Pachymic Acid Research Articles

Pharmacological profiles and therapeutic applications of pachymic acid (Review).

Poria cocos is a saprophytic fungus that grows in diverse species of Pinus. Its sclerotium, called fu-ling or hoelen, has been used in various traditional Chinese medicines and health foods for thousands of years, and in several modern proprietary traditional Chinese medicinal products. It has extensive clinical indications, including sedative, diuretic, and tonic effects. Pachymic acid (PA) is the main lanostane-type triterpenoid in Poria cocos. Evidence suggests that PA has various biological properties such as cytotoxic, anti-inflammatory, antihyperglycemic, antiviral, antibacterial, sedative-hypnotic, and anti-ischemia/reperfusion activities. Although considerable advancements have been made, some fundamental and intricate issues remain unclear, such as the underlying mechanisms of PA. The present study aimed to summarize the biological properties and therapeutic potential of PA. The biosynthetic, pharmacokinetic, and metabolic pathways of PA, and its underlying mechanisms were also comprehensively summarized.

Pachymic acid prevents neuronal cell damage induced by hypoxia/reoxygenation via miR‑155/NRF2/HO‑1 axis

Pachymic acid (PA) plays a neuroprotective role during cerebral ischemia/reperfusion. However, the protective mechanisms of PA in cerebral ischemia/reperfusion have been not fully determined. This investigation aims to explore the neuroprotective role of PA in ischemia/reperfusion via miR‑155/NRF2/HO‑1 axis. The N2a cell line was induced by hypoxia/reoxygenation (H/R) to simulate the neuronal damage that occurs during cerebral ischemia/reperfusion. PA was used to treat H/R‑induced N2a cells. An MTT assay was used to determine cell viability. The protein levels of Bcl‑2, Bax, heme oxygenase‑1 (HO‑1) and nuclear factor E2‑related factor 2 (NRF2) were measured via Western blot analysis. The level of apoptosis of N2a cells was determined by flow cytometry. The expression levels of miR‑155 and NRF2 were quantified by real‑time PCR. PA treatment inhibits the increase in apoptosis induced by H/R and also enhances the viability of cells exposed to H/R. PA reverses the increased expression of miR‑155 caused by H/R. Furthermore, H/R does not change the expression of HO‑1 and NRF2, but PA upregulates the expressions of HO‑1 and NRF2. Additionally, NRF2 is the target of miR‑155. Inhibiting miR‑155 contributes to increased cell viability and decreased apoptosis via targeting the NRF2/HO‑1 pathway. Overall, PA prevents neuronal cell damage induced by hypoxia/reoxygenation via miR‑155/NRF2/HO‑1 axis.

Antitumor activity of pachymic acid in cervical cancer through inducing endoplasmic reticulum stress, mitochondrial dysfunction, and activating the AMPK pathway.

Pachymic acid has various pharmacological effects, including anti-inflammatory, antioxidant, immunomodulatory, and antitumor. However, the role of pachymic acid in cervical cancer remains unclear. So, we investigated the effects of pachymic acid in cervical cancer and elucidated the underlying mechanisms. We treated HeLa cells and normal cervical epithelial cells (HUCECs) with pachymic acid (0, 10, 20, 40, 80, or 160 μM) for 72 h, and found the cell activity was decreased in cells treated with 160 μM pachymic acid for 48 h or 80 μM pachymic acid for 72 h, while HUCECs viability without effect. Next, we observed that endoplasmic reticulum (ER) related gene expression, mitochondrial membrane potential (MMP) changes, ATP depletion, reactive oxygen species (ROS) generation and apoptosis were increased. Moreover, we observed that cytochrome C (Cytc) expression was increased and apoptosis-inducing factor (AIF) was decreased in the cytoplasm of pachymic acid-treated HeLa cells. Tauroursodeoxycholic acid (TUDCA) of ER stress inhibitor reversed the effects of pachymic acid on HeLa cells. Phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) of the AMPK pathway key protein was upregulated in pachymic acid-induced HeLa cells. Finally, we subcutaneously implanted HeLa cells into female nude mice and treated them with pachymic acid (50 mg/kg) for 3 weeks (5 days/week), and observed in pachymic acid induced xenograft mice, tumor growth was suppressed, cell apoptosis, ER-related gene expression, and ROS levels in tumor tissues were increased. Therefore, these findings demonstrated that pachymic acid plays an anti-tumor activity in cervical cancer through inducing ER stress, mitochondrial dysfunction, and activating the AMPK pathway.

