1.The effect of Biejiajian Pills on regulating the EGFR/MAPK/ERK pathway in MHCC-97H liver cancer cells
Mengsi WU ; Hua LIU ; Nianhua TAN ; Yaoyao LI ; Lin DING ; Yu XIA ; Yang CHEN ; Bin CHEN
Journal of Beijing University of Traditional Chinese Medicine 2024;47(3):394-406
Objective We aimed to investigate the effects of Biejiajian Pills on MHCC-97H hepatoma cells and whether Biejiajian Pills regulate the epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway through miR-885-5p.Methods SPF SD rats (n = 10) were randomly divided into the blank group and the Biejiajian Pills (1.1 g/kg) group to prepare blank and Biejiajian Pills-containing serum. MHCC-97H cells in the logarithmic growth phase were divided into the model group, blank serum groups with different concentrations (5%, 10%, 15%, and 20%), the serum containing Biejiajian Pills group, and the blank group without cells. Cell proliferation was detected by the CCK-8 assay, and the optimal intervention time and concentration of drug-containing serum were screened. MHCC-97H cells were divided into the blank control group (no intervention), the Biejiajian Pills-containing serum group (20% Biejiajian Pills-containing serum), the miR-885-5p mimics group (transfected with miR-885-5p mimics), the miR-NC group (transfected with miR-885-5p NC), and the Biejiajian Pills-containing serum + miR-885-5p mimics group (treated with 20% Biejiajian Pills-containing serum and transfected with miR-885-5p mimics). Cells in each group were cultured for 72 hours. A dual luciferase reporter assay was conducted to verify the targeting relationship between miR-885-5p and EGFR. Cell proliferation was detected by the CCK-8 assay, cell migration and invasion abilities were detected by the cell scratch assay and the Transwell invasion assay. Annexin V-APC/PI double staining was performed to detect the apoptosis level, and real-time fluorescence quantitative PCR (RT-qPCR) analysis was conducted to determine the mRNA expression levels of miR-885-5p, EGFR, MEK, and ERK1/2. The expression levels of EGFR, p-EGFR, MEK, p-MEK, ERK1/2, p-ERK1/2, matrix metalloproteinase 1 (MMP1), and CyclinD1 were determined by Western blotting analysis. The subcutaneous tumor model of MHCC-97H hepatoma cells in nude mice was established by subcutaneous injection to observe the inhibitory effect of Biejiajian Pills of different doses(0.55,1.1,2.2 g/kg).Results The optimal concentration and intervention time of Biejiajian Pills-containing serum were 20% and 72 hours, respectively. Meanwhile, the dual luciferase reporter assay showed that miR-885-5p could directly target EGFR. No statistical significances between the blank control group and the miR-NC group were observed (P>0.05). Compared with the blank control group, the proliferation rates of MHCC-97H hepatoma cells in the Biejiajian Pills-containing serum group, the miR-885-5p mimics group, and the Biejiajian Pills-containing serum + miR-885-5p mimics group were significantly decreased (P<0.01), and their migration and invasion abilities were significantly decreased (P<0.05, P<0.01). At the same time, the protein expression levels of CyclinD1 and MMP1, which are closely related to cell proliferation and invasion, were significantly downregulated (P<0.05, P<0.01). The proportions of late apoptotic cells and the proportion of total apoptotic cells were significantly increased (P<0.01). In the Biejiajian Pills-containing serum group, the miR-885-5p mimics group, and the Biejiajian Pills-containing serum + miR-885-5p mimics group, miR-885-5p mRNA was significantly upregulated (P<0.01) and EGFR, MEK, and ERK1/2 were significantly downregulated at the mRNA level (P<0.05, P<0.01). EGFR, MEK, and ERK1/2 phosphorylation was inhibited (P<0.01), and the Biejiajian Pills-containing serum + miR-885-5p mimics group showed the best effect (P<0.05, P<0.01). The subcutaneous liver tumor model in nude mice verified that Biejiajian Pills can inhibit tumor growth in a dose-dependent manner. Conclusion Biejiajian Pills can promote apoptosis of MHCC-97H hepatoma cells and inhibit their proliferation, invasion, and migration. The mechanism may be related to the targeted regulation of the EGFR/MAPK/ERK signaling pathway by miR-885-5p.
