Zuojin Pill (ZJP), a traditional Chinese medicine prescription composed of Rhizoma Coptidis and Euodiae Fructus in the ratio of 6:1 (w/w), has been widely used for the treatment of gastric disorders. However, an in-depth understanding of in vivo metabolism and distribution profiles of protoberberine alkaloids (PBAs) and indole alkaloids (IDAs) in ZJP is lacking. In this study, a method using ultra-high performance liquid chromatography coupled with quadruple time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) was developed to systematically screen the alkaloids and their metabolites in rat plasma and various tissues after oral administration of ZJP. Furthermore, bioinformatics and molecular docking analyses were conducted to elucidate the contribution of the alkaloids and metabolites enriched in the stomach to the therapeutic effect of ZJP on gastritis. A total of 33 compounds, including 7 prototype alkaloids and 26 metabolites, were chemically defined or tentatively identified in this work. The metabolic pathways of PBAs (hydroxylation, oxidation, reduction, demethylation, demethylenation, glucuronide conjugation, sulfate conjugation) and IDAs (hydroxylation, glucuronide conjugation) were revealed. Notably, 7 prototype alkaloids and 18 metabolites were detected in the stomach, indicating their propensity for gastric distribution. These alkaloids and metabolites showed strong affinities with the 7 hub targets associated with gastritis, such as CCR7, CXCR4, IL6, IFNG, CCL2, TNF, and PTPRC, and could be considered the potential active substances of ZJP for treating gastritis. In conclusion, this study clarified the gastric distribution propensity of PBAs and IDAs and their metabolites, as well as their favorable binding interactions with gastritis-related targets, which could provide essential data for the further study of the pharmacodynamic material basis and gastroprotective mechanism of ZJP.

UNASSIGNED: The Zuojin Pill (ZJP) is widely used for treating chronic atrophic gastritis (CAG) in clinical practice, effectively ameliorating symptoms such as vomiting, pain, and abdominal distension in patients. However, the underlying mechanisms of ZJP in treating CAG has not been fully elucidated.
UNASSIGNED: This study aimed to clarify the characteristic function of ZJP in the treatment of CAG and its potential mechanism.
UNASSIGNED: The CAG model was established by alternant administrations of ammonia solution and sodium deoxycholate, as well as an irregular diet. Therapeutic effects of ZJP on body weight, serum biochemical indexes and general condition were analyzed. HE staining and AB-PAS staining were analyzed to characterize the mucosal injury and the thickness of gastric mucosa. Furthermore, network pharmacology and molecular docking were used to predict the regulatory mechanism and main active components of ZJP in CAG treatment. RT-PCR, immunohistochemistry, immunofluorescence and Western blotting were used to measure the expression levels of apoptosis-related proteins, gastric mucosal barrier-associated proteins and PI3K/Akt signaling pathway proteins.
UNASSIGNED: The results demonstrated that ZJP significantly improved the general state of CAG rats, alleviated weight loss and gastric histological damage and reduced the serum biochemical indicators. Network pharmacology and molecular docking found that ZJP in treating CAG by inhibiting inflammation, suppressing apoptosis, and protecting the gastric mucosal barrier via the PI3K/Akt signaling pathway. Further experiments confirmed that ZJP obviously modulated the expression of key proteins involved in gastric mucosal cell apoptosis, such as Bax, Bad, Apaf-1, cleaved-caspase-3, cleaved-caspase-9, Cytochrome C, Bcl-2, and Bcl-xl. Moreover, ZJP significantly reversed the protein expression of Occludin, ZO-1, Claudin-4 and E-cadherin.
UNASSIGNED: Our study revealed that ZJP treats CAG by inhibiting the PI3K/Akt signaling pathway. This research provided a scientific basis for the rational use of ZJP in clinical practice.

BACKGROUND: Zuojin Pill (ZJP), composed of Coptis chinensis Franch. and Euodia ruticarpa (A. Juss.) Benth. in a mass ratio of 6:1, is a famous traditional Chinese medicine (TCM) formula recorded in \"Danxi\'s Experiential Therapy\", an ancient medical book from the Ming Dynasty of China. It is used to treat liver fire invading the stomach, which is caused by liver stagnation transforming into fire and disharmony between the liver and stomach.
OBJECTIVE: To develop a systematic strategy to screen hepatoprotective components from TCM using ZJP as a model sample.
METHODS: A CCl4-induced mouse model of acute liver injury was used for the verification of the hepatoprotective effects of ZJP. UPLC-Q-Exactive Plus Orbitrap MS/MS was used for the identification of the components in mouse serum after intragastric administration of ZJP. The hepatoprotective activities of the components found in mouse serum were tested in primary cultured mouse hepatocytes induced by CCl4.
RESULTS: Nine components with significant hepatoprotective activity including berberine, epiberberine, coptisine, palmatine, jatrorrhizine, rutaecarpin, dehydroevodiamine, evocarpine and chlorogenic acid were successfully screened out.
CONCLUSIONS: Our developed strategy has the advantages of high efficiency and low cost, and would provide a powerful tool for screening potential hepatoprotective components from TCM.