Fuzi decoction ameliorates pain and cartilage degeneration of osteoarthritic rats through PI3K-Akt signaling pathway and its clinical retrospective evidence

BackgroundOsteoarthritis (OA) is a difficult disease but the clinic lacks effective therapy. As a classic formula of traditional Chinese medicine (TCM), Fuzi decoction (FZD) has been clinically applied for treating OA-related syndromes, but its anti-OA efficacy and mechanism remain unclear. PurposeTo experimentally and clinically determine the anti-OA efficacy of FZD and clarify the underlying mechanism. MethodsUPLC/MS/MS was applied to identify the main components of FZD. A monoiodoacetate (MIA)-induced OA rat model was employed to evaluate the in vivo efficacy of FZD against OA, by using pain behavior assessment, histopathological observation, and immunohistochemical analysis. Primary rat chondrocytes were isolated to determine the in vitro effects of FZD by using cell viability assay, wound healing assay, and real-time PCR (qPCR) analysis on anabolic/catabolic mRNA expressions. RNA sequencing (RNA-seq) and network pharmacology analysis were conducted and the overlapping data were used to predict the mechanism of FZD, followed by verification with qPCR and Western blot assays. Finally, a retrospective analysis was performed to confirm FZDʹs efficacy and safety in OA patients. ResultsThe UPLC/MS/MS result showed that FZD contained atractylenolide I, benzoylhypaconitine, benzoylmesaconitine, benzoylaconitine, hypaconitine, mesaconitine, aconitine, lobetyolin, paeoniflorin, and pachymic acid. The in vivo data showed that FZD restored the cartilage degeneration in MIA-induced OA rats by ameliorating pain behavior parameters, recovering histopathological alterations, benefitting cartilage anabolism (up-regulating Col2 expression), and suppressing catabolism (down-regulating MMP13 and Col10 expressions). The in vitro data showed that FZD increased cell viability and wound healing capacity of chondrocytes, and restored the altered expressions of anabolic and catabolic genes of chondrocytes. The overlapping results of RNA-seq and network pharmacology analysis suggested that PI3K/Akt signaling mediated the anti-OA mechanism of FZD, which was verified by qPCR and Western blot experiments. Clinically, the anti-OA efficacy and safety of FZD were confirmed by the retrospective analysis on OA patients. ConclusionThe scientific innovation of this study was the determination of anti-OA efficacy of FZD by experimental and clinical evidence and the discovery of its mechanism by integrated RNA-seq, network pharmacology, and molecular experiments, which suggests FZD as a promising TCM agency for OA treatment.

Effect of Fushengong Decoction on PTEN/PI3K/AKT/NF-κB Pathway in Rats With Chronic Renal Failure via Dual-Dimension Network Pharmacology Strategy.