2.Mechanism of Radix Paeoniae Rubra-Radix Aconiti Lateralis in Treatment of Acute-on-chronic Liver Failure Based on Macrophage Polarization
Nianhua TAN ; Yunan CAO ; Chenqin TANG ; Jie PENG ; Bin CHEN
Chinese Journal of Experimental Traditional Medical Formulae 2022;28(15):35-41
ObjectiveTo observe the therapeutic effect of Radix Paeoniae Rubra-Radix Aconiti Lateralis on acute-on-chronic liver failure (ACLF) rats and its effect on M1/M2 macrophage polarization. MethodMale SD rats were randomly divided into normal group, model group, positive group (lactulose, 1.8 g·kg-1) and traditional Chinese medicine (TCM) group (Radix Paeoniae Rubra-Radix Aconiti Lateralis, 5.85 g·kg-1), six in each group. The ACLF rat model was established by subcutaneous and tail vein injection of bovine serum albumin combined with intraperitoneal injection of D-galactosamine+lipopolysaccharide. Then the modeled rats were intervened with corresponding drugs for one week. The normal group and model group were given the same dose of distilled water. The histopathological changes of liver tissue were observed by hematoxylin-eosin (HE) staining. The mRNA and protein expression levels of CD86, inducible nitric oxide synthase (iNOS), CD206 and arginase 1 (Arg1) were detected by real-time polymerase chain reaction (Real-time PCR), Western blot and immunohistochemistry. ResultCompared with the conditions in the normal group, pseudolobule formation in liver tissue and morphological changes and necrosis of hepatocytes were observed in ACLF rats, accompanied by a large number of inflammatory cell infiltration. Moreover, the mRNA and protein expression levels of CD86, iNOS were up-regulated(P<0.01). Compared with the model group, the treatment groups had improved necrosis and inflammatory infiltration of hepatocytes, down-regulated mRNA and protein expression of CD86 and iNOS (P<0.01) and up-regulated mRNA and protein expression of CD206 and Arg1 (P<0.05, P<0.01), with the up regulation in the TCM group better than that in the positive group. ConclusionACLF rats had unbalanced M1/M2 macrophage polarization, and the imbalance shifted towards M1. Radix Paeoniae Rubra-Radix Aconiti Lateralis inhibited the activation of M1 macrophages and reduced the inflammatory response of liver failure by promoting the polarization of liver macrophages towards M2.
3.Effect of Yinchenhao Tang Combined with Yinchen Zhufu Tang on Treg/Th17 Cells in Vitro from Patients with Hepatitis B Virus-related Acute-on-chronic Liver Failure
Menghui ZENG ; Shan DU ; Nianhua TAN ; Jie PENG ; Bin CHEN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(16):43-51
ObjectiveTo study the regulatory effects of Yinchenhao Tang combined with Yinchen Zhufu Tang on the expression of regulatory T (Treg)/helper T 17 (Th17) cells cultured in vitro from the patients with hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF). MethodsFresh peripheral blood was collected from the patients with HBV-ACLF for the separation of peripheral blood mononuclear cells (PBMCs). Immunomagnetic beads were used to isolate primary Treg and naive CD4+ T cells. After in vitro expansion, naive CD4+ T cells were induced to differentiate into Th17 cells. Rats were treated with the clearing method (Yinchenhao Tang), warming method (Yinchen Zhufu Tang), and combination of clearing method with warming method (Yinchenhao Tang combined with Yinchen Zhufu Tang, also known as Wenyang Jiedu Huayu Prescription), respectively, and then the medicated plasma samples were collected. Meanwhile, blank plasma was collected from the rats treated with normal saline. Cells were classified into blank, clearing method (5.04 g·kg-1), warming method (6.21 g·kg-1), and combination of clearing method with warming method (17.1 g·kg-1) groups and treated with corresponding plasma. The frequency of Treg/Th17 cells was detected by flow cytometry. The level of transforming growth factor-β (TGF-β) was measured by the enzyme-linked immunosorbent assay. The cytometric bead array (CBA) was employed to measure the levels of interleukin-10 (IL-10), interleukin-17A (IL-17A), tumor necrosis factor-α (TNF-α), and interleukin-23 (IL-23). The mRNA and protein levels of Forkhead box P3 (FoxP3) and retinoic acid-related orphan receptor-gamma t (ROR-γt) were determined by Real-time PCR and Western blot, respectively. ResultsCompared