BACKGROUND: The complex bidirectional communication between the gastrointestinal tract and the brain is associated with mental disorders such as depression; serotonin, as a crucial neurotransmitter in the communication system between the central nervous system and the gastrointestinal tract, has effects on regulating gastrointestinal motility and sensation and improving psychosomatic status. Zuojin pill is used as a traditional Chinese medicine formula for the treatment of gastrointestinal disorders. This study explored the effects of Zuojin pill on the improvement of depression and gastrointestinal function in CUMS mice via TPH2 and its mechanism.
OBJECTIVE: The aim of this study was to investigate whether Zuojin pill could improve depression and concomitant gastrointestinal dysfunction, and to reveal whether Zuojin pill could work through the regulation of the tryptophan hydroxylase 2 (TPH2) pathway.
METHODS: The CUMS model was established to observe the effects of Zuojin pill on depression-like behavior and gastrointestinal function in mice. Nissler staining and HE staining were used to observe the structure of hippocampal neurons and intestinal mucosa respectively. 5-HT levels in serum, hippocampus, and intestinal tissues were measured by ELISA, and TPH2 expression in hippocampus and intestinal nerves was observed by WB and immunofluorescence. In order to investigate the protective effect and mechanism of Zuojin pill on PC12 cells, CORT used an in vitro model to produce PC12 cell damage.
RESULTS: Our study showed that Zuojin pill ameliorated depression-like behavior and gastrointestinal dysfunction in CUMS mice, elevated BDNF, 5-HT, and TPH2 expression in the hippocampus, and restored the ratio of dopaminergic and GABAergic neurons between intestinal muscles. In vitro experiments showed that Zuojin pill exerted a protective effect on neurons by regulating TPH2 ubiquitination and thus inhibiting CORT-induced apoptosis of PC12 cells.
CONCLUSIONS: Zuojin pill improves chronic unpredictable stress-induced depression-like behavior and gastrointestinal dysfunction in mice via the TPH2/5-HT pathway. Therefore, TPH2 may be a potential therapeutic target for depression with gastrointestinal dysfunction.

Current quality control methods for Zuojin Pill (ZJP) lack comprehensiveness and practicability. This study aimed to develop a comprehensive strategy for the quality evaluation of ZJP and the prediction of potential bioactive components in ZJP. First, an HPLC method with excellent separation of main components was developed and was used to establish the chromatographic fingerprint of ZJP. Similarities were calculated by comparing 28 batches of ZJPs with the reference fingerprint and the resulting similarity values were all greater than 0.976. The 28 samples were classified into different groups according to their origins by Hierarchical Cluster Analysis, Principal component analysis, and orthogonal partial least squares discriminant analysis. Based on the classification, eight quality markers (Q-Markers) affecting the quality of ZJP were discovered. Then, using berberine as an internal standard substance, quantitative analysis of multi-components by single marker method (QAMS) for the determination of eight Q-markers was developed. The results showed that there was no significant difference between QAMS and external standard method (P＞0.05). Finally, using an off-line antioxidant system and partial least-squares model (PLS), the fingerprint-efficacy relationship of ZJP was constructed to explore and predict the bioactive components in ZJP. The present study strategy could be also applied to comprehensive quality study of other TCMs.