Overview: The treatment of chronic renal failure (CRF) with traditional Chinese medicine has attracted much attention, but its mechanism is not clear. Network pharmacology is an effective strategy for exploring the interaction mechanisms between Chinese herbs and diseases, however, it still needs to be validated in cell and/or animal experiments due to its virtual screening characteristics. Herein, the anti-CRF mechanism of the Fushengong decoction (FSGD) was investigated using a dual-dimension network pharmacological strategy combined with in vivo experiment. Methods: The traditional Chinese medicine systems pharmacology (TCMSP) database (https://tcmspw.com) and UHPLC-MS/MS technology were used to identify the effective compounds of FSGD in theory and practice, such as quercetin, formononetin, and pachymic acid. The putative targets of FSGD and CRF were obtained from the Swisstarget prediction platform and the Genecards database, respectively. The common target pathways between FSGD and CRF were got from the dual-dimension network pharmacology analysis, which integrated the cross-common targets from the TCMSP components-Swisstarget-Genecards-Venn platform analysis in theory, and the UHPLC-MS/MS identified effective ingredients-Swisstarget screening, such as TNF and PI3K/AKT. Furthermore, system molecular determinations were used to prove the dual-dimension network pharmacology study through CRF rat models, which were constructed using adenine and treated with FSGD for 4 weeks. Results: A total of 121 and 9 effective compounds were obtained from the TCMSP database and UHPLC-MS/MS, respectively. After dual-dimension network pharmacology analysis, the possible mechanism of PTEN/PI3K/AKT/NF-κB pathway was found for FSGD in CRF. In vivo experiments indicated that FSGD can play a role in protecting renal function and reducing fibrosis by regulating the PTEN/PI3K/AKT/NF-κB pathway. These findings provide a reference for FSGD in CRF. Conclusion: Based on the theoretical and practical dual-dimension network pharmacology analysis for FSGD in CRF, the possible molecular mechanism of PTEN/PI3K/AKT/NF-κB was successfully predicted, and these results were verified by in vivo experiments. In this study, the dual-dimension network pharmacology was used to interpret the key signal pathway for FSGD in CRF, which also proved to be a smart strategy for the study of effective substances and pharmacology in FSGD.

Rapid Determination of Pachymic Acid Content by Near-Infrared Spectroscopy

In this paper, a rapid model for the determination of pachymic acid content in Poria was established by partial least squares (PLS) regression and near-infrared spectroscopy (NIR). During the research, a total of 108 batches of Poria samples from different producing regions were used, while their corresponding pachymic acid contents by high-performance liquid chromatography (HPLC) were adopted as reference. These samples were divided randomly into calibration sets for model establishment and validation sets for model validation. The test results from the calibration set showed that the best preprocessing method of the NIR spectra model was the standard normal variate (SNV) + second derivatives (SD), and the most suitable number of principal factors was 9. In this model, the coefficient of determination of the calibration set ( r c 2 ) and validation set ( r v 2 ) was 0.915 and 0.917, respectively. Meanwhile, the root mean square error of calibration (RMSEC) and the root mean square error of validation (RMSEP) with the calibration set were 0.051 mg/g and 0.054 mg/g, respectively. These results indicated this model could rapidly and reliably predict the pachymic acid content in Poria and increase the determination efficiency of pachymic acid in Poria. This is conducive to promote the development of industry.

Histologic evaluation of dentin bridge formation by pachymic acid and biodentine in human tooth culture model

Histologic evaluation of dentin bridge formation by pachymic acid and biodentine in human tooth culture model

Simultaneous Determination of Ten Bioactive Components from Shenling Baizhu San in Rat Plasma by UHPLC-MS/MS: Application to a Comparative Pharmacokinetic Study in Normal and Two Models of Ulcerative Colitis Rats.

Shenling Baizhu San, a traditional formula, has a long history of treating spleen asthenic diarrhea by invigorating the spleen and dispelling dampness in China. A rapid and accurate UHPLC-MS/MS method was developed and fully validated for the simultaneous determination of ten active constituents in rat plasma: panaxadiol, ginsenoside Rg1, atractylenolide I, atractylenolide III, pachymic acid, neferine, nuciferine, diosgenin, platycodin D, and isoliquiritigenin. The plasma samples were pretreated by the protein precipitation method with acetonitrile. The analytes and puerarin (internal standard) were determined with high selectivity and sensitivity (LLOQ, 0.31–0.68 ng·mL−1) within 10 minutes. The validation parameters, including intra-/interday precisions, accuracy, recovery, matrix effect, and stability, were within acceptable ranges. The validated method was successfully applied to the pharmacokinetics study of ten components in normal and two rat models of ulcerative colitis (i.e., spleen deficiency with dampness retention-ulcerative colitis (SDDR-UC) rats and pure-ulcerative colitis (P-UC) rats). The pharmacokinetic parameters were significantly different among the three groups of rats. Overall, the absorption of the components was shown as follows: normal group > SDDR-UC group > P-UC group. The study could provide a scientific basis for further studies on pharmacokinetics and clinical differential application of SDDR-UC and P-UC patients.

Functional Analysis of Sterol O-Acyltransferase Involved in the Biosynthetic Pathway of Pachymic Acid in Wolfiporia cocos.

