Background

Gastric cancer (GC) is a prevalent malignancy, ranking fifth in terms of new cases in 2020 and fourth in terms of mortality, causing approximately 800,000 deaths [1, 2]. Even with various treatment options like surgery, chemotherapy, radiotherapy, drugs, and immunotherapy, the prognosis for patients with GC remains unsatisfactory [3]. Therefore, the identification of alternative and effective treatments for GC are of paramount importance.

Having been practiced for over 5000 years, Traditional Chinese Medicine (TCM) has gained recognition as a credible and promising alternative therapy for cancer. Gexia-Zhuyu Tang (GZT) is a TCM Formula (TCMF) that shows promise in treating chronic diseases [4]. It is composed of 12 Chinese herbal medicines, including Peach kernel, Safflower, Angelica sinensis, Chuanxiong, Red peony, Peony bark, YanhuSuo, Wulingzhi, Wuyao, Fructus Aurantii, ** between the M and H groups, accounting for 6.33%, while the H group had 15888 unique OTUs, accounting for 53.77% (Fig. 5A). At the phylum level, the phyla Firmicutes, Bacteroidetes, and Proteobacteria were relatively abundant (Fig. 5B). With high-dose modified GZT treatment, the abundance of Bacteroidetes, Proteobacteria, and Tenericutes increased, and the abundance of Firmicutes decreased. At the genus level, the relative abundance of Bacteroides, Lactobacillus, and Oscillospira ranked in the top three. In the H group, the abundance of Bacteroides decreased compared to the M group, while the abundance of Coprococcus and Psychrobacter increased (Fig. 5B). The heatmap was generated for the top 50 genera based on the mean abundance. The high-dose modified GZT treatment reduced the abundance of Desulfovibrio, Helicobacter, and Facklamia, while increased the abundance of Alistipes, Sutterella, Paraprevotella, and Psychrobacter (Fig. 5C).

Fig. 5
figure 5

The modified GZT treatment alters the community of intestinal flora in mice with GC. A Venn diagram showed the overlap** OTUs in different groups. B Average relative abundance of gut microbiota at the phylum and genus level, respectivly, in different regions of the Venn diagram. C Differences in gut microbiota in the Model, Lower dose, and Higher dose group at the genus level were demonstrated by heatmaps

Full size image

By analyzing the iconic species, notable differences in ASV/out were found between the M group and the H group (Additional file 1: Figure S3A). The abundance of Desulfovibrionaceae and Desulfovibrio was significantly higher in the M group compared to the H group (Additional file 1: Figure S3B). The analysis of marker species showed significant differences in the abundance of species between the M group and the H group (Additional file 1: Figure S3C). The taxonomic hierarchy, from phylum to genus, in each group of samples was shown in Additional file 1: Figure S1D. At the order, family, and genus levels, the marker species in the modified GZT high-dose treatment group were Turicibacterales, Turicibacteraceae, and Turicibacter.

The PCA method was used to study the degree of dispersion of the differential species. The results demonstrated a distinct trend of separation between the M and H groups, with the ratio of the difference in species abundance composition to the total difference for all samples at 43.3% (Additional file 1: Figure S4A). At the same time, the results of OPLS-DA indicated that the abundance composition of the differential species between the M and H groups exhibited a separation trend with only a few overlap** individual parts and a high overall separation, indicating a better classification effect (Additional file 1: Figure S4B).

Modified GZT inhibits the GC development by regulating intestinal flora

The Paraprevotella, Psychrobacter, Trematoda, Alistipes, Coprococcus, Oscillospira, and Sutterella are not the predominant bacterial community in GC. Therefore, the study opted for a more comprehensive analysis by using the entire fecal microbiota for the FMT experiment, which provided insights into how GZT treatment affected the gut microbiota and its potential impact on gastric cancer in mice. Fecal microorganisms from donor mice were extracted and transferred to recipient mice to evaluate the impact of FMT on GC progression in mice (Fig. 6A). The results indicated that a noteworthy decrease was observed in both the tumor volume and weight in the high-dose of the modified GZT (FMT) group compared to the Model (FMT) group (Fig. 6B–D). Moreover, there was a significant reduction in immune cell infiltration and inflammatory cells in the Higher Dose (FMT) group compared to the Model (FMT) group (Fig. 6E). IHC assay results showed a significant increase in GSDMD expression in the Higher Dose (FMT) group compared with the Model (FMT) group (Fig. 6F). Furthermore, modified GZT (FMT) decreased the number of Ki67-positive cells (Fig. 6G). The expression levels of metastasis and invasion-associated proteins CD147, VEGF, and MMP-9 were significantly lower in the Higher Dose (FMT) group than in the Model (FMT) group (Fig. 6H).

