated and utilized to generate the weight coefficient for the bioeffect of each constituent. By exploring the kinetic parameters of the active compounds in a pharmacokinetic study, the exposure levels of these pharmacologically active compounds were determined by area under the curve (AUC0→∞) calculations. Finally, by calculating the effect–constituent index (ECI) for each compound, five key active components (cryptochlorogenic acid, chlorogenic acid, isochlorogenic acid C, salvianolic acid B, and neochlorogenic acid), which possess both pharmacological activities and higher exposure levels, were revealed to be the key effective substances of QSG. Conclusions: This study is the first to combine pharmacological activities with in vivo exposure for investigating the effective components of QSG. The identification of key active components provides a foundation for improving the quality control of QSG in clinics. The efficacy- and in vivo exposure-oriented integrated method could provide reliable references for other traditional Chinese medicines (TCMs).

Purpose:The effect of metabolic factors on cardiovascular risk in obstructive sleep apnea (OSA) is unclear. This study aimed to investigate the effect of metabolic factors on the left ventricular diastolic function in patients with OSA. Patients and Methods:This cross-sectional study included a total of 478 patients with OSA from September 2018 to September 2023. After propensity score matching, wherein 193 patients with OSA with metabolic syndrome (MS) were 1:1 matched to patients with OSA without MS by sex and age, data from 386 patients were ultimately analyzed. Furthermore, all patients were divided into mild, moderate, and severe OSA groups according to their sleep apnea-hypopnea index (AHI). Measurements included nocturnal polysomnography, biochemical testing, and transthoracic echocardiography data. Results:The AHI in the MS group was higher (30.24±21.69 vs 23.19±17.65, p<0.001) and the lowest oxygen saturation at night was lower (77.67±9.23 vs 80.59±9.26, p<0.001) than those in the non-MS group. Additionally, the left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), end-diastolic ventricular septal thickness (IVST), left ventricular end-diastolic posterior wall thickness (LVPWT), left atrial internal diameter (LAD), and E peak to A peak velocity ratio (E/A) in the MS group were higher than those in the non-MS group (P<0.05). The E peak to e' peak velocity ratio (E/e') in the MS group was higher than that in the non-MS group (12.02±3.68 vs 11.13±3.12, P=0.011) and was positively correlated with the diagnosis of MS and metabolic factors (r=0.115, p=0.024; r=0.131, p=0.010, respectively). Patients with five metabolic factors had a significantly higher risk of E/e' elevation than patients in the non-MS group (odds ratio=4.238, p=0.007). Conclusion:MS may be related to OSA severity and left ventricular diastolic dysfunction. An increase in metabolic factors may increase the risk of diastolic dysfunction. Among metabolic factors, blood pressure may be the most important.

The advancement of graph neural networks (GNNs) has made it possible to accurately predict metabolic sites. Despite the combination of GNNs with XGBOOST showing impressive performance, this technology has not yet been applied in the realm of metabolic site prediction. Previous metabolic site prediction tools focused on bonds and atoms, regardless of the overall molecular skeleton. This study introduces a novel tool, named D-CyPre, that amalgamates atom, bond, and molecular skeleton information via two directed message-passing neural networks (D-MPNN) to predict the metabolic sites of the nine cytochrome P450 enzymes using XGBOOST. In D-CyPre Precision Mode, the model produces fewer, but more accurate results (Jaccard score: 0.497, F1: 0.660, and precision: 0.737 in the test set). In D-CyPre Recall Mode, the model produces less accurate, but more comprehensive results (Jaccard score: 0.506, F1: 0.669, and recall: 0.720 in the test set). In the test set of 68 reactants, D-CyPre outperformed BioTransformer on all isoenzymes and CyProduct on most isoenzymes (5/9). For the subtypes where D-CyPre outperformed CyProducts, the Jaccard score and F1 scores increased by 24% and 16% in Precision Mode (4/9) and 19% and 12% in Recall Mode (5/9), respectively, relative to the second-best CyProduct. Overall, D-CyPre provides more accurate prediction results for human CYP450 enzyme metabolic sites.

