3.1. Identification of the Main Components of JPTQ Decoction
The JPTQ Decoction is an improved formula of the classic formula "Si Jun Zi Decoction". Some studies have confirmed the therapeutic effect of Sijunzi Decoction in tumor diseases[36, 37]. Its clinical application has obviously improved the quality of life of BC patients, prolonged the survival time of patients, and reduced the incidence of lung metastases. Before further exploring the mechanism of JPTQ decoction, we preliminarily identified the main components of JPTQ decoction by HPLC-MS. Fig. 1A shows the (Electrospray ionization, ESI)(+) mode and ESI(-) mode total ion chromatogram (TIC) respectively.
In the secondary mass spectrum analysis table of JPTQ decoction, the relative content of a compound was judged based on the matching degree of multiple databases greater than 80 and the peak area size, and the top 12 compounds (Table-3) with different content abundance under different modes were selected (Fig. 1B). These compounds are shown in Table-3. Although JPTQ has not traditionally been used to treat BC, some of its active compounds, such as resveratrol , genistein , galangin , and quinic acid , have been shown to have effective anti-tumor and anti-metastatic effects in different models. These anti-cancer compounds become the material basis for the effectiveness of the formula.
Table 3 JPTQ decoction Mass Spectrometry compounds identification
(Top 12, sorted by relative abundance)
3.2. JPTQ Decoction Retard the Proliferation of in-situ Tumor and Promoted its Necrosis and Apoptosis
On the day of starting the JPTQ intervention (Day 0), baseline Biofluorescence values were measured between the two groups (Fig. 2A), and no statistical difference was found (P=0.34, Fig. 2D).
After two weeks of JPTQ decoction intervention, biofluorescence imaging (Fig. 2B) was performed again. The biofluorescence value of mice in the JPTQ group was weaker than that in the model group, but there was no statistical difference (P=0.4706) (Fig. 2E). The 3rd biofluorescence imaging (Fig. 2C) was evaluated again at the end of the experiment (4 weeks). The biofluorescence value of the JPTQ group was significantly weaker than that of the model group, with a statistical difference (Fig. 2F, P=0.049). We measured the tumor volume and body weight changes of tumor-bearing mice per three days. Within 4 weeks of drug intervention, the tumor volume of JPTQ group was smaller than that of the model group (Fig. 2G). Till the end of the experiment, the mean tumor volume and weight of JPTQ group was still smaller than that of the model group (Fig. 2I,J), on the 28th day of sacrifice, although it was not as different as the bioluminescence value at the end of the experiment (Fig. 2H, P=0.3045). The weight change curves of the two groups were also measured and recorded per three days. In the experiment, with the extension of tumor bearing time, the weight of mice in both groups decreased, but the weight loss rate of mice in the model group was significantly higher than that in the JPTQ group (Fig. 2K). At the end of the fourth week, the weight of mice in the JPTQ group was significantly higher than that in the model group (Fig. 2M) (P=0.0313). Excessive splenomegaly indicates that it has entered the stage of negative immune regulation, which is closely related to tumor immune escape . The spleen weight of JPTQ group decreased significantly (Fig. 2L) (P=0.0333). Compared with the model group, the tumor tissue in JPTQ group had less inflammatory infiltration and more necrosis (Fig. 2N).
we also studied the anti-proliferation and anti-angiogenesis effects of JPTQ. The expression of proliferation protein Ki-67 and angiogenesis marker VEGF protein in tumor samples of TNBC bearing mice was studied by IHC assay. In the JPTQ group, the expression of Ki-67 and VEGF were significantly lower than that in the model group (Fig. 2O,P, P<0.0001, P=0.0139). For verify the pro-apoptotic effect of JPTQ, we studied the expression levels of pro-apoptotic protein cleved-caspase 3 and anti-apoptotic protein Bcl-2 in tumor tissues. The expression of cleved-caspase 3 of JPTQ group was significantly increased compared with that in the model group (Fig. 2Q, P=0.0489), while the expression of Bcl-2 was significantly decreased (Fig. 2R, P=0.1275). These results were consistent with the results of transcriptome analysis, indicating that the apoptotic pathway is one of the pathways of the therapeutic effect of JPTQ decoction.
3.3. JPTQ Decoction Significantly Reduced Lung Metastasis in TNBC Bearing Mice
4T1 BC cells is TNBC cell lines with high metastatic characteristics [38–40]. The isolated lung tissue biofluorescence imaging results were as follows Fig. 3A, 6 mice in the model group had different degrees of lung metastasis, and only one mice in the JPTQ group had slightly lung metastasis. The statistical analysis of biofluorescence imaging values of the two groups showed that there was significant difference between the two groups (Fig. 3B, P=0.0476), compared with JPTQ group, the model group has higher biofluorescence value, which means more serious lung metastasis.