with the blank group, the combination of clearing method with warming method group showed decreased frequency of Treg and Th17 cells, lowered levels of Treg cytokines (TGF-β and IL-10) and Th17 cytokines (TNF-α, IL-17A, and IL-23), and down-regulated mRNA and protein levels of FoxP3 and ROR-γt (P<0.01). Compared with the clearing method group, the combination of clearing method with warming method group showed decreased Treg cell frequency and down-regulated mRNA and protein levels of FoxP3. Meanwhile, the combination group showed decreased Th17 cell frequency, lowered levels of TGF-β, IL-10, TNF-α, IL-17A, and IL-23, and down-regulated mRNA and protein levels of ROR-γt (P<0.05, P<0.01). Compared with the warming method group, the combination of clearing method with warming method group showed decreased frequency of Treg cells and down-regulated mRNA and protein levels of FoxP3. Meanwhile, the combination group showed decreased Th17 cell frequency, declined levels of TGF-β, IL-10, TNF-α, IL-17A, and IL-23, and down-regulated mRNA and protein levels of ROR-γt (P<0.05). ConclusionThe combination of clearing method with warming method can down-regulate the expression of specific cytokines of Treg and Th17 cells, inhibit the over activation of Treg and Th17 cells, and reduce the secretion of cytokines such as TGF-β, IL-10, TNF-α, IL-17A, and IL-23, thereby alleviating inflammation and improving the prognosis of the patients with liver failure.
4.Differences in Expression of Intestinal Flora in Patients with Different TCM Syndrome Types of "Yang Huang-Yin-Yang Huang-Yin Huang" in Hepatitis B Virus-related Acute-on-chronic Liver Failure
Shan DU ; Menghui ZENG ; Nianhua TAN ; Jie PENG ; Bin CHEN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(16):62-69
ObjectiveTo investigate the differential expression of intestinal flora in patients with different traditional Chinese medicine (TCM) syndrome types (Yang Huang syndrome, Yin-Yang Huang syndrome, and Yin Huang syndrome) of hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) and clarify the biological basis of jaundice and Yin Huang syndrome in liver failure. MethodsA total of 20 cases of HBV-ACLF patients were included in the Yang Huang group, 20 cases in the Yin-Yang Huang group, 16 cases in the Yin Huang group, and 20 healthy adult volunteers. 16S rRNA gene sequencing was used to detect the diversity, species distribution, and differences of the subjects' intestinal flora, and bioinformatics analysis was conducted. ResultsCompared with those in the healthy control group, the species richness and diversity of intestinal flora in the HBV-ACLF Yang Huang group, Yin-Yang Huang group, and Yin Huang group were significantly reduced, and there were significant differences in the composition of intestinal flora compared with healthy volunteers. However, there were no significant differences in the species richness, diversity, and composition of intestinal flora among the three groups. LEfSe analysis showed that compared with the healthy control group, the HBV-ACLF Yang Huang group showed significant enrichment of Staphylococcus aureus(P<0.01). Yin-Yang Huang group showed significant enrichment of s_Ileibacterium valens(P<0.05,P<0.01), and the Yin Huang group showed significant enrichment of Enterococcus faecium and Streptococcus sali varius(P<0.05). These strains may be biomarkers between the three groups of patients and the healthy control group. Compared with that in the Yin-Yang Huang group, Tyzzerella_nexilis was significantly enriched in the Yang-Huang group, and Streptococcus lactiae was significantly enriched in the Yin-Yang Huang group. Compared with that in the Yang-Huang group and the Yin-Yang Huang group, Enterococcus faecalis was significantly enriched in the Yin Huang group. The above strains may be biomarkers among the three groups of patients, and Enterococcus faecium may be a biomarker for the transition from the Yang Huang group to the Yin Huang group. ConclusionsThere are significant differences in the intestinal flora between patients with HBV-ACLF Yang Huang syndrome, Yin-Yang Huang syndrome, and Yin Huang syndrome. Enterococcus faecium is significantly enriched in the Yin Huang syndrome group, suggesting that dysbiosis of the intestinal flora may be the biological basis for jaundice and Yin Huang syndrome in liver failure.