ETHNOPHARMACOLOGICAL RELEVANCE:Da Chuanxiong Formula (DCX) is a traditional herbal compound composed of Gastrodia elata Bl. and Ligusticum chuanxiong Hort, which could significantly enhance blood circulation and neuroprotection, showing promise in treating Vascular Cognitive Impairment (VCI).AIM OF STUDY:This study aims to elucidate the potential of DCX in treating VCI and its underlying mechanism.MATERIALS AND METHODS:Firstly, the cognitive behavior level, blood flow changes, and brain pathology changes were evaluated through techniques such as the Morris water maze, step-down, laser speckle, coagulation analysis, and pathological staining to appraise the DCX efficacy. Then, the DCX targeting pathways were decoded by merging metabolomics with transcriptomics. Finally, the levels of reactive oxygen species (ROS), Fe2+, and lipid peroxidation related to the targeting signaling pathways of DCX were detected by kit, and the expression levels of mRNAs or proteins related to ferroptosis were determined by qPCR or Western blot assays respectively.RESULTS:DCX improved cognitive abilities and cerebral perfusion significantly, and mitigated pathological damage in the hippocampal region of VCI model rats. Metabolomics revealed that DCX was able to call back 33 metabolites in plasma and 32 metabolites in brain samples, and the majority of the differential metabolites are phospholipid metabolites. Transcriptomic analysis revealed that DCX regulated a total of 3081 genes, with the ferroptosis pathway exhibiting the greatest impact. DCX inhibited ferroptosis of VCI rates by decreasing the levels of ferrous iron, ROS, and malondialdehyde (MDA) while increasing the level of superoxide dismutase (SOD) and glutathione (GSH) in VCI rats. Moreover, the mRNA and protein levels of ACSL4, LPCAT3, ALOX15, and GPX4, which are related to lipid metabolism in ferroptosis, were also regulated by DCX.CONCLUSION:Our research findings indicated that DCX could inhibit ferroptosis through the ACSL4/GPX4 signaling pathway, thereby exerting its therapeutic benefits on VCI.

BackgroundNonalcoholic steatohepatitis (NASH) is a prominent cause of liver-related death that poses a threat to global health and is characterized by severe hepatic steatosis, lobular inflammation, and ballooning degeneration. To date, no Food and Drug Administration-approved medicine is commercially available. The Chaihu Guizhi Ganjiang Decoction (CGGD) shows potential curative effects on regulation of blood lipids and blood glucose, mitigation of organism inflammation, and amelioration of hepatic function. However, the overall regulatory mechanisms underlying its effects on NASH remain unclear.PurposeThis study aimed to investigate the efficiency of CGGD on methionine- and choline-deficient (MCD)-induced NASH and unravel its underlying mechanisms.MethodsA NASH model of SD rats was established using an MCD diet for 8 weeks, and the efficacy of CGGD was evaluated based on hepatic lipid accumulation, inflammatory response, and fibrosis. The effects of CGGD on the intestinal barrier, metabolic profile, and differentially expressed genes (DEGs) profile were analyzed by integrating gut microbiota, metabolomics, and transcriptome sequencing to elucidate its mechanisms of action.ResultsIn MCD-induced NASH rats, pathological staining demonstrated that CGGD alleviated lipid accumulation, inflammatory cell infiltration, and fibrosis in the hepatic tissue. After CGGD administration, liver index, liver weight, serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST) contents, liver triglycerides (TG), and free fatty acids (FFAs) were decreased, meanwhile, it down-regulated the level of proinflammatory mediators (TNF-α, IL-6, IL-1β, MCP-1), and up-regulated the level of anti-inflammatory factors (IL-4, IL-10), and the expression of liver fibrosis markers TGFβ, Acta2, Col1a1 and Col1a2 were weakened. Mechanistically, CGGD treatment altered the diversity of intestinal flora, as evidenced by the depletion of Allobaculum, Blautia, norank_f_Erysipelotrichaceae, and enrichment of the probiotic genera Roseburia, Lactobacillus, Lachnoclostridium, etc. The colonic histopathological results indicated that the gut barrier damage recovered in the CGGD treatment group, and the expression levels of colonic short-chain fatty acids (SCFAs)-specific receptors FFAR2, FFAR3, and tight junction (TJs) proteins ZO-1, Occludin, Claudin-1 were increased compared with those in the model group. Further metabolomic and transcriptomic analyses suggested that CGGD mitigated the lipotoxicity caused by glycerophospholipid and eicosanoid metabolism disorders by decreasing the levels of PLA2G4A, LPCAT1, COX2, and LOX5. In addition, CGGD could activate the inhibitory lipotoxic transcription factor PPARα, regulate the proteins of FABP1, APOC2, APOA2, and LPL to promote fatty acid catabolism, and suppress the TLR4/MyD88/NFκB pathway to attenuate NASH.ConclusionOur study demonstrated that CGGD improved steatosis, inflammation, and fibrosis on NASH through enhancing intestinal barrier integrity and alleviating PPARα mediated lipotoxicity, which makes it an attractive candidate for potential new strategies for NASH prevention and treatment.

