BACKGROUND: Liver cancer represents a most common and fatal cancer worldwide. Xianglian Pill (XLP) is an herbal formula holding great promise in clearing heat for treating diseases in an integrative and holistic way. However, due to the complex constituents and multiple targets, the exact molecular mechanisms of action of XLP are still unclear.
OBJECTIVE: This study is focused on hepatocellular carcinoma (HCC), the most common type of liver cancer. The aim of this study is to develop a fast and efficient model to investigate the anti-HCC effects of XLP, and its underlying mechanisms.
METHODS: HepG2, Hep3B, Mahlavu, HuH-7, or Li-7 cells were employed in the studies. The ingredients were analyzed using liquid chromatography tandem mass spectrometry (LC-MS). RNA sequencing combined with network pharmacology was used to elucidate the therapeutic mechanism of XLP in HCC via in silico and in vitro studies. An approach was constructed to improve the accuracy of prediction in network pharmacology by combining big data and omics.
RESULTS: First, we identified 13 potential ingredients in the serum of XLP-administered rats using LC-MS. Then the network pharmacology was performed to predict that XLP demonstrates anti-HCC effects via targeting 94 genes involving in 13 components. Modifying the database thresholds might impact the accuracy of network pharmacology analysis based on RNA sequencing data. For instance, when the matching rate peak is 0.43, the correctness rate peak is 0.85. Moreover, 9 components of XLP and 6 relevant genes have been verified with CCK-8 and RT-qPCR assay, respectively.
CONCLUSIONS: Based on the crossing studies of RNA sequencing and network pharmacology, XLP was found to improve HCC through multiple targets and pathways. Additionally, the study provides a way to optimize network pharmacology analysis in herbal medicine research.

Antibiotic-associated diarrhea (AAD) is a common side effect of antibiotic therapy, characterized by intestinal inflammation which reduces the quality of life of patients. Xianglian Pill (XLP) has long been used to treat abdominal pain, diarrhea, bacillary dysentery and enteritis. Studies found that XLP has curative effect on AAD; however, the chemical constituents and mechanism of XLP have not been fully elucidated because of the lack of in vitro and in vivo studies. In this study, ultra-high performance liquid chromatography mass spectrometry method (UPLC-Q-Exactive-Orbitrap-HRMS) was used to examine the components of the XLP. Then, the binding between active compounds and the key targets was studied using network pharmacology and molecular docking. A comparative tissue distribution study was established for the simultaneous determination of the 10 active components in healthy and AAD mouse models. Forty-six components were characterized from XLP. According to the network pharmacology degree value, a prediction was made that encompassed 42 components and 14 core targets, which were intricately involved in crucial biological pathways, such as the AGE-RAGE signaling, cellular senescence, and MAPK signaling. Tissue distribution analysis showed that the 10 components were widely distributed in the heart, liver, spleen, lungs, kidneys, small intestine, and large intestine of mice, with varying concentrations in healthy and AAD mice. Molecular docking analysis also indicated that the active compounds in the tissue distribution could bind tightly to key targets of network pharmacological studies. This study provides a reference for further investigations of the relationships between the chemical components and pharmacological activities of XLP.