Objective: Chronic nonatrophic gastritis (CNG) is the most common digestive disease. In China, Zuojin pill (ZJP) is considered an effective medicine formula for CNG. However, its efficacy and mechanism have never been explored. In order to understand how and why ZJP demonstrates therapeutic effect on CNG, a clinical trial was conducted. Metabolomics was used to explore its deep mechanism. Methods: A total of 14 patients with CNG were recruited from October 2020 to March 2021 (ChiCTR2000040549). The endoscopy and histopathological changes were evaluated as efficacy. Serum samples were prepared and detected by performing widely targeted metabolome using UPLC. Multivariate statistical analysis was conducted to identify potential differential metabolites and signaling pathways. Last, the signal-related inflammatory factors containing COX-2, IL-4, and IL-17 were confirmed via immunohistochemical staining and enzyme-linked immunosorbent assay. Results: ZJP was able to alleviate several indexes of mucosal injury under endoscopy and histology. Erosion and bile reflux, but not red plaques and hemorrhage, were downregulated by ZJP. In addition, it could remarkably alleviate active chronic inflammation. A total of 14 potential metabolites, namely, hypoxanthine, adipic acid, D-ribono-1,4-lactone, L-sepiapterin, imidazoleacetic acid, sebacate, ADP-ribose, 4-hydroxybenzyl alcohol, 11,12-EET, 15-OxoETE, 12-OxoETE, (±)8-HETE, glycyrrhizinate, and DL-aminopimelic acid, were discriminated by metabolomics. Moreover, certain amino acid metabolism got significance during the disease progress and treatment. The related inflammatory factors including COX-2, IL-4, and IL-17 were inhibited by ZJP in both mucosa and serum. Conclusion: All these results indicated that ZJP partially acts as an inflammatory suppressor to regulate comprehensive metabolism disorders. This might be an important mechanism of ZJP in the treatment of CNG.

Venlafaxine (VEN), a first-line antidepressant, and Zuojin Pill (ZJP), a common herbal medicine consisting of Rhizoma Coptidis and Fructus Evodiae, are high likely co-administered in China. ZJP could significantly inhibit VEN pharmacokinetics in vitro and in rats through suppression of CYP2D6 activity. To date, however, no clinical study has demonstrated the clinical relevance. Here, the VEN pharmacokinetics at a single dose of VEN with or without co-administration of ZJP was compared. ZJP had a weak herb-drug interactions (HDI) on the pharmacokinetics of VEN. The geometric means of Cmax and AUC0-∞ of VEN increased by 36.7% and 34.6%, respectively, and the corresponding 90% confidence intervals (CIs) of geometric mean ratios (GMRs) exceed outside bioequivalent range of 0.80-1.25. However, the corresponding 90% CIs of GMRs of these parameters for ODV were within the range. Since ODV exposure (AUC), approximately 3.4-fold higher than that of VEN, hardly changed, the systemic exposure of VEN active moiety (VEN + ODV) with ZJP increased slightly (≤8.5%) compared with that of VEN alone. In addition, the incidence of VEN-related side effects, especially gastrointestinal relevance, was significantly reduced with ZJP. Therefore, rational concomitant use of VEN and ZJP might have low risk of HDI and be promising in clinical practice.

BACKGROUND: Zuojin pill (ZJP), a classical Chinese medicine formula, has been widely applied in Chinese clinical practice for the treatment of gastric injury such as acute gastric lesion, acute gastric mucosal injury, chronic unpredictable mild stress, gastroesophageal reflux disease, etc, thereby exerting anti-chronic atrophic gastritis (CAG) effects in traditional Chinese herbal medicine.
OBJECTIVE: This study was aimed to explore the therapeutic effects and molecular mechanisms of ZJP on Helicobacter pylori (H. pylori)-induced CAG based on the comprehensive approaches.
METHODS: Sprague-Dawley rats were infected with H. pylori for 8 weeks to establish CAG model. Then, rats in the ZJP groups received doses of 0.63, 1.26, and 2.52 g/kg ZJP for 4 weeks. Therapeutic effects of ZJP on serum indices and the histopathology of the gastric were analyzed in vivo. Moreover, GES-1 cells were infected with H. pylori to establish gastric epithelial cell injury model in vitro. Cell viability and gastric epithelial cell morphology were detected by a high-content screening (HCS) assay. Furthermore, the relative mRNA and protein expression of JMJD2B/COX-2/VEGF axis and HMGB1/NF-κB signaling pathway in vivo and in vitro were determined by RT-PCR and Western Blotting, respectively.
RESULTS: The results showed that the therapeutic effects of ZJP on CAG rats were presented in down-regulation serum biochemical indices and alleviating histological damage of gastric tissue. ZJP could dose-dependently decrease the serum IL-6, MCP-1, PGE2, TNF-α, and VEGF level and significantly improved gastric tissue inflammatory lesions. Besides, ZJP has an effect on increasing cell proliferation of GES-1 cells, ameliorating H. pylori-induced gastric epithelial cell damage. It was found that ZJP has a down-regulating effect on inflammatory reaction and could inhibit the relative mRNA and protein expression of JMJD2B/COX-2/VEGF axis and HMGB1/NF-κB signaling pathway in vivo and in vitro, including JMJD2B, COX-2, VEGF, VEGFR1, and VEGFR2, which in turn reduced the damage of gastric mucosal cells.
CONCLUSIONS: The results suggested that ZJP exerts therapeutic effects on H. pylori-induced CAG by inhibiting the JMJD2B/COX-2/VEGF axis and HMGB1/NF-κB signaling pathway. These findings deeply explained why ZJP could be used to treat CAG clinically and clarified its pharmacological effect and potential mechanism in the treatment of CAG.