目的 采用超高效液相色谱-四极杆飞行时间质谱(UPLC-Q-TOF-MS)对速效救心丸的化学成分进行研究.方法 采用Agilent Poroshell 120 EC C18柱(150 mmX3.0mm,2.7 μm),以0.1％甲酸水-乙腈为流动相梯度洗脱,体积流量为0.8mL·min-1(分流比1:2);采用Dual AJS ESI源在正、负离子模式下分别采集数据.构建化合物库,根据化合物精确相对分子质量及二级碎片离子信息结合参考文献数据,鉴定速效救心丸的主要化学成分.结果 从速效救心丸中分离和鉴定出90个化学成分,其中苯酞类化合物47个,有机酸类化合物11个,生物碱类化合物13个,萜类化合物4个,其他类化合物15个.结论 该方法快速、准确、便捷,为阐明速效救心丸的药效物质基础及进一步研究其体内代谢过程提供了科学的数据支撑和方法参考.

Objective: To explore the possible mechanisms of cholestasis induced by Polygoni Multiflori Radix (PM).Methods: Low and high doses of water extract of PM were given to mice by gavage for 8 weeks. The serum biochemical indexes of aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutamyltransferase (GGT) alkaline phosphatase (ALP) and so on were detected in the second, fourth, sixth, and eighth weeks after administration. At the end of the eighth week of administration, the bile acid metabolic profiles of liver and bile were screened by high-performance liquid chromatography tandem triple quadrupole mass spectrometry (HPLC-QQQ-MS/MS). Liver pathological changes were observed by hematoxylin and eosin staining. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the mRNA transcription of the target genes and Western blotting (WB) was used to the detect target protein expression.Results: Biochemical tests results showed the values of ALP and GGT were two and three times greater than the normal values respectively, and the value of R was less than 2. Histopathology also showed that PM caused lymphocyte infiltration, a small amount of hepatocyte necrosis and nuclear fragmentation in mouse liver. The proliferation of bile duct epithelial cells was observed in the high group. These results indicated that PM may lead to cholestatic liver injury. HPLC-QQQ-MS/MS analysis with the multivariate statistical analysis revealed significant alterations of individual bile acids in liver and gallbladder as compared to those of the control group. RT-qPCR showed that the transcription of Fxr, Shp, Bsep, Bacs, Mdr2, and Ugt1a1 were downregulated and that of Cyp7a1, Mrp3, and Cyp3a11 was significantly upregulated in the treatment group. WB demonstrated that PM also markedly downregulated the protein expression of FXR, BSEP, and MDR2, and upregulated CYP7A1.Conclusion: PM inhibited the expression of FXR, which reduced the expression of MDR2 and BSEP, leading to the obstruction of bile acids outflow, and increased the expression of CYP7A1, resulting in an increase of intrahepatic bile acid synthesis, which can lead to cholestasis.