Pachymic acid from Wolfiporia cocos possesses important medicinal values including anti-bacterial, anti-inflammatory, anti-viral, invigorating, anti-rejection, anti-tumor, and antioxidant activities. However, little is known about the biosynthetic pathway from lanostane to pachymic acid. In particular, the associated genes in the biosynthetic pathway have not been characterized, which limits the high-efficiency obtaining and application of pachymic acid. To characterize the synthetic pathway and genes involved in pachymic acid synthesis, in this study, we identified 11 triterpenoids in W. cocos using liquid chromatography tandem mass spectrometry (LC-MS/MS), and inferred the putative biosynthetic pathway from lanostane to pachymic acid based on analyzing the chemical structure of triterpenoids and the transcriptome data. In addition, we identified a key gene in the biosynthetic pathway encoding W. cocos sterol O-acyltransferase (WcSOAT), which catalyzes tumolusic acid to pachymic acid. The results show that silence of WcSOAT gene in W. cocos strain led to reduction of pachymic acid production, whereas overexpression of this gene increased pachymic acid production, indicating that WcSOAT is involved in pachymic acid synthesis in W. cocos and the biosynthesis of W. cocos pachymic acid is closely dependent on the expression of WcSOAT gene. In summary, the biosynthetic pathway of pachymic acid and the associated genes complement our knowledge on the biosynthesis of W. cocos pachymic acid and other triterpenoids, and also provides a reference for target genes modification for exploring high-efficiency obtaining of active components.

Pachymic Acid Ameliorates Pulmonary Hypertension by Regulating Nrf2-Keap1-ARE Pathway.

Pulmonary hypertension (PH) is a severe pulmonary vascular disease that eventually leads to right ventricular failure and death. The purpose of this study was to investigate the mechanism by which pachymic acid (PA) pretreatment affects PH and pulmonary vascular remodeling in rats. PH was induced via hypoxia exposure and administration of PA (5 mg/kg per day) in male Sprague-Dawley rats. Hemodynamic parameters were measured using a right ventricular floating catheter and pulmonary vascular morphometry was measured by hematoxylin-eosin (HE), α-SMA and Masson staining. MTT assays and EdU staining were used to detect cell proliferation, and apoptosis was analyzed by TUNEL staining. Western blotting and immunohistochemistry were used to detect the expression of proteins related to the Nrf2-Keap1-ARE pathway. PA significantly alleviated hypoxic PH and reversed right ventricular hypertrophy and pulmonary vascular remodeling. In addition, PA effectively inhibited proliferation and promoted apoptosis in hypoxia-induced pulmonary artery smooth muscle cells (PASMCs). Moreover, PA pretreatment inhibited the expression of peroxy-related factor (MDA) and promoted the expression of antioxidant-related factors (GSH-PX and SOD). Furthermore, hypoxia inhibited the Nrf2-Keap1-ARE signaling pathway, while PA effectively activated this pathway. Most importantly, addition of the Nrf2 inhibitor ML385 reversed the inhibitory effects of PA on ROS generation, proliferation, and apoptosis tolerance in hypoxia-induced PASMCs. Our study suggests that PA may reverse PH by regulating the Nrf2-Keap1-ARE signaling pathway.

Progress in pharmacodynamic basis, mechanism, and prediction of Q-markers of Zhenwu Decoction in treatment of chronic heart failure

Zhenwu Decoction(ZWD) has a history of more than 1 800 years in traditional Chinese medicine(TCM), which is used to treat various diseases characterized by Yangqi deficiency and exuberant water and dampness. It is currently the classic prescription for the treatment of chronic heart failure(CHF). This study provides a basis for the treatment of CHF with ZWD by elaborating the traditional efficacy, theoretical basis, and underlying mechanism of the prescription. Based on the research methods and judgment basis of quality markers(Q-markers) of Chinese medicine, the Q-markers of ZWD in the treatment of CHF were predicted from the aspects of transfer and traceability, specificity, effectiveness, compatibility environment, measurability, and processing. Demethyl-coclaurine,benzoylaconine, atractylenolide Ⅲ, paeoniflorin, 6-gingerol, 8-gingerol, pachymic acid, and dehydrotumulosic acid can be used as Q-markers of ZWD for treating CHF. The result provides a reference for exploring the pharmacodynamic substances of ZWD in the treatment of CHF.

