Published journal: Frontiers in Endocrinology
Impact factor: 6.055
Author unit: The First Affiliated Hospital of Henan University of Traditional Chinese Medicine
Baiqu Biotech provides services: discovery of metabolomics Classic-hydrophilic version + intestinal flora sequencing
Research background
Cancer-related fatigue (CRF) is the most common adverse effect after cancer treatment. CRF is more common in breast cancer (BC) survivors than other cancer survivors. Studies have found that the incidence of CRF in patients receiving chemotherapy is 70%-100%, and the fatigue of patients is significantly more severe than that of patients not receiving chemotherapy. Chemotherapy can activate peripheral pro-inflammatory cytokines and cause central nervous system inflammation by disrupting the permeability of the physiological barrier, which in turn leads to dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis, ultimately leading to CRF. The tight junction proteins ZO-1, occludin and claudin-5 can regulate cell permeability and maintain tight junction structure and barrier function. Numerous studies have shown that changes in cortisol (CORT), corticotropin-releasing hormone (CRH) and adrenocorticotropin (ACTH) of the HPA axis play an important role in the development of fatigue. In addition, acupuncture can modulate gut microbiota disturbances and restore homeostasis by interfering with various aspects of the gut-brain axis. Therefore, the author studied the effect of acupuncture on fatigue behavior, intestinal flora, intestinal inflammation, neuroinflammatory response, intestinal barrier and HPA axis and the changes in serum metabolites in the CRF mouse model after breast cancer chemotherapy, in order to elucidate the effects of acupuncture and moxibustion. Anti-fatigue mechanisms of therapy.
Materials and Methods
1 Animals and Cells
Forty 6-8 week old SPF BALB/c female mice were adaptively fed for 1 week. 4T1 luciferase mouse breast cancer cells were cultured in RPMI-1640 medium.
2 Grouping and Modeling
Using the random grouping method, 40 female mice were randomly divided into 4 groups (n=10/group): (1) Control group, fed normally without any intervention; (2) Model group, received subcutaneous tumor transplantation and environmental protection. Phosphoramide chemotherapy treatment (CTX); (3) Acu group, after successful modeling, all mice received acupuncture treatment at the corresponding treatment point; (4) Sham group, mice were stimulated at non-acupoints near the corresponding treatment point.
3 experiments
Do forced swimming (FST) and open-air test (OFT) for fatigue behavior verification, and take intestinal contents, intestinal tissue, hippocampal tissue and serum samples of mice for 16S, WB, ELASA and serum metabolites and other related tests .
Figure 1. Experimental design
Research results
1 Anti-fatigue effect of acupuncture and moxibustion on breast cancer after BC chemotherapy
First, FST and OFT experiments were used to explore the fatigue behavior of mice in each group after tumor chemotherapy and acupuncture intervention. The results showed that the forced swimming immobility time increased in the model group compared with the control group (Fig. 2C). In the OFT experiment, both the total distance traveled and the time spent in the central area decreased in the model group (Fig. 2D,E). Compared with the model group, the immobility time of forced swimming in the acupuncture group was significantly reduced (Fig. 2C), and the total traveling distance and time spent in the central area were significantly increased (Fig. 2D, E), while the sham acupuncture group and the model group had There was no difference in terms of FST and OFT. At the same time, compared with the sham acupuncture group, the forced swimming immobility time was also significantly shortened, and the total traveling distance and time spent in the central area were significantly increased in the acupuncture group (Fig. 2C-E). These data suggest that acupuncture treatment has an anti-fatigue effect on CRF mice after BC chemotherapy.
Figure 2. Anti-fatigue effect of acupuncture and moxibustion on breast cancer after BC chemotherapy
2 Acupuncture treatment regulates the species abundance of intestinal flora in CRF mice after BC chemotherapy
The authors used 16S rRNA sequencing to examine changes in the gut microbiota following chemotherapy in mice with cancer-associated fatigue. The results showed that the Shannon index and Simpson index of the control group were significantly higher than those of the model group and the sham acupuncture group, meanwhile, the Shannon index and Simpson index of the acupuncture group also increased significantly (Fig. 3A,B). The results of PCoA showed that the samples of the model group and the sham acupuncture group were completely separated from the control group, and the samples of the acupuncture group were relatively close to those of the control group (Figure 3C). The above results indicated that the richness, diversity and species differences of gut microbiota in cancer-related fatigue (CRF) mice were significantly changed after chemotherapy, and acupuncture treatment could effectively reverse this change.
Figure 3. Acupuncture treatment modulates the species abundance of intestinal flora in CRF mice after BC chemotherapy
Then the author counted the top 30 relative abundance species in each group at the phylum level, and drew an abundance histogram. It can be seen that at the phylum level, compared with the control group, the abundance of Proteus significantly increased in the model group and the sham acupuncture group, while the abundance of Bacteroides and Patellar bacteria decreased significantly. Compared with the model group and the sham acupuncture group, the abundance of Firmicutes, Bacteroides, and Patellar bacteria in the acupuncture group was significantly increased, and the abundance of Proteobacteria was significantly decreased. At the genus level, compared with the control group, the abundance of Burkholderia, Escherichia coli, Shigella, and Streptococcus in the model group was higher, while Lactobacillus, unclassified Bacteroides, segmented filamentous Bacteria and Clostridium were low. Compared with the model group and the sham acupuncture group, the abundance of Burkholderia, Escherichia coli, Shigella, and Streptococcus in the acupuncture group decreased, while Lactobacillus, Bacteroides unclassified, segmented filamentous bacteria and unclassified Clostridium UCG-014 increased in abundance (Fig. 3E). According to LEfSe analysis, the abundance of Lactobacillus in the acu group was higher than that in the model group and the sham acupuncture group (Fig. 3F,G). In addition, according to the species abundance table, a significant difference analysis between groups was performed based on the genus level. The authors found that the level of Lactobacillus was significantly increased after acupuncture (Fig. 3H,I). The above results indicate that acupuncture treatment can improve the imbalance of intestinal flora and increase the content of beneficial bacteria in BC-related fatigue mice after chemotherapy.