本文建立了超高效液相色谱-四极杆-飞行时间串联质谱(UHPLC-Q-TOF-MS/MS)法联合分子网络策略快速分析紫菀中肽类成分.采用ACQUITY UPLC?HSS T3柱(100 mm×2.1 mm×1.8μm),以乙腈-水为流动相进行梯度洗脱,在正离子模式下,收集紫菀75％ 乙醇提取物的M S/M S数据,并创建GNPS分子网络.根据标准品、精确相对分子质量、碎片离子等信息,共鉴定出紫菀中43个肽类成分,包括31个环肽类和12个直链肽类,其中分别有16个和8个成分可能为潜在的新型环肽类和直链肽类化合物.UHPLC-Q-TOF-MS/MS联合分子网络策略能够快速、准确、全面地鉴定紫菀中的肽类成分,可为进一步分离紫菀化学成分和研究药效物质基础提供依据.

目的 研究速效救心丸对心肌缺血大鼠的保护作用及对血浆代谢物的影响,初步探讨其干预心肌缺血的代谢途径和可能机制.方法 采用垂体后叶素建立大鼠心肌缺血模型,通过病理组织切片和肌酸激酶(creatine kinase,CK)、丙氨酸转氨酶(alanine aminotransferase,ALT)、天冬氨酸转氨酶(aspartate aminotransferase,AST)、羟丁酸脱氢酶(hydroxybutyrate dehydrogenase,HBDH)、乳酸脱氢酶(lactate dehydrogenase,LDH)和超氧化物歧化酶(superoxide dismutase,SOD)生化指标评估速效救心丸对心肌缺血大鼠的保护作用;采用超高效液相色谱-四级杆飞行时间质谱联用(UHPLC-QTOF/MS)技术对血浆进行代谢组学研究,筛选潜在生物标志物并富集代谢通路.结果 速效救心丸可明显改善心肌缺血大鼠心脏组织病理变化;显著降低血浆CK、AST、HBDH、LDH活性(P＜0.05、0.01),升高SOD活性(P＜0.05、0.01).代谢组学分析共筛选到39个潜在生物标志物,涉及丙氨酸、天冬氨酸和谷氨酸代谢、三羧酸循环、谷氨酰胺和谷氨酸代谢等7条通路.结论 速效救心丸能够有效改善模型大鼠的心肌缺血损伤,可能通过影响能量代谢及鞘脂代谢等相关通路发挥作用.