Pachymic Acid Attenuated Doxorubicin-Induced Heart Failure by Suppressing miR-24 and Preserving Cardiac Junctophilin-2 in Rats.

Defects in cardiac contractility and heart failure (HF) are common following doxorubicin (DOX) administration. Different miRs play a role in HF, and their targeting was suggested as a promising therapy. We aimed to target miR-24, a suppressor upstream of junctophilin-2 (JP-2), which is required to affix the sarcoplasmic reticulum to T-tubules, and hence the release of Ca2+ in excitation–contraction coupling using pachymic acid (PA) and/or losartan (LN). HF was induced with DOX (3.5 mg/kg, i.p., six doses, twice weekly) in 24 rats. PA and LN (10 mg/kg, daily) were administered orally for four weeks starting the next day of the last DOX dose. Echocardiography, left ventricle (LV) biochemical and histological assessment and electron microscopy were conducted. DOX increased serum BNP, HW/TL, HW/BW, mitochondrial number/size and LV expression of miR-24 but decreased EF, cardiomyocyte fiber diameter, LV content of JP-2 and ryanodine receptors-2 (RyR2). Treatment with either PA or LN reversed these changes. Combined PA + LN attained better results than monotherapies. In conclusion, HF progression following DOX administration can be prevented or even delayed by targeting miR-24 and its downstream JP-2. Our results, therefore, suggest the possibility of using PA alone or as an adjuvant therapy with LN to attain better management of HF patients, especially those who developed tolerance toward LN.

Untargeted Metabolomics and Targeted Quantitative Analysis of Temporal and Spatial Variations in Specialized Metabolites Accumulation in Poria cocos (Schw.) Wolf (Fushen).

Poria cocos (Schw.) Wolf is a saprophytic fungus that grows around the roots of old, dead pine trees. Fushen, derived from the sclerotium of P. cocos but also containing a young host pine root, has been widely used as a medicine and food in China, Japan, Korea, Southeast Asian countries, and some European countries. However, the compound variations at the different growth periods and in the different parts of Fushen have not previously been investigated. In this study, an untargeted metabolomics approach based on ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) and targeted quantitative analysis was utilized to characterize the temporal and spatial variations in the accumulation of specialized metabolites in Fushen. There were 119 specialized metabolites tentatively identified using the UPLC-Q/TOF-MS. The nine growth periods of Fushen were divided into four groups using partial least squares discrimination analysis (PLS-DA). Four different parts of the Fushen [fulingpi (FP), the outside of baifuling (BO), the inside of baifuling (BI), and fushenmu (FM)] were clearly discriminated using a PLS-DA and orthogonal partial least squares discrimination analysis (OPLS-DA). Markers for the different growth periods and parts of Fushen were also screened. In addition, the quantitative method was successfully applied to simultaneously determine 13 major triterpenoid acids in the nine growth periods and four parts. The quantitative results indicated that the samples in January, March, and April, i.e., the late growth period, had the highest content levels for the 13 triterpenoid acids. The pachymic acid, dehydropachymic acid, and dehydrotumulosic acid contents in the FM were higher than those in other three parts in March, whereas the poricoic acid B, poricoic acid A, polyporenic acid C, dehydrotratrametenolic acid, dehydroeburicoic acid, and eburicoic acid in FP were higher beginning in October. These findings reveal characteristics in temporal and spatial distribution of specialized metabolites in Fushen and provide guidance for the identification of harvesting times and for further quality evaluations.

Potential therapeutic efficacy of pachymic acid in chronic kidney disease induced in rats: role of Wnt/β-catenin/renin-angiotensin axis.