UNASSIGNED: Pancreatic cancer is one of the most malignant tumors worldwide. Zuojin pills (ZJP), a traditional Chinese medicine (TCM) formula, which can treat a variety of cancers. However, the active compounds present in ZJP and the potential mechanisms through which ZJP acts against pancreatic cancer have not been thoroughly investigated.
UNASSIGNED: Data on pancreatic cancer-related genes, bioactive compounds, and potential targets of ZJP were downloaded from public databases. Bioinformatics analysis, including protein-protein interaction (PPI), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, was conducted to identify important components, potential targets, and signaling pathways through which ZJP affects pancreatic cancer. The results of this analysis were verified by in vitro experiments.
UNASSIGNED: The network pharmacology analysis results showed that 41 compounds and 130 putative target genes of ZJP were associated with anti-pancreatic cancer effects. ZJP may exert its inhibitory effects against pancreatic cancer by acting on key targets such as JUN, TP53, and MAPK1. Moreover, KEGG analysis indicated that the anti-pancreatic cancer effect of ZJP was mediated by multiple pathways, such as the PI3K-AKT, IL-17, TNF, HIF-1, and P53 signaling pathways. Among these, the PI3K-AKT signaling pathway, which included the highest number of enriched genes, may play a more important role in treating pancreatic cancer. The in vitro results showed that ZJP significantly inhibits the cell cycle and cell proliferation through the PI3K/AKT/caspase pathway and that it can induce apoptosis of pancreatic cancer cells, consistent with the results predicted by network pharmacological methods.
UNASSIGNED: This study preliminarily investigated the pharmacological effects of ZJP, which appear to be mediated by multiple compounds, targets and pathways, and its potential therapeutic effect on pancreatic cancer. Importantly, our work provides a promising approach for the identification of compounds in TCM and the characterization of therapeutic mechanisms.

BACKGROUND: Zuojin Pill (ZJP) has been a classic prescription for the treatment of gastrointestinal diseases in China since ancient times. But its effect on non-steroidal anti-inflammatory drugs (NSAIDs) induced gastric injury (GI) is still uncharted.
OBJECTIVE: This study aims to investigate the therapeutic effect and molecular mechanism of ZJP on indomethacin (IDO) induced gastric injury.
METHODS: GI was induced in rat by oral administration of 5 mg/kg IDO. Then the rats were treated with ZJP (1.26, 2.52, 5.04 g/kg, ig). The changes of food intake, body weight, gastric pH and general state observation were carried out to determine the improvement of ZJP in IDO-induced GI: HE staining and AB-PAS staining was analyzed to characterize the thickness of gastric mucosa and micro mucosal injury; in order to elucidate the effect of ZJP on IDO-induced inflammatory injury, the inflammatory infiltration of gastric tissue was observed by MPO immunohistochemical method, and the contents of TNF-α, IL-6 and IL-10 were measured. Furthermore, the regulatory mechanism of ZJP in treating IDO-induced GI was predicted with the help of network pharmacology, and the expression levels of key proteins ERK, p-ERK, P38, p-P38, JNK, p-JNK were determined to elucidate the molecular mechanism of ZJP.
RESULTS: Current data strongly demonstrated that ZJP alleviated food intake reduction, weight loss and gastric injury caused by IDO and made gastric pH and mucosal thickness return to normal. In addition, ZJP could reduce the level of MPO to alleviate the inflammatory infiltration of gastric tissue. Simultaneously, ZJP could down regulate the expression of TNF-α and IL-6 and up regulate the expression of IL-10 to reduce the damage caused by inflammatory, and create a healing environment. Furthermore, ZJP could significantly inhibit the phosphorylation of ERK, p38 and JNK, which leaded to the increase of inflammatory factors and the damage of gastric mucosa.
CONCLUSIONS: ZJP improved local inflammation by inhibiting MAPK signaling pathway, and had a good therapeutic effect on IDO-induced GI. This study has reference significance for the study of ZJP in the prevention and treatment of NSAID induced gastric injury. In addition, ZJP may be a new treatment option for the prevention and treatment of NSAID induced gastric disease.