The HE pathological analysis of lung tissue also found that the model group had larger tumor infiltration area and more metastatic lesions (Fig. 3D,E). In Fig. 3D, extensive alveolar wall thickening was found in the lung tissue of the model group, accompanied by a large number of lymphocytes and neutrophils infiltration (black arrow), a large number of tumor cells (green arrow) were found in the tissue, bleeding can be seen around the local blood vessels (yellow arrow). In Fig. 3E, in JPTQ group, the thickening of alveolar wall was improved, and the infiltration of lymphocytes and neutrophils was decreased (black arrow),a small amount of tumor cell mass (green arrow) was found in the tissue, local perivascular bleeding decreased (yellow arrow). After sacrificing mice, there was no statistically significant difference in lung tissue weighing between the two groups, but the mean lung weight of the JPTQ group was lower than that of the model group (Fig. 3C, P=0.5637), which was considered to be because the model group had more severe tumor load accompanied by inflammatory infiltration and edema.
We also analyzed whether the reduction of lung metastasis is related to the inhibition of proliferation and promotion of apoptosis. WB analysis showed that the expression of cleaved caspase-3 was significantly increased (Fig. 3F, P=0.0428) and the expression of Bcl-2 was significantly decreased (Fig. 3G, P=0.0168) in pulmonary metastases of JPTQ group. In addition, immunohistochemical analysis revealed that the expression of Ki-67 and VEGF in JPTQ group was significantly decreased compared with model group (Fig. 3H,I, P<0.0001, P=0.0170 respectively).
3.4. Transcriptome Analysis of Tumor Tissue and Lung Tissue in Tumor Bearing Mice
For explore the specific mechanism of JPTQ decoction in reducing tumor proliferation and progression and lung metastasis, we sequenced the transcriptome of lung and tumor tissue of two groups of mice. A total of 191 differentially expressed Genes (DEGs) were screened out in tumor tissues, including 163 up-regulated genes and 28 down-regulated genes (Fig. 4A).
KEGG pathway analysis indicated the PI3K-Akt, PPAR, Estrogen signaling pathway, ECM-receptor interaction and other pathways were significantly enriched (Fig. 4B).The Gene Ontology (GO) analysis revealed obvious enrichment of immune-related functions in biological processes (BP) category, such as immune response, immunoglobulin generation, and B cell receptor signaling pathway (P<0.05 respectively) (Fig. 4C). In the cellular component (CC) category, the greatest numbers of genes were found in the “Extracellular regions” and “extracellular matrix”(P<0.05 respectively) (Fig. 4D). In the molecular function (MF) category, the“Protein binding”(Fig. 4E) was regulated signifcantly(P<0.05).
A total of 207 differentially expressed genes were identified in lung tissues, including 98 up-regulated genes and 109 down regulated genes (Fig. 4F). KEGG pathway analysis showed that adhesion plaque, chemokine signaling pathway, IL-17 signaling pathway, etc. were significantly enriched (Fig. 4G, P<0.05 respectively). The Go analysis was also performed, in the category of BP, inflammation, immune function, etc. were significantly enriched (Fig. 4H). In the MF category, the “ Integrin, Chemokine Activation, EGFR Binding, FATZ binding, etc. (Fig. 4J) was regulated signifcantly (P<0.05). In the CC category, focal adhesion, etc. were significantly enriched (Fig. 4I).
EMT process changes, angiogenesis, EGFR overexpression and other mechanisms will also aggravate the malignant phenotype of BC. RNA sequencing of tumor bearing lung tissue and tumor tissue indicated that EMT process related genes, proliferation, anti apoptosis related genes and angiogenesis related genes were down regulated (See Supplementary Tables S1and S2).
3.5. JPTQ Significantly Improved the Tumor Immune Microenvironment of Tumor Bearing Mice
The escape of tumor immune to aggravate the occurrence and development of tumor diseases . The transcriptome analysis of lung and tumor tissues indicated that the pathways or biological processes related to immune function were significantly enriched. So,we analyzed the changes in the immune microenvironment of the experimental mice by FCM. We analyzed the of overall immune status of tumor-bearing mice, that is, the proportion of CD4+ T cells in the peripheral blood, CD4+ T cells in mice in the JPTQ treatment group were significantly increased, compared with the model group (40.95% VS 55.83%, Fig. 5A,B, P=0.0384). On the basis of these findings, we further analyzed the difference of the effector Th1/2 cells in the spleen of two groups of tumor-bearing mice, and the FCM analysis results indicated that although no difference was observed in the proportion of Th2 cells between the two groups (0.7% VS 0.93%, P=0.0572, Fig. 5E), but the Th1 cells were significantly increased in the JPTQ group,compared with the model group (2.333% VS 1.167% P=0.0095, Fig. 5D). The imbalance of Th1/2 cells was improved to some extent (Fig. 5C).