Chronic kidney disease (CKD) is a major public health problem associated with high mortality. The therapeutic effects of pachymic in CKD management and its underlying mechanisms have not been studied. Therefore, we aimed to investigate the possible inhibitory effect of PA on renal Wnt/β-catenin signalling in CKD. CKD was induced in rats by doxorubicin (DOX; 3.5 mg/kg i.p., twice weekly for 3 weeks). Rats were treated orally with PA (10 mg/kg/day), LOS (10 mg/kg/day) or their combination (PA + LOS) for 4 weeks starting after the last dose of DOX. DOX-induced renal injury was characterized by high serum cystatin-C, and urine albumin/creatinine ratio, renal content of podocin and klotho were decreased. Tumour necrosis factor-α, interleukin-6, interleukin-1β, Wnt1, active β-catenin/total β-catenin ratio and fibronectin along with mRNA expression of RENIN, ACE and AT1 were increased in renal tissues. Treatment with either PA or LOS ameliorated all DOX-induced changes. The combined treatment was more effective in improving all changes than monotherapy. These results suggest a new therapeutic benefit of PA in ameliorating CKD in rats through its up-regulatory effect on renal klotho thereby preventing Wnt/β-catenin reactivation and RAS gene expression. PA/LOS combination provided an additional inhibition of Wnt/β-catenin signalling and its downstream targets.

Pachymic acid inhibits inflammation and cell apoptosis in lipopolysaccharide (LPS)-induced rat model with pneumonia by regulating NF-κB and MAPK pathways.

Pneumonia is a common infectious disease with high morbidity and mortality. It is caused by a variety of pathogenic microorganisms that infect the lung parenchyma. Anti-infective drugs are one of the preferred choices for the treatment of pneumonia. Pachymic acid (PA) is a lanolin triterpene compound from Poria cocos, which has antiemetic, anti-inflamma-tory, and anticancer properties. Although PA inhibits inflammatory response in a variety of diseases, its role in pneumonia is not clear. In this study, we established that PA improved histopathological changes in the lungs of rats with pneumonia. PA inhibited the expression of inflammatory cytokines in the serum of rats having pneumonia. In addition, PA inhibited the apoptosis of cells from rat lung tissues. Mechanically, PA inhibited inflammation and cell apoptosis via NF-κB and MAPK pathways. Therefore, PA could serve as a promising drug for treating pneumonia.

An UPLC - Q - Orbitrap method for pharmacokinetics and tissue distribution of four triterpenoids in rats after oral administration of Poria cocos ethanol extracts

Poria cocos (Schw.) Wolf, is a fungus that is widely used as medicine and dietary supplement in China. But its action mechanism is still not very clear. In this paper, a rapid, specific and sensitive high performace liquid chromatography coupled with hybrid quadrupole - orbitrap mass sepctrometry (UPLC - Q - Orbitrap MS) method has been developed and validated to simultaneously determine of four triterpenoids including Dehydrotumulosic acid (DTA), Dehydropachymic acid (DPA), Pachymic acid (PA), Dehydrotrametenolic acid (DMA) from Poria cocos in rat plasma and tissues. The analyte was extracted from rat plasma and tissue homogenates by protein precipitation with acetonitrile using glibenclamide as the internal standard (IS). Chromatographic separation was carried out on ACQUITY UPLC BEH - C18 column (2.1 mm × 50 mm, 1.7 μm) with a mobile phase composed of acetonitrile - water (containing 1.0 mmol/L ammonium acetate) using gradient elution at a flow rate of 0.2 mL/min. Electrospray ionization (ESI −) under negative ion mode was used, and its quantization was performed with multiple reaction monitoring (MRM) mode. The method was fully validated and successfully applied to pharmacokinetics and tissue distribution study in rats after oral administration of ethanol extracts of Poria cocos. Compared with that of plasma exporsure, triterpenoids could be detected in various tissues with a relatively high degree of tissue distribution. After oral administration, the concentration orders in seven different tissues were ranked as DTA > PA > DPA > DMA in intestine and stomach, wheras DTA > DMA > PA > DPA in heart, liver, spleen, lung and kidney tissues, which is speculated that DPA, PA may be converted into DMA in vivo. In conclusion, this results may provide a material basis for study of the pharmacological actions of triterpenoids in Poria cocos.