3 Acupuncture treatment improves intestinal barrier, intestinal inflammation and neuroinflammation in CRF mice after BC chemotherapy
In the author's study, it was found that compared with the model group and the sham acupuncture group, the expression levels of tight junction protein ZO-1, occludin and tight junction transmembrane protein-5 were significantly increased in the acupuncture group (Fig. 4A, B) . In addition, CRF significantly increased the protein expression levels of proinflammatory factors IL-1β, IL-6, and TNF-α in the gut (Fig. 4C, D) and hippocampus (Fig. 4E, F) compared with the control group, and The levels of three pro-inflammatory factors were significantly suppressed after acupuncture treatment (Fig. 4C–F). The results showed that acupuncture treatment could improve intestinal barrier function and reduce intestinal and hippocampal inflammatory responses in breast cancer mice after BC chemotherapy.
Figure 4. Acupuncture treatment improves intestinal barrier, intestinal inflammation and neuroinflammation in CRF mice after BC chemotherapy
4. Acupuncture treatment improves HPA axis dysfunction after BC chemotherapy in mice
In this study, ELISA was used to detect the protein content changes of HPA axis-related factors in serum. Studies have shown that, compared with the control group, the levels of CRH and CORT were significantly reduced and the level of ACTH was increased in the model group. Compared with the model group, the expression levels of CRH and CORT were increased, while the expression of ACTH was inhibited in the acupuncture group. And the levels of CRH, CORT and ACTH in the sham acupuncture group and the model group did not change significantly (Fig. 5A-C). The results showed that acupuncture therapy exerted an anti-fatigue effect by improving HPA axis dysfunction.
Figure 5. Acupuncture treatment improves HPA axis dysfunction after BC chemotherapy in mice
5 Effect of acupuncture and moxibustion on serum metabolites and metabolic pathways of CRF mice after BC chemotherapy
The changes of serum metabolites in each group were analyzed based on LC-MS non-target technology. The results showed that PCA analysis did not show significant differences between groups (Fig. 6A). The OPLS-DA score chart showed that the metabolites in the model group were significantly different from those in the control group (Figure 6B), and the metabolites in the acupuncture group were also significantly different from those in the model group and the sham acupuncture group (Figure 6C, D). At the same time, there was good model fit and predictability among the groups (Fig. 6E–G). In addition, the authors combined volcano map analysis and found that compared with the model group and the sham acupuncture group, the significantly up-regulated metabolites in the acupuncture group were more than the significantly down-regulated metabolites. (Fig. 6H, I).
Then the author further screened out 8 main differential metabolites, red alginate, N-methylnicotinamide, 4-hydroxy-1 indole-3-acetonitrile, β-glycerophosphate, geranyl acetate, The levels of styrene oxide, serotonin, and acetylephrine were higher than those in the model group and the sham acupuncture group, and these major differential metabolites were also highly expressed in the control group (Fig. 6J). In addition, the authors analyzed differential metabolism-related metabolic pathways using the KEGG database and found that phenylalanine, tyrosine, and tryptophan biosynthesis, taurine and hypotaurine metabolism, phenylalanine metabolism, and β-alanine Acid metabolism is the main differential metabolic pathway after acupuncture treatment. Collectively, acupuncture treatment can modulate changes in serum metabolites and metabolic pathways in BC cancer-associated fatigue mice after BC chemotherapy.
Figure 6. Effects of acupuncture and moxibustion on serum metabolites and metabolic pathways in CRF mice after BC chemotherapy
6 Correlation analysis between intestinal flora and intestinal barrier, intestinal and hippocampal inflammation, HPA axis and serum metabolites
Finally, the authors used the Spearman analysis method to analyze the correlation between the main differential flora and intestinal barrier, intestinal inflammation, neuroinflammation, HPA axis function changes and serum differential metabolites between the model group and the control group. The results showed that Burkholderia, Escherichia coli, Shigella, and Streptococcus were negatively associated with intestinal tight junction proteins, CORT, and CRH, while Lactobacillus and Clostridium unclassified-UCG-014 were associated with intestinal Tight junction protein, CORT and CRH were positively correlated. Streptococcus, Escherichia coli, Shigella, and Burkholderia were positively correlated with inflammatory factors, ACTH, while Lactobacillus and unclassified Clostridium UCG-014 were negatively correlated with inflammatory factors, ACTH (Fig. 7B-D) . In addition, Lactobacillus, segmented filamentous bacteria, and unclassified Clostridium UCG-014 were positively correlated with 8 differential metabolites, while Burkholderia, Escherichia coli, Shigella, and Streptococcus were positively correlated with 8 differential metabolites. Metabolites were negatively correlated (Fig. 7E).
Figure 7. Correlation analysis between intestinal flora and intestinal barrier, intestinal and hippocampal inflammation, HPA axis and serum metabolites
Analysis conclusion
The results of the study showed that acupuncture treatment can regulate the intestinal microecological balance and neuroinflammation in the CRF mouse model after breast cancer chemotherapy by regulating the intestinal flora, improving the intestinal barrier and HPA axis dysfunction, and inhibiting intestinal inflammation and neuroinflammation. Changes in serum metabolites relieve fatigue symptoms. The above results indicate that the anti-fatigue mechanism of acupuncture treatment may be closely related to the gut microbiota-gut-brain axis. In addition, this study provides a new reference for the basic and clinical research of cancer-associated fatigue CRF after breast cancer chemotherapy.