BACKGROUND: Previous studies have revealed that a high-fat diet (HFD) promotes the progression of colorectal cancer (CRC) in close association with disturbances in the intestinal flora and metabolic disorders. Xianglian pill (XLP) is a well-established traditional prescription with unique advantages in controlling intestinal flora imbalance and inflammation. However, its therapeutic effects on HFD-related CRC remain largely unknown.
OBJECTIVE: The primary objective of this research was to investigate the anticancer mechanism of XLP in countering HFD-related CRC.
METHODS: The protective effect of XLP was evaluated using azoxymethane (AOM) and dextran sulfate sodium (DSS)-induced CRC model of mice exposed to a HFD. The degree of colorectal carcinogenesis, including body weight, colon length, and histopathology, was measured in mice treated with XLP and untreated mice. The effect of XLP on gut microbiota and its metabolites was detected using 16S rDNA and liquid chromatography/mass spectrometry analysis. Furthermore, a \"pseudo-sterile\" mouse model was constructed using antibiotics (Abx) to verify whether the gut microbiota and metabolites play a role in the pathogenesis of CRC.
RESULTS: XLP inhibited colorectal tumorigenesis in a dose-dependent fashion. Our findings also highlighted that XLP protected the integrity of the intestinal barrier by reducing the expression of pro-inflammatory cytokines, such as IL-6 and TNF-α, as well as the infiltration of pro-inflammatory macrophages. Mechanistically, XLP inhibited the TLR4/MyD88 pathway. Notably, the XLP treatment increased the proportion of probiotics (particularly Akkermansia) and significantly reduced fecal deoxycholic acid (DCA), a microbiota-derived metabolite of bile acids (BA) closely related to Muribaculaceae. Furthermore, after Abx treatment, XLP showed no clear antitumor effects on CRC. Simultaneously, DCA-supplemented feedings promoted colorectal tumorigenesis and provoked obvious colonic inflammation, M1 macrophage infiltration, and colonic injury. In vitro, the results of RAW-264.7 macrophages and normal intestinal epithelial cells treated with DCA corroborated our in vivo findings, demonstrating consistent patterns in inflammatory responses and intestinal barrier protein expression.
CONCLUSIONS: Our findings suggest that XLP inhibits colorectal cancer associated with HFD via inactivating TLR4/MyD88 by remodeling gut microbiota composition and BA metabolism.

BACKGROUND: Perioperative or postoperative adjuvant chemotherapy based on 5-fluorouracil (5-FU) is a common first-line adjuvant therapy for gastric cancer (GC). However, drug resistance and the side effects of 5-FU have reduced its efficacy. Among these side effects, gastrointestinal (GI) toxicity is one of the most common. Xianglian Pill (XLP) is a Chinese patent medicine that is commonly used for the treatment of diarrhoea. It can reduce inflammation and has a protective effect on the intestinal mucosa. Recent studies have shown that many components of XLP can inhibite tumor cell growth. However, the therapeutic effect of XLP in combination with 5-FU on GC is unclear.
OBJECTIVE: To investigate whether the combination of XLP and 5-FU can enhance anti-GC activity while reducing GI toxicity.
METHODS: XLP was administered orally during intraperitoneal injection of 5-FU in GC mice model. Mice were continuously monitored for diarrhea and xenograft tumor growth. After 2 weeks, the mice were sacrificed and serum was collected to determine interleukin-6 levels. Pathological changes, the expression of pro-inflammatory factors and p38 mitogen-activated protein kinase (MAPK) in GI tissue were determined by Western blot analysis. Pathological changes, apoptosis levels and p38 MAPK expression levels in xenograft tissues were also determined.
RESULTS: The results showed that XLP could alleviate GI mucosal injury caused by 5-FU, alleviated diarrhea, and inhibited the expression of nuclear factor (NF)-κB and myeloid differentiation primary response-88. Besides, XLP could promote the 5-FU-induced apoptosis of GC cells and enhance the inhibitory effect of 5-FU on tumor xenografts. Further study showed that XLP administration could regulate the expression of p38 MAPK.
CONCLUSIONS: XLP in combination with 5-FU could alleviate its GI side effects and enhance its inhibitory effect on xenograft tumor. Moreover, these effects were found to be related to the regulation of the p38 MAPK/NF-κB pathway.