Ethnopharmacological relevance: Shengyu Decoction (SYD), a classical Chinese medicine formula, is good at nourishing blood, promoting blood circulation, and soothe the nerves. SYD can improve cognitive ability. This decoction is suitable for treating vascular cognitive impairment (VCI). however, its active ingredients and possible mechanism have not been investigated. Aim of the study: This study was conducted to observe the effects of SYD on improving the cognitive abilities of rats with VCI, to explore its active ingredients and mechanism. Materials and methods: The rats with VCI model were established by bilateral common carotid artery occlusion (BCCAO), and the effects of SYD (5, 2.5 g/kg) on the cognitive abilities of VCI rats were evaluated using the Morris water maze (MWM) and neurological assessment. The pathological changes of hippocampal CA1 were observed by H &E and Nissl staining. The effect of SYD on cerebral blood flow (CBF) was evaluated by Laser Speckle Contrast Imager. The expression of CD31 in the cerebral cortex was measured by immunofluorescence (IF) to evaluate the number of cerebral micro vessels. The levels of IL-6, IL-1 beta, and TNF-alpha in the hippocampus were determined using an ELISA kit, and the active components in the plasma and brain tissues of rats after SYD administration were analyzed using UPLC-Q-TOF-MS/MS. The interaction network of the compound-target pathway was established using the SWISS Target, GO, and DAVID databases. The expression of AKT/HIF-1 alpha/ VEGF and p38 MAPK signaling pathway in the brain tissues was determined using western blotting (WB). Results: SYD (2.5, 5 g/kg) significantly improved the cognitive abilities of VCI rats in the MWM and neurological assessment. H&E and Nissl staining showed that SYD significantly ameliorated the pathological hippocampal CA1 area and increased the number of Nissl bodies. The Laser Speckle Contrast Imager showed that the cortical CBF of VCI rats in the SYD group was significantly increased, and the IF results showed that CD31 expression was significantly increased in the SYD group. The ELISA results showed that the contents of IL-6, IL-1 beta, and TNF-alpha in SYD were significantly reduced. A total of 29 compounds were found in the plasma and brain tissues of the rats treated with SYD. Network pharmacology revealed 99 targets for the treatment of VCI. Pathway enrichment analysis showed that the HIF-1 and MAPK signaling pathways might be important for SYD to ameliorate VCI. WB showed that the expressions of AKT, HIF-1 alpha, and VEGF in the brain tissues of rats were significantly increased; in addition, NF-kappa B and p38 MAPK were significantly reduced in the SYD group. Conclusion: SYD can improve the cognitive abilities of VCI rats. The mechanism of action of its active ingredients improves cognitive impairment by affecting the AKT/HIF-1 alpha/VEGF and p38 MAPK/NF-kappa B signaling pathways, promoting cerebrovascular generation, and ameliorating neuroinflammation.

在中药学专业实验课程独立考核以及给予独立学分的背景下,对北京中医药大学2020级中药学专业本科生开展分析化学实验课程多元化成绩评价的实践,在实践的基础上构建了易于执行的多元化成绩评价方式,由注重学习过程的"平时成绩"和"终结性实验考核"成绩评价两部分以及下属多个评价指标组成.将该实验课程成绩的多元化评价方案在2020级的400名中药学专业的学生中实践,结果表明该方案能够让多个实验组实验考核评分具有可比性,同时实验考核成绩的区分度好;形成性评价对学生实验过程做出发展性评价,引导学生在过程反馈中修正自己的基本操作,提升学生基本操作的组合应用能力和整合思辨思维,为进一步设计实验新方案并实施奠定基础,督促学生的整体"递进式"实验过程.同时,在实验教学过程中细化了形成性评价指标的评价标准,使得教师有章可循,在实验考核时利用了在线考核方式,减轻了教师的工作量,有利于该多元化评价方案的长期应用.该课程成绩评价实践可以为中药学专业的其他实验课程的成绩评价起到示范作用.

目的 建立高效液相色谱-三重串联四极杆质谱联用(HPLC-QQQ-MS/MS)测定芪参颗粒中38种主要成分含量的方法,明确其化学组成,并对制剂批次间一致性进行分析.方法 HPLC采用Zorbax Eclipse Plus C18(50 mmX2.1 mm,1.8μm)色谱柱,0.1％甲酸水溶液-0.1％甲酸乙腈溶液梯度洗脱,质谱对正(11个离子对)、负(22个离子对)离子进行动态多反应监测(dynamic multi-response detection,dMRM),实现多批次芪参颗粒中38种主要成分的含量测定.通过主成分分析(principal component analysis,PCA)和正交偏最小二乘-判别分析(orthogonal partial least squares-discriminant analysis,OPLS-DA),筛选影响批次间一致性的成分.结果 建立的HPLC-MS/MS方法在15 min内完成了 38个成分的同时定量分析,12批次芪参颗粒样品中38个化合物的总量为34.923～47.148 mg/g,其中臣药丹参中的丹酚酸B的量最高(16.718 mg/g),其次是金银花中的绿原酸(3.550 mg/g),君药黄芪中黄芪甲苷、毛蕊异黄酮苷葡萄糖苷的质量分数分别为0.136、0.753 mg/g,黑顺片中3种双酯型生物碱的质量分数均低于0.003 mg/g,其余化合物的质量分数在0.004～2.935 mg/g.PCA结合OPLS-DA筛选出了 5个对批次一致性影响最大的成分,依次为黄芪甲苷、黄芪皂苷Ⅲ、隐丹参酮、丹参酮ⅡA和二氢丹参酮Ⅰ.结论 所建立的分析方法专属性强,灵敏度高,为芪参颗粒质量控制和一致性分析提供了科学方法和依据.