Pachymic acid alleviates TNBS-induced intestine mucosal injury in mice

Objective: Pachyman might reduce the intestinal dysfunction and inflammation. Therefore, in this study, we aimed to explore the effect of pachymic acid on repairing the intestine mucosal injury in mice. Materials and Methods: Male BALB/c mice were randomly divided into six groups: control group, trinitrobenzenesulfonic acid (TNBS)-induced group, pachymic acid (50, 100, and 200 mg/kg) groups, and mesalazine group. In addition to the mice in the control group, all mice in other groups were induced by TNBS, an inducer of colitis, to establish the intestine mucosal injury model. To study the effect of pachymic acid on the intestine mucosal injury in mice, we assessed the disease activity index (DAI) and the morphology colon specimen in mice. Histopathological examination was also used to access the effects of different concentrations of pachymic acid on the intestinal mucosal injury in mice. Enzyme-linked immunosorbent assay (ELISA) tests were performed to detect the level of glutathione (GSH), myeloperoxidase (MPO), (MDA), and pro-inflammatory factors (tumor necrosis factor-α [TNF-α], interleukin (IL)-1 β, and IL-8) to evaluate the effect of pachymic acid on the biomarkers of oxidative stress and pro-inflammatory factors. At the in vitro level, the human colon carcinoma cells (Caco-2) were induced by TNBS to establish the in vitro intestinal mucosal injury model. After treatment with the pachymic acid, the cell viability was examined by methyl thiazolyl tetrazolium assay, and the cell apoptosis was detected by flow cytometric analysis. The expression levels of apoptosis-related proteins were analyzed by Western blot analysis. Meanwhile, the secretion level of TNF-α, IL-1 β, and IL-8 in the supernatant of Caco-2 cells was detected by ELISA kits. Results: Pachymic acid was found to reduce the DAI of intestine mucosal injury in treated mice and improved the hyperemia, edema, and necrosis in intestinal mucosal tissue. Pachymic acid decreased the secretion levels of pro-inflammatory factors TNF-α, IL-1 β, and IL-8, as well as the oxidative stress markers, MPO and MDA. In contrast, the serum levels of GSH were found to be increased. After being challenged by TNBS, the cell viability of Caco-2 cells was inhibited, while after treated with pachymic acid, inhibitory effect was partially blocked. Similarly, the TNBS-induced Caco-2 cells' apoptosis was abolished by pachymic acid. Conclusion: Pachymic acid repaired the intestine mucosal injury and attenuated inflammatory response in experimental mice. It provides an alternative therapeutic strategy for intestine mucosal injury and highlights the protective effects of traditional Chinese medicine in these diseases.

Systems pharmacological study based on UHPLC-Q-Orbitrap-HRMS, network pharmacology and experimental validation to explore the potential mechanisms of Danggui-Shaoyao-San against atherosclerosis

Ethnopharmacological relevanceAtherosclerosis (AS) plays an important role in the pathogenesis of cardiovascular and cerebrovascular diseases. Danggui-Shaoyao-San (DSS) is not only a representative Chinese formula to treat gynecological disorder, but also found its use in AS-related diseases. However, the active ingredients and the anti-AS effects are vague yet. Aim of the studyAn integrated strategy combined ultrahigh-performance liquid chromatography quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap-HRMS), network pharmacology and experiments was carried out to investigate the potential materials and pharmacological mechanisms of DSS for AS. Materials and methodsFirst, UHPLC-Q-Orbitrap-HRMS was applied to identify the active compositions of DSS. Then, the putative targets of DSS relevant to AS were predicted from TCMSP and BATMAN, which were further determined through bioinformatic analyses, including protein-protein interactions (PPI), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG). Finally, Western blot, qPCR and ELISA were carried out for target validation in human umbilical vein endothelial cells (HUVECs). ResultsA total of 37 active ingredients of DSS, connecting 47 key targets were identified. The functional enrichment showed that DSS may treat AS through regulating a series of signaling pathways which involving inflammatory responses, immune systems and metabolism. The in vitro experiment revealed that DSS ameliorated AS mainly through anti-inflammatory effects, by reducing the levels of vascular cell adhesion molecule-1 (VCAM1), intercellular adhesion molecule-1 (ICAM1), IL-6, TNF-α, cyclooxygenase-2 (Cox-2) and IL-1β. DSS also inhibited the phosphorylation of IκB-α, NF-κB (p65), p38 and JNK in lipopolysaccharide (LPS)-induced HUVEC injury model. Moreover, as the main bioactive compounds of DSS, paeoniflorin (PF), ferulic acid (FA) and pachymic acid (PA) inhibited IL-6 and TNF-α secretion as well as IκB-α, NF-κB (p65), p38 and JNK activation. All these findings were consistent with the predicted targets and pathways. ConclusionCollectively, the basic pharmacological effects and relevant mechanisms of DSS in the treatment of AS were revealed. The results suggest that DSS is a potential drug for AS treatment, and PF, FA, PA may be the core compositions contributing to the pharmacological function of this formula.