BACKGROUND: Gastric cancer (GC) affects people\'s quality of life because of its high incidence rate and mortality. The Xianglian Pill (XLP) is a traditional Chinese medicine (TCM) prescription used to treat gastrointestinal (GI） diseases. Its anti-tumor effect has been found in recent years, but it\'s bioactive compounds and mechanism of action in treating GC are remain unknown.
OBJECTIVE: This study reveals the bioactive compounds and mechanisms of XLP in the treatment of GC through network pharmacology analysis and experimental verification.
METHODS: The main compounds in XLP were searched and the active compounds with anti-GC activity were selected. Compounds targets and GC- related targets were predicted, and common targets were obtained. Subsequently, a protein-protein interaction (PPI) network of common targets is constructed, while GO and KEGG enrichment analyses were performed on common targets. Finally, the anti-GC effects of active compounds in XLP were verified in GC cell lines MGC-803 and HGC-27 by wound healing assay, cell cycle assay, cell apoptosis assay and western blotting (WB) assay.
RESULTS: A total of 33 active compounds of XLP were obtained. MTT assay showed that dehydrocostus lactone (DHL) and berberrubine (BRB) had lower IC50 value in GC cells HGC-27 and MGC-803, and has a less inhibitory effect on normal gastric epithelial cells. Further, 73 common targets were obtained after the total target of DHL and BRB intersected with GC. Among them, CASP3, AKT1, SRC, STAT3,and CASP9 were the most associated genes in the PPI network. GO and KEGG enrichment analyses indicated that apoptosis played a major role in the biological processes and signaling pathways involved. Moreover, the in vitro experiment revealed that DHL and BRB inhibited GC cell viability via inducing cell cycle arrest at G2/M phase, and promoting cell apoptosis by up-regulating the caspase3 expression and down-regulating the expression of Bcl2/Bax.
CONCLUSIONS: DHL and BRB are the two main anti-GC active compounds in XLP, and their mechanism is mainly to inhibit cell cycle and promote cell apoptosis.

BACKGROUND: Xianglian pill (XLP), a traditional Chinese formula, is widely used as treatment for ulcerative colitis (UC) in China. However, the mechanism of its therapeutic effect is still unclear.
OBJECTIVE: Our previous studies showed a low oral bioavailability and a predominant distribution of major XLP ingredients in the gut. In the present study, we aimed to explore the mechanism of action of XLP on UC with respect to the regulation of gut microecology.
METHODS: UC model rats established using 5% dextran sulfate sodium were treated with XLP. After the treatment period, bodyweight, colon length, histopathology, and inflammatory changes were evaluated. Further, changes in gut microbiota structure were detected via 16S rRNA sequencing, and microbial metabolites in feces were analyzed via a metabolomic assay. Antibiotic intervention and fecal microbiota transplantation were also employed to explore the involvement of gut microbiota, while the level of regulatory T cells (Tregs) in mesenteric lymph nodes was determined via flow cytometry. Transcriptome sequencing was also performed to determine colonic gene changes.
RESULTS: XLP alleviated colonic injury, inflammation, and gut microbial dysbiosis in UC model rats and also changed microbial metabolite levels. Particularly, it significantly decreased succinate level in the tyrosine pathway. We also observed that fecal microbiota derived from XLP-treated rats conferred resilience to UC model rats. However, this therapeutic effect of XLP on UC was inhibited by succinate. Moreover, XLP increased the level of anti-inflammatory cellular Tregs via gut microbiota. However, this beneficial effect was counteracted by succinate supplementation. Further, XLP induced the differentiation of Treg possibly by the regulation of the PHD2/HIF-1α pathway via decreasing microbial succinate production.
CONCLUSIONS: Our findings indicated that XLP exerts its therapeutic effects on UC mainly via the gut microbiota-succinate-Treg differentiation axis.

Xianglian pill (XLP) is a typical traditional Chinese herbal medicine prescription composed of Coptidis Rhizoma and Aucklandiae Radix. It has been used to treat gastrointestinal disease for centuries. In the present study, the potential mechanisms of XLP in the treatment of ulcerative colitis (UC) were predicted by integrative pharmacology-based approach. Then, the main compounds of XLP were detected by liquid chromatography-mass spectrometry (LC-MS/MS). Finally, we verified the mechanism of XLP in the treatment of UC in a dextran sulfate sodium (DSS) model. C57BL/6 mice were randomly divided into the control group, DSS group, 5-aminosalicylic acid (5-ASA) group which was used as the positive drug control, XLP low, medium, and high dose group, with 10 mice per group. Except for the control group, acute colitis model was induced in the other mice by administering 3% DSS for consecutive 7 days. Mice in 5-ASA and XLP groups were administered with 5-ASA (50 mg/kg) or XLP (0.8, 1.6, 3.2 g/kg) via oral gavage once per day respectively. Body wight and disease activity index were assay during drug intervention. On day 8, all animals in this experiment were sacrificed and colon tissues were collected for analysis after measurement of the length. The results showed that XLP alleviate DSS -induced acute colitis in mice, including inhibition the secretion of pro-inflammatory cytokines, repairing the dysfunction of intestinal epithelial barrier, enhanced autophagy, and blocked the activation of PI3K/Akt/mTOR pathway. Furthermore, inhibiting autophagy by 3-methyladenine attenuated the protective effects of XLP on colitis. The underlying mechanism may be that Xianglian pill promote autophagy by blocking the activation of PI3K/Akt/mTOR signaling pathway.