5.Changes of Treg/Th17 Cell Expression in Peripheral Blood of Patients with Hepatitis B Virus-related Acute-on-chronic Liver Failure in Five Types of Traditional Chinese Medicine Syndrome
Shan DU ; Menghui ZENG ; Nianhua TAN ; Jie PENG ; Bin CHEN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(16):70-76
ObjectiveTo study the expression differences of regulatory T cells (Treg) and T helper 17 cells (Th17) in the peripheral blood of patients with hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) in five types of traditional Chinese medicine (TCM) syndrome. MethodsA total of 144 patients with HBV-ACLF were included and divided into five types of TCM syndrome, including 34 cases of heat-toxin amassment syndrome, 44 cases of dampness-heat amassment syndrome, 27 cases of Qi-deficiency and stasis jaundice syndrome, 21 cases of spleen-kidney Yang deficiency syndrome, and 18 cases of liver-kidney Yin deficiency syndrome. Meanwhile, 30 healthy volunteers were included as controls. The frequency of Treg and Th17 cells in the peripheral blood of subjects in each group was detected by flow cytometry, and the Treg/Th17 ratio was calculated. Cytometric bead array (CBA) was used to detect the levels of cytokines interleukin (IL)-10, transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), IL-17A, and IL-23. Real-time fluorescence quantitative PCR (Real-time PCR) detected the mRNA expression of forkhead box P3 (FoxP3) and retinoic acid-related orphan receptor gamma t (ROR-γt). Results(1) Compared with that in the healthy control group, the frequency of Treg and Th17 cells in patients with various TCM syndrome types of HBV-ACLF increased (P<0.05). Compared with that in the heat-toxin amassment syndrome group, the frequency of Treg and Th17 cells decreased in the dampness-heat amassment syndrome group (P<0.05), while the frequency of Treg and Th17 cells increased in the Qi-deficiency and stasis jaundice syndrome group, spleen-kidney Yang deficiency syndrome group, and liver-kidney Yin deficiency syndrome group (P<0.05). Compared with that in the dampness-heat amassment syndrome group, the frequency of Treg and Th17 cells increased in the dampness-heat amassment syndrome group, spleen-kidney Yang deficiency syndrome group, and liver-kidney Yin deficiency syndrome group (P<0.05). Compared with that in the Qi-deficiency and stasis jaundice syndrome group, the frequency of Treg and Th17 cells increased in the spleen-kidney Yang deficiency syndrome group (P<0.05), while the frequency of Treg and Th17 cells decreased in the liver-kidney Yin deficiency syndrome group (P<0.05). Compared with the spleen-kidney Yang deficiency syndrome group, the frequency of Treg and Th17 cells decreased in the liver-kidney Yin deficiency syndrome group (P<0.05). (2) Compared with that in the healthy control group, the Treg/Th17 cell ratio in patients with various TCM syndromes of HBV-ACLF decreased (P<0.05). Compared with that in the heat-toxin amassment syndrome group, the Treg/Th17 cell ratio increased in the dampness-heat amassment syndrome group (P<0.05), while it decreased in the Qi-deficiency and stasis jaundice syndrome group, spleen-kidney Yang deficiency syndrome group, and liver-kidney Yin deficiency syndrome group (P<0.05). Compared with that in the dampness-heat amassment syndrome group, the Treg/Th17 cell ratio decreased in the Qi-deficiency and stasis jaundice syndrome group, spleen-kidney Yang deficiency syndrome group, and liver-kidney Yin deficiency syndrome group (P<0.05). Compared with that in the Qi-deficiency and stasis jaundice syndrome group, the Treg/Th17 cell ratio increased in the spleen-kidney Yang deficiency syndrome group and liver-kidney Yin deficiency syndrome group (P<0.05). Compared with the spleen-kidney Yang deficiency syndrome group, the Treg/Th17 cell ratio in the liver-kidney Yin deficiency syndrome group increased (P<0.05). (3) Compared with those in the healthy control group, the levels of Treg-related cytokines IL-10 and TGF-β, as well as Th17-related cytokines TNF-α, IL-17A, and IL-23, were elevated in patients with various TCM syndrome types of HBV-ACLF (P<0.05). There was no significant difference in TNF-α levels among different TCM syndrome types. Compared with those in the heat-toxin amassment syndrome group, the levels of IL-10, TNF-β, IL-17A, and IL-23 in the dampness-heat amassment syndrome group, Qi-deficiency and stasis jaundice syndrome group, spleen-kidney Yang deficiency syndrome group, and liver-kidney Yin deficiency syndrome groups increased (P<0.05). Compared with those in the dampness-heat amassment syndrome group, the levels of IL-10, TGF-β, IL-17A, and IL-23 increased in the Qi-deficiency and stasis jaundice syndrome group, spleen-kidney Yang deficiency syndrome group, and liver-kidney Yin deficiency