Bioinformatics and experimental findings reveal the therapeutic actions and targets of pachymic acid against cystitis glandularis.

Pachymic acid (PA), a bioactive ingredient isolated from Poria cocos Wolf, is reported with potential benefits of anti-inflammatory, anti-oxidative actions. It is reasoned that PA may play the potential benefits against cystitis glandularis (CG), an inflammation of the bladder tissue. In this study, we aimed to apply the network pharmacology and molecular docking analyses to reveal concrete anti-CG targets and mechanisms of PA, and then the bioinformatic findings were verified by using clinical and animal samples. The methodological data from network pharmacology approach showed that 303 and 243 reporting targets of CG and PA, and other 31 shared targets of CG and PA were identified. Subsequently, all top targets of PA against CG were screened out, including cyclooxygenase-2, epidermal growth factor receptor, tumor antigen p53 (TP53), tumor necrosis factor-alpha (TNF), interleukin-1 (IL-1) beta, proto-oncogene c-jun. Molecular docking data demonstrated that PA exerted potent bonding capacities with TNF, TP53 proteins in CG. In human study, the findings suggested that overactivated TNF-α expression and suppressed TP53 activation were detected in CG samples. In animal study, PA-treated mice showed reduced intravesical IL-1, IL-6 levels, and lactate dehydrogenase content, downregulated TNF-α and upregulated TP53 proteins in bladder samples. Taken together, our bioinformatics and experimental findings identify the key anti-CG biotargets and mechanisms of PA. More markedly, these pivotal pharmacological targets of PA against CG have been screened out and verified by using computational and experimental analyses.

Screening the Q-markers of TCMs from RA rat plasma using UHPLC-QTOF/MS technique for the comprehensive evaluation of Wu-Wei-Wen-Tong Capsule.

The appropriate selection of quality marker (Q-marker) for performing the comprehensive quality evaluation of traditional Chinese medicines (TCMs) has much more significance. Wu-Wei-Wen-Tong Capsule (WWWTC), a TCMs prescription, is mainly utilized to treat rheumatoid arthritis (RA) in China. However, the comprehensive quality control for WWWTC has not been achieved because of lacking system analysis for the Q-marker. In this study, a dual wavelength, 203 and 270 nm, was selected based on the feature of 15 Q-markers, and a reliable UHPLC-UV fingerprinting approach was established, achieving the comprehensive quality evaluation of WWWTC. First, we identified 91 prototypes in rat plasma after administering a set amount of WWWTC by using UHPLC-QTOF/MS technique and selected them as the candidate Q-markers. Next, based on the "five principles" of Q-marker selection, 15 absorbed components among them including coumarin, cinnamic acid, cinnamaldehyde, cinnamic alcohol, and 2-methoxycinnamaldehyde derived from Monarch medicine of Cmnamomi Mmulus; epimedin C, icariin, baohuoside I, and anhydroicaritin derived from Monarch medicine Epimedii Folium; germacrone, the sesquiterpene compound in Minister medicine Rhizoma Wenyujin Concisum; pachymic acid, the tetracyclic triterpenoid acids in Assistant medicine Poria; baicalin, baicalein, wogonin, and wogonoside in Guide medicine Scutellariae Radix, respectively, were seriously chosen as the Q-markers, indicating preferable pharmacological effect on RA, characterization of transitivity and traceability as well as measurable components in WWWTC. The effective and meaningful strategy displayed a unique perspective for the exploration of Q-markers in the quality evaluation and further ensured efficacy and safety of the TCMs.