Treatment with JPTQ also reduced the incidence of lung metastasis in tumor-bearing mice, so we also analyzed the local immune microenvironment in the lung. The MDSCs is divided into M-MDSCs and PMN-MDSCs. The two cell subsets perform different immunosuppressive functions [22, 23]. By FCM analysis, it was found that the proportion of M-MDSC in the lung single cell suspension of mice in the model and JPTQ group were 2.602% and 1.911% respectively (Fig. 5F), The lung M-MDSC in the mice of JPTQ group was significantly reduced (Fig. 5G, P=0.001). The proportion of PMN-MDSC in single-cell lung suspension in the model group and the JPTQ group was 14.70% (Fig. 5F) and 18.64% respectively(Fig. 5F), and no difference was observed between the two groups (Fig. 5H, P=0.2834).
3.6. JPTQ Decoction Reduced the Tumor Proliferation and Occurrence of Lung Metastasis in Tumor-Bearing Mice by Regulating the EMT Process
EMT is the key link of malignant transformation of many tumor diseases, which can promote the occurrence, proliferation and distant metastasis of tumor. Transcriptome analysis of tumor samples showed that EMT related genes such as E-cadherin were up-regulated, snail was down regulated, but Vimentin and MMP-9 were not changed (See Supplementary Tables S1 and S2).
The q-PCR was used to verify the results of transcriptomics.In tumor tissues, the results showed that E-cadherin, Vimentin and MMP9 expression was up-regulated (Fig. 6A,B,D, P=0.001, P=0.0002 and P=0.0127 respectively), Snail expression was down regulated (Fig. 6C, P༜0.0001).
The results of IHC showed that E-cadherin was significantly increased in JPTQ group (Fig. 6I, P=0.0403, Fig. 6Q), Vimentin and MMP-9 was not significantly different between the two groups (Fig. 6J,Q, P=0.698; Fig. 6L,Q, P=0.568), Snail was significantly decreased in JPTQ group (Fig. 6K, P=0.0371, Fig. 6Q). WB was used to detect EMT-associated proteins expression status (Fig. 6S, Fig. 6U). WB results of tumor tissue showed that the expression of E-cadherin in the JPTQ group was significantly higher than that in the model group (P=0.0196), and Vimentin had no significant difference between the two groups (P=0.1917), but there was a down-trend in JPTQ group. Snail was significantly down regulated in JPTQ group (P=0.0463), and MMP-9 showed a downward trend, but there was no statistical significance (P=0.3152). The q-PCR revalidation, WB, and immunohistochemical results were basically the same as transcriptome results. Combined with the above conclusions, we can conclude that the inhibition of JPTQ decoction on the proliferation of in-situ tumors may be partly due to the regulation of EMT process.
Transcriptome analysis of lung tissue in JPTQ group showed that the expressions of EMT related genes such as E-cadherin, Snail and MMP-9 were higher than those in model group, while Snail expression was lower than that in model group (supplementary material). q-PCR results showed that compared with the model group, the expression of E-cadherin and Vimentin increased (Fig. 6E, F P=0.0303, P༜0.0001), snail and MMP-9 decreased (Fig. 6G, H, P=0.0001, P=0.0003) in JPTQ group. The results of IHC showed that E-cadherin in JPTQ group was significantly higher than that in model group (Fig. 6M, P=0.004, Fig. 6R), and Vimentin in JPTQ group was not different from that in model group (Fig. 6N, P=0.3039, Fig. 6R). MMP-9 and Snail was significantly down regulated (Fig. 6O,P, P=0.038, P=0.0471, Fig. 6R). In the WB results of lung tissue(Fig. 6T), E-cadherin was significantly increased in the JPTQ group compared with the model group (Fig. 6V, P=0.0492), while Vimentin was insignificant in the two groups (Fig. 6V, P=0.2120), but the JPTQ group showed a decreasing trend. Snail and MMP-9 were significantly down-regulated in the JPTQ group (Fig. 6V, P=0.0299, P=0.0273). Combined with the results of IHC and WB, it was considered that MMP-9 showed a down-trend in the lung tissue of mice in the JPTQ group. As mentioned above, JPTQ realizes the regulation of EMT process through the regulation of targets such as E-cadherin, Snail and MMP-9, thereby reducing the occurrence of lung metastasis.