syndrome group (P<0.05). Compared with those in the Qi-deficiency and stasis jaundice syndrome group, the levels of IL-10, TGF-β, IL-17A, and IL-23 in the spleen-kidney Yang deficiency syndrome group increased (P<0.05), while those in the liver-kidney Yin deficiency syndrome group decreased (P<0.05). Compared with those in the spleen-kidney Yang deficiency syndrome, the levels of IL-10, TGF-β, IL-17A, and IL-23 in the liver-kidney Yin deficiency syndrome group decreased (P<0.05). (4) Compared with that in the healthy control group, the mRNA of Treg/Th17 cell specific transcription factors FoxP3 and ROR-γt were elevated in patients with various TCM syndrome types of HBV-ACLF (P<0.05). Compared with that in the heat-toxin amassment syndrome group, the mRNA of FoxP3 and ROR-γt increased in the Qi-deficiency and stasis jaundice syndrome group, spleen-kidney Yang deficiency syndrome group, and liver-kidney Yin deficiency syndrome group (P<0.05). Compared with that in the dampness-heat amassment syndrome group, the mRNA of FoxP3 and ROR-γt increased in the Qi-deficiency and stasis jaundice syndrome group, spleen-kidney Yang deficiency syndrome group, and liver-kidney Yin deficiency syndrome (P<0.05). Compared with that in the Qi-deficiency and stasis jaundice syndrome group, the mRNA of FoxP3 and ROR-γt in the spleen-kidney Yang deficiency syndrome group increased (P<0.05), and it decreased in the liver-kidney Yin deficiency syndrome group (P<0.05). Compared with that in the spleen-kidney Yang deficiency syndrome group, the mRNA of FoxP3 and ROR-γt decreased in the liver-kidney Yin deficiency syndrome group (P<0.05). ConclusionThe frequency and ratio of Treg/Th17 cells, as well as the expression of related cytokines and specific receptors in peripheral blood of patients with HBV-ACLF in five types of TCM syndromes are different, which has certain reference value for TCM syndrome differentiation and treatment of patients with HBV-ACLF.
6.Mechanism of Paeoniae Radix Rubra and Aconiti Lateralis Radix Praeparata in Treatment of Acute-on-chronic Liver Failure Based on Bioinformation Analysis and Experimental Validation
Xiaoling TIAN ; Yu ZHANG ; Shan DU ; Mengsi WU ; Nianhua TAN ; Bin CHEN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(1):156-165
ObjectiveTo explore the mechanism of action of Paeoniae Radix Rubra and Aconiti Lateralis Radix Praeparata (CSFZ) in the treatment of acute-on-chronic liver failure (ACLF) through network pharmacology, molecular docking, and animal experiments. MethodsNetwork pharmacology was used to identify potential targets and related signaling pathways for the treatment of ACLF with CSFZ. Molecular docking was used to examine the binding activity of the core components with corresponding key targets. An ACLF rat model was established by subcutaneous and tail vein injections of bovine serum albumin combined with lipopolysaccharide (LPS) + D-galactosamine (D-GalN) intraperitoneal injection. A normal control group (NC), a model group, a CSFZ group (CSFZ, 5.85 g·kg-1), and a hepatocyte growth-promoting granule group (HGFG, 4.05 g·kg-1) were set up in this study. Pathological changes in rat liver tissue were observed using hematoxylin and eosin (HE) and Masson staining. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of interleukin-6 (IL-6), B-cell lymphoma-2 (Bcl-2), Caspase-3, and albumin (ALB). Real-time quantitative polymerase chain reaction (Real-time PCR) and Western blot were used to measure the mRNA and protein expression levels of phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), phosphorylated PI3K (p-PI3K), and phosphorylated Akt (p-Akt). ResultsNetwork pharmacology screening identified 49 active ingredients of CSFZ, 103 action targets, and 3 317 targets related to ACLF. Among these, 74 targets overlapped with CSFZ drug targets. Key nodes in the protein-protein interaction (PPI) network included Akt1, tumor necrosis factor (TNF), IL-6, Bcl-2, and Caspase-3. Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis identified multiple signaling pathways, with the PI3K/Akt signaling pathway being the most frequent. Molecular docking showed that the core components of the drug exhibited good binding activity with the corresponding key targets. Animal experiments confirmed that CSFZ significantly improved liver tissue pathological damage in ACLF rats, reduced the release of inflammatory factors and liver cell apoptosis, and upregulated the expression levels of the PI3K/Akt signaling pathway. ConclusionThrough network pharmacology, molecular docking, and in vivo experiments, this study confirms the effect of CSFZ in reducing liver cell inflammatory damage and inhibiting liver cell apoptosis. The specific mechanism may be related to its involvement in regulating the PI3K/Akt signaling pathway.