Ethnopharmacological relevance: Danhe granule (DHG) is used by Chinese doctors to treat blood stasis, phlegm and dampness. Its lipid-lowering ability has been investigated in our previous research. However, the anti-liver inflammatory and fibrotic effects and mechanism of action of DHG in non-alcoholic steatohepatitis (NASH) have not been explored.Aim of the study: To evaluate the ameliorative effects of DHG on liver inflammation and fibrosis in a methionine/ choline-deficient (MCD) diet-induced NASH rat model, and its underlying mechanism. Materials and methods: Sprague-Dawley rats were fed an MCD diet for two weeks and then treated with or without DHG by oral gavage for eight weeks. Their body weight and liver index were measured. The serum alanine aminotransferase (ALT) and aspartate transaminase (AST) activities as well as the liver triglyceride (TG) and free fatty acid (FFA) levels were tested using reagent kits. Inflammatory cytokines, including Tnf-alpha, Il-beta and Il-6, and fibrosis genes, including Acta2, Col1a1, Col1a2 and Tgf-beta were examined by real-time quantitative PCR (RTqPCR). Hematoxylin-eosin (H&E), Oil Red O, Masson's and Sirius Red staining were used to observe liver changes. The plasma and liver ceramide levels were analyzed using HPLC-QQQ-MS/MS. The expression of serine palmitoyl-CoA transferase (Spt), ceramide synthase 6 (Cers6), dihydroceramide desaturase 1 (Des1), glucosylceramide synthase (Gcs), and ceramide kinase (Cerk) mRNA was assayed by RT-qPCR, while the protein expression of CerS6, DES1, GCS, CerK, and casein kinase 2 alpha (CK2 alpha) was tested by western blotting (WB). CerS6 degradation was evaluated using a cycloheximide (CHX) assay in vitro.Results: The liver index decreased by 20% in DHG groups and the serum ALT and AST decreased by approximately 50% and 30%, respectively in the DHG-H group. The liver Oil Red O staining, TG, and FFA changes showed that DHG reduced hepatic lipid accumulation by approximately 30% in NASH rats. H&E, Masson's and Sirius Red staining and the mRNA levels of Tnf-alpha, Il-beta, Il-6, Acta2, Col1a1, Col1a2 and Tgf-beta revealed that DHG alleviated liver inflammation and fibrosis in NASH rats. The ceramide (Cer 16:0), and hexosylceramide (HexCer 16:0, HexCer 18:0, HexCer 22:0, HexCer 24:0 and HexCer 24:1) levels decreased by approximately 17-56% in the plasma of the DHG-M and H rats. The Cer 16:0 content in the liver decreased by 20%, 50%, and 70% with the DHG-L, M, and H treatments; additionally, the dhCer 16:0, Cer 18:0, HexCer 18:0, HexCer 20:0 Cer 22:0-1P, Cer 24:0-1p, Cer 24:1-1p, and Cer 26:1-1p levels decreased in the DHG groups. The mRNA and protein expression levels of DES1, GCS, Cerk, CerS6, and CHX assay indicated that DHG decreased the mRNA and protein expression levels of CerK and reduced CerS6 protein expression by promoting its degradation. Additionally, DHG attenuated the protein expression of CK2 alpha which could increase CerS6 enzymatic activity by phosphorylating its C-terminal region.Conclusion: DHG ameliorated the levels of liver FFA and TG and inflammation and fibrosis in MCD-induced rats, which were associated with decreasing ceramide species in the plasma and liver by reducing the expression levels of CerS6 and CerK.