BACKGROUND: Ulcerative colitis (UC) is a chronic inflammatory bowel disease with high morbidity, which leads to poor quality of life. The Xianglian pill (XLP) is a classical Chinese patent medicine and has been clinically proven to be an effective treatment for UC.
OBJECTIVE: The pharmacological mechanism of the key bioactive ingredients of XLP for the treatment of UC was investigated by a network pharmacology and pharmacokinetics integrated strategy.
METHODS: Network pharmacology was used to analyze the treatment effect of nine quantified XLP ingredients on UC. Key pathways were enriched and analyzed by protein-protein interaction and Kyoto Encyclopedia of Genes and Genomes analyses. The effect of XLP on Th17 cell differentiation was validated using a mouse model of UC. The binding of nine compounds with JAk2, STAT3, HIF-1α, and HSP90AB1 was assessed using molecular docking. A simple and reliable ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed for the simultaneous quantification of nine ingredients from XLP in plasma and applied to a pharmacokinetic study following oral administration.
RESULTS: Nine compounds of XLP, including coptisine, berberine, magnoflorine,berberrubine, jatrorrhizine, palmatine, evodiamine, rutaecarpine, and dehydrocostus lactone, were detected. Network pharmacology revealed 50 crossover genes between the nine compoundsand UC. XLP treats UC mainly by regulating key pathways of the immune system, including Th17 cell differentiation, Jak-Stat, and PI3K-Akt signaling pathways. An in vivo validation in mice found that XLP inhibits Th17 cell differentiation by suppressing the Jak2-Stat3 pathway, which alleviates mucosal inflammation in UC. Molecular docking confirmed that eight compounds are capable of binding with JAk2, HIF-1α, and HSP90AB1, further confirming the inhibitory effect of XLP on the Jak2-Stat3 pathway. Moreover, apharmacokinetic study revealed that the nine ingredients of XLP are exposed in the plasma and colon tissue, which demonstrates its pharmacological effect on UC.
CONCLUSIONS: This study evaluates the clinical treatment efficacy of XLP for UC. The network pharmacology and pharmacokinetics integrated strategy evaluation paradigm is efficient in discovering the key pharmacological mechanism of herbal formulae.

BACKGROUND: Xianglian Pill (XLP), a traditional Chinese pharmaceutical preparation for the treatment of gastrointestinal disease, possessing anti-inflammatory, anti-microbial and analgesic activities, may represent a promising candidate for the treatment of antibiotic-associated diarrhea (AAD).
OBJECTIVE: This study aimed to unravel the underlying mechanism of XLP on the amelioration of AAD.
METHODS: AAD was induced by intragastric administration of a mixture of cefuroxime and levofoxacin (300 mg/kg. bw + 200 mg/kg. bw) for five consecutive days. Then AAD mice were treated with XLP at the dose of 500, 1000 and 2000 mg/kg. bw, respectively for 5 days. The physical manifestations, diarrhea status were monitored during the drug delivery. Histopathology of colon, intestinal microbiota, inflammatory cytokines, tight junction protein and short chain fat acids (SCFAs) were determined.
RESULTS: Mice received cefuroxime and levofoxacin for 5 days developed medium to severe diarrhea. XLP treatment, however, mitigated the diarrhea status. Further evaluation revealed that XLP promoted the recovery of mucosa, maintained the integrity of tight junction, attenuated the inflammatory disorders, restored intestinal microbiota and increased SCFAs level in feces.
CONCLUSIONS: XLP ameliorates AAD by restoring intestinal microbiota and attenuating mucosal damage.