Fig. 6
figure 6

The modified GZT treatment inhibits tumor growth in vivo by regulating intestinal flora. A Schematic diagram of fecal gavage experiment in BALB/c mice. B Photographs of tumors in mice. C Tumor volume growth curve. D Tumor weight change curve. E Histopathological changes observed by HE staining (200 × , scale bar: 100 μm); F The expression level of GSDMD was assessed by IHC (magnification: 200 × , scale bar: 50 μm). G Ki67 immunohistochemical staining was used to detect cell proliferation (200 × , scale bar: 100 μm). H Western blot was used to detect the protein expression levels of CD147, VEGF and MMP-9 in the Model (FMT) and the Higher Dose (FMT) group. *P < 0.05; **P < 0.01 vs. Model group

Full size image

Modified GZT promotes pyroptosis by regulating gut microbiota

Next, we used FMT to detect whether gut microbiota altered by modified GZT had therapeutic benefits for GC. Fecal microbiota from Model or higher dose modified GZT-treated mice were transplanted into GC mice recipients. ELISA analysis showed that the levels of TNF-α, IL-1β, IL-18, and LDH levels were significantly lower in the Model (FMT) group compared to the Control group. In contrast, the levels of TNF-α, IL-1β, IL-18, and LDH levels in the Higher Dose (FMT) group were significantly higher than that in the Model (FMT) group (Fig. 7A, B). Furthermore, the expression of NLRP3, ASC, and caspase-1 proteins were increased in the Higher Dose (FMT) group than that in the Model (FMT) group (Fig. 7C).

Fig. 7
figure 7

The modified GZT inhibits pyroptosis by regulating gut microbiota. A–B ELISA assay was used to detect the expression levels of TNF-α, IL-1β, IL-18 and LDH. C Western blot was used to detect the protein expression of NLRP3, ASC, and caspase-1. *P < 0.05; **P < 0.01 vs. Control group; ##P < 0.01 vs. Model group

Full size image

Discussion

GC is a malignancy that originates from the inner lining of the stomach with a high morbidity and mortality rate [36]. H. pylori infection, diet, family history, and alcohol consumption are all high risk factors for GC [37], of which H. pylori infection is responsible for approximately 70% of all GC cases worldwide [38]. Advances in molecular biology and sequencing technologies have enabled researchers to study the gut microbiome in greater depth and breadth in GC, not just H. Pylori. 16S rRNA sequencing has proven to be a powerful tool for exploring the diversity and composition of microbial communities. In the gut microbiome, 16S rRNA sequencing has been employed to study the effects of diet, drug therapy, and disease on microbial diversity and composition [39]. In the current study, we demonstrated that modified GZT inhibited the growth, proliferation, metastasis, invasion, and promoted pyroptosis of GC. Through 16S rRNA sequencing analysis, we detected alterations in the gut microbiota following modified GZT treatment. This study demonstrates that modified GZT effectively suppresses GC progression by modulating the gut microbiota.

First, we analyzed the metabolites of modified GZT by HPLC–MS/MS. The results showed that the metabolites of modified GZT mainly included dihydroxybenzoic acid, 4-methylcatechol, ailanthone, and 18β-glycyrrhetinic acid. In GC 3,4-dihydroxybenzoic acid promotes cell apoptosis through JNK/p38 MAPK signaling activation and exerts anti-tumor effects [40]. The 18β-glycyrrhetinic acid exhibits properties with potential benefits, including antitumor, anti-inflammatory, and antiviral effects [41,42,43]. Furthermore, 18β-glycyrrhetinic acid impedes the development and advancement of GC by downregulating COX-2 expression and suppressing Wnt-1 expression through miR-149–3 upregulation [44]. It inhibits the migration and invasion of GC cells through the ROS/PKC-α/ERK pathway [45]. 4-methylcatechol induces apoptosis via the intrinsic mitochondrial pathway and demonstrates cytotoxic effects on metastatic malignant melanoma cells [46]. Ailanthone possesses anti-inflammatory, anti-malarial, and anti-tumor effects. It can inhibit the progression of multiple tumors, including acute myeloid leukemia, lung, breast, melanoma, and gastric cancer [47]. These findings all suggest that modified GZT may have an important role in inhibiting tumor development.

Then we explored the effects of modified GZT on GC. The results revealed that modified GZT significantly inhibited tumor growth in a dose-dependent manner. The Ki-67 marker is a recognized parameter for measuring proliferation in malignant tumors [48]. IHC results showed that modified GZT reduced the number of Ki67-positive cells, inhibiting the proliferation of GC tumor tissue cells. CD147, which is highly expressed on the extracellular surface of numerous types of tumor tissue, potentially promotes tumor metastasis through its regulation of cellular substrate and adhesion mechanisms [49]. The role of CD147 in promoting tumor invasiveness has been recently confirmed in various human malignancies, including GC [33]. Angiogenesis plays a crucial role in the metastasis of solid tumors, with VEGF standing out as the most potent mediator of angiogenesis [50]. MMP-9, a member of the matrix metalloproteinase family, is involved in GC metastasis and invasion [51]. In the present study, we found that both low and high doses of modified GZT suppressed the expression of CD147, VEGF, and MMP-9, implying that modified GZT inhibited the metastasis and invasion of GC tumors. In addition, IHC staining demonstrated that modified GZT significantly increased the expression level of GSDMD. GSDMD is a widely investigated pore-forming protein in pyroptosis, and it is a substrate for activated caspase-1/4/5/11 [52]. Further investigation showed that modified GZT greatly promoted the protein expression levels of NLRP3, ASC and caspase-1, and the concentrations of TNF-α, IL-1β, IL-10 and LDH. Subsequent FMT showed that modified GZT inhibited the development of GC by modulating the gut microbiota.