7.Mechanism of Xiayuxue Tang in Inhibiting Hepatocellular Carcinoma Cells by Regulating YAP1/SIRT5 Signaling Axis to Mediate Succinate Metabolism and Succinylation
Linzhu LU ; Qianqian GUO ; Xuefei TIAN ; Bin CHEN ; Nianhua TAN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(16):52-61
ObjectiveTo investigate the inhibitory effects and mechanisms of Xiayuxue Tang (XYXT) on hepatocellular carcinoma cells through the regulation of succinate metabolism and succinylation modification. MethodsXYXT-medicated serum was prepared. The effects of different concentrations of succinate on the proliferation of HepG2 and MHCC97H cells were observed using the cell counting kit-8 (CCK-8) assay, and the experimental concentrations for subsequent tests were determined. Further CCK-8 assays were performed to evaluate the effects of XYXT-medicated serum of different concentrations (5%, 10%, and 15%) on cell proliferation. Flow cytometry, scratch test, and Transwell assay were employed to analyze the effect of 10% XYXT-medicated serum on the cell cycle, migration, invasion, and apoptosis. The changes in succinate metabolism and succinylation modification were examined based on succinate content assays, succinate dehydrogenase (SDH) activity detection, and western blot. The expressions of Yes-associated protein 1 (YAP1) and sirtuin 5 (SIRT5) were detected via Real-time PCR and western blot. Molecular docking was applied to validate the binding between XYXT’s main components and target proteins. ResultsCompared with the control group, 1-2 mmol·L-¹ succinate significantly promoted HepG2 and MHCC97H cell proliferation (P<0.01). XYXT-medicated serum (5%, 10%, and 15%) markedly inhibited the proliferation of both cell lines compared with the blank group (P<0.05, P<0.01). Treatment with 10% XYXT-medicated serum arrested the cell cycle at the stage prior to DNA synthesis (G0/G1) (P<0.01), suppressed migration (P<0.01) and invasion (P<0.05, P<0.01), and promoted apoptosis of HepG2 and MHCC97H cells (P<0.01). Co-treatment with XYXT and succinate reversed the inhibitory effects of XYXT on proliferation, migration, and invasion of HepG2 and MHCC97H cells (P<0.05, P<0.01). The proportion of cells at G0/G1 phase decreased (P<0.01), and the apoptosis rate decreased (P<0.01). In terms of succinate metabolism, compared with the blank serum group, the 10% XYXT-medicated serum reduced succinate levels of HepG2 and MHCC97H cells (P<0.05, P<0.01), enhanced SDH activity (P<0.01), and downregulated succinylation modification. In terms of YAP1/SIRT5 pathway, compared with the blank serum group, the 10% XYXT-medicated serum significantly downregulated the mRNA and protein expressions of YAP1 in HepG2 and MHCC97H cells (P<0.05, P<0.01) while significantly upregulated the mRNA and protein expressions of SIRT5 (P<0.05, P<0.01). Molecular docking confirmed that there was a good binding ability between YAP1 and SIRT5, as well as between XYXT's main active components and YAP1 and SIRT5. ConclusionXYXT suppresses hepatocellular carcinoma cells by modulating the YAP1/SIRT5 signaling axis to intervene in succinate metabolism and succinylation modification.