Composition and species abundance of intestinal flora are significantly altered in GC. Studies have shown that as the progression from gastritis to intestinal metaplasia and GC occurs, there is a gradual decline in both diversity and abundance of gut microbiota [23, 53]. In GC, the abundance of Fusobacterium, Leptotrichia, Veillonella, Campylobacter and Haemophilus is notably elevated [54]. In comparison to patients with intestinal metaplasia and gastritis, those with GC exhibit a significantly higher abundance of Lactobacillus, Clostridium, and Lachnospiraceae [55]. The fecal microbiota promotes inflammation, affects cell proliferation and immune regulation, and disrupts DNA integrity, ultimately contributing to carcinogenesis [56]. The most common Helicobacter species is H. Pylori. The mechanism through which H. pylori infection induces GC mainly involves chronic gastritis, releasing gastrin and histamine, damaging DNA, and activating the proliferation of gastric epithelial cells through multiple pathways such as the induction of the PI3K/Akt pathway [57]. The increased abundance of Lachnospiracea in GC may be related to inflammatory regulation [58]. In our study, we found that after high-dose modified GZT treatment, the abundances of many gut flora species and the community composition were significantly changed. At the genus level, the abundance of several genera including Bacteroides, Desulfovibrio, Helicobacter, and Facklamia decreased, while the abundance of Paraprevotella, Psychrobacter, Trematoda, Alistipes, Coprococcus, Oscillospira, and Sutterella increased.

Numerous studies have shown that TCMF can regulate intestinal flora and affect the development of intestinal cancer. A previous study has found that the intestinal flora diversity, microbial composition, and abundance in patients with precancerous lesions of GC are altered after treatment with Weifuchun capsules [59]. Quxie Capsules elevate the abundance of Actinobacteria and Lachnospiraceae, while decrease the abundance of Bacteroides, Bacteroidetes, Escherichia Shigella, and Gammaproteobacteria, which may help protect against colorectal cancer tumors and enhance immunity [60]. Curcumin, a compound generated by the roots of the Curcuma longa plant, may have anti-carcinogenic qualities via preserving the diversity of gut bacteria [61]. Antibiotics are extensively employed in the realm of Western medicine for the management of a broad spectrum of ailments [62]. Nevertheless, their usage is concomitant with an alteration in the composition and a reduction in the diversity of the human microbiota [63, 64]. Investigation has revealed a progressive increase in the risk of GC associated with the cumulative number of penicillin courses administered [65]. Antibiotic administration not only influences the resistome of the subject to whom it is given, but also the whole population owing to selection for resistance to its function [66]. Prior research has demonstrated that the use of antibiotics in conjunction with prebiotics, probiotics, and synbiotics assists the gut microbiota in resisting H. pylori infections and lowers the proportion of drug-resistant bacteria [67]. Therefore, we hypothesize that modified GZT combined with antibiotic therapy plays a role in preserving gut microbiota homeostasis and decreasing drug resistance.

Inflammasomes in the gastrointestinal tract are associated with intestinal flora homeostasis and infection [68]. Studies have shown that TCM can reduce H. pylori-induced gastritis by decreasing the expression of IL-8, TNF-α, IL-6, iNOS, and IFN-γ [69]. These findings suggest that there is an important correlation between TCM and intestinal flora, inflammation, and pyroptosis. In this study, we found that high dose modified GZT facilitated the expression proinflammatory factors TNF-α, IL-1β, IL-18, and caspase-1-dependent pyroptosis by modulating intestinal flora. Caspase-1 activates primarily the pro-inflammatory factors IL-1β and IL-18 [70]. Moreover, studies have shown that some pathogen-associated molecular patterns, carried by bacteria, are delivered to the host cytoplasm where they activate inflammasomes [68]. Thus, we speculate that modified GZT promotes caspase-1-dependent pyroptosis by inhibiting harmful bacteria, which in turn enhances the release of proinflammatory factors.

However, there are also limitations in this investigation. Firstly, we relied on previous studies for GZT ingredients by HPLC analysis and did not conduct HPLC analysis of modified GZT in this study due to scientific limitations. Secondly, for the assessment of pyroptosis, we determined the expression levels of GSDMD, inflammatory factors, and pyroptosis-related proteins using IHC assay. However, morphological analysis was not performed through electron microscopy due to scientific limitations. Additionally, in the evaluation of modified GZT effect on the metastasis and invasion of GC, we solely assessed the protein expression levels of CD147, VEGF, and MMP-9, which will be further validated by in vitro experiments in our subsequent exploration.

Conclusion

In this study, we found that the modified GZT inhibited the growth, proliferation, metastasis, and invasion of GC by regulating the intestinal flora. Furthermore, modified GZT also promoted pyroptosis of GC. The current investigation revealed a novel therapeutic strategy and theoretical foundation for utilizing TCMF in the treatment of GC.