Part Ⅱ：Cistanche Tubulosa Phenylethanoid Glycosides Induce Apoptosis Of Hepatocellular Carcinoma Cells By Mitochondria-Dependent And MAPK Pathways And Enhance Antitumor Effect Through Combination With Cisplatin
Mar 05, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
We further evaluated the immunostimulatory effect of CTPG(Cistanche Tubulosa Phenylethanoid Glycosides) on mice. After i.p. injection, s.c. injection and i.g. administration of CTPG (200 and 400mg/kg), there was no significant change in the state of mice compared with the untreated group. The bodyweight of mice also had no significant difference among CTPG-treated groups and untreated groups (Figure 7A). The heart, liver, spleen, lung, kidney, and thymus indexes of mice were similar among all groups except the spleen indexes of mice in the 400mg/ kg CTPG i.p. injection group (Table 1). The results suggested that the selected doses of CTPG had no side effects in mice. The spleen is an important immune defense organ in mammals, and its development directly affects the individual’s immune function and the ability to resist diseases. The immune organ index reflects the development and immune function of immune organs. The spleen contains a variety of immune cells, including T cells, B cells, natural killer cells (NK cells), macrophages, dendritic cells (DCs), and others, which play an important role in the immune system.30 It had been reported that polysaccharides can increase the number of immune cells to enhance the body’s immunity.31 As i.p. injection of CTPG significantly increased the spleen index (increased 0.55 times), we further analyzed the numbers of various immune cells in the spleen. The results showed that i.p. injection of CTPG significantly increased the numbers of T cells (CD3+CD19− cells) (increased 0.49 times), B cells (CD3−CD19+ cells) (increased 0.49 times), and NK cells (CD3−CD19−CD49b+ cells) (increased 1.63 times) compared with the untreated group. CD4+ and CD8+ T cells, as the main subtypes of T cells, play an important role in the antigen-specific cellular immune response.32 Therefore, we further analyzed the number and activation status of CD4+ and CD8+ T cells in the spleen. The results indicated that i.p. injection of CTPG not only significantly increased the numbers of CD4+ and CD8+ T cells (increased 0.55 and 0.34 times, respectively) but also enhanced the activation state of CD4+ T cells (CD4+CD44+ cells) (increased 0.84 times) (Figure 7B), these results suggesting that CTPG had immune enhancement function.
The Combination of CTPG(Cistanche Tubulosa Phenylethanoid Glycosides) and Cisplatin Exhibited Potent Antitumor Effect on H22 Tumor Mouse Model
It is generally accepted that chemotherapy or radiotherapy can lead to poor prognosis and induce numerous adverse events, which may be due to the suppression of the immune system and damage to normal tissues and organs. Polysaccharides from TCM have immune enhancement effects and alleviate the damage of immune organs and immune cells caused by chemotherapy, for example, chitosan33 and Lycium barbarum polysaccharides.34 The above result showed that CTPG(Cistanche Tubulosa Phenylethanoid Glycosides) could ameliorate the side effects of cisplatin on the immune system. Therefore, the antitumor effect of CTPG combined with cisplatin was evaluated in the H22 tumor mouse model. As shown in Figure 8A, CTPG alone, cisplatin alone, and the combination of CTPG and cisplatin (CTPG+cisplatin) significantly suppressed H22 tumor growth compared with the control group. CTPG+cisplatin showed a stronger inhibitory effect than that of CTPG and cisplatin alone. On the 20th day, the tumor volumes in the control, cisplatin alone, CTPG alone, and CTPG+cisplatin groups were about 1707.8, 722.5, 1033.8, and 367.5mm3, respectively. On the same day, the tumors were collected for visual observation (Figure 8B) and weight comparison (Figure 8C). CTPG+cisplatin showed better therapeutic effects on the H22 tumor mouse model than CTPG or cisplatin alone. The tumor weight in the CTPG+cisplatin group was lower than that in other groups. These results suggest that the anti-tumor activity of CTPG and cisplatin results in synergistic effects.
The body weights of mice were measured to reflect the health status of mice. The body weight remarkably decreased after cisplatin treatment, suggesting cisplatin-induced severe systemic toxicity.35 In comparison, CTPG combined with cisplatin significantly ameliorated body weight loss induced by cisplatin, while CTPG(Cistanche Tubulosa Phenylethanoid Glycosides) alone had no significant effect on mice body weight (Figure 8D). The organs including the spleen, heart, liver, lung, kidney, and thymus were also isolated to calculate organ indexes. Compared with the untreated group, spleen indexes were significantly decreased and increased by cisplatin and CTPG, respectively. CTPG + cisplatin recovered spleen indexes compared with cisplatin alone. Other organ indexes showed no significant difference among all groups (Table 2).CTPG(Cistanche Tubulosa Phenylethanoid Glycosides) Combined with Cisplatin Enhanced the Immunity of Tumor Mice.
The above results showed that CTPG(Cistanche Tubulosa Phenylethanoid Glycosides) enhanced the immunity of mice and inhibited the apoptosis of splenocytes induced by cisplatin. We further investigated whether the antitumor effect of CTPG+cisplatin was correlated with enhanced immunity. In the process of tumor occurrence and development, the body’s immune system plays an important immune surveillance function, and T cell-mediated cellular immunity plays a key role. CD4+ T cells have the function of assisting cellular immunity and humoral immune response, and CD8+ T cells are the main effector cells of an immune response, which can specifically kill target cells and play an important role in tumor immunity and antiviral immunity.32 The spleens of tumor-bearing mice were collected to detect the numbers of T cells and B cells by flow cytometry. The frequencies and numbers of T cells including CD4+ and CD8+ T cells in the CTPG+cisplatin group were significantly higher than those in the control (increased 0.55 and 0.7 times, respectively) and cisplatin groups (increased 0.92 and 1.16 times, respectively) (Figure 9A and B). Compared with the control group, CTPG alone also partly increased the numbers of CD4+ and CD8+ T cells although there was no significant difference. The frequencies and numbers of B cells had no significant difference among all groups.
Myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) expand in tumors and inhibit antitumor immune responses; they suppress the activation and expansion of CD4+ T cells, the function of effector T cells, the activation and/or proliferation, and cytokine formation of CD4+ and CD8+ T cells, the B-cell proliferation and immunoglobulin production and class switch, the cytotoxic functions of NK and natural killer T cells (NKT), and the function and maturation of DCs.36-38 The frequencies and numbers of MDSCs (CD11b+Gr-1+), macrophages (CD11b+Gr-1−), and Tregs (natural Tregs: CD4+CD25+Foxp3+; induced Tregs: CD4+CD25−Foxp3+) in the spleens of tumor mice were analyzed by flow cytometry. Compared with the control group, the frequencies and numbers of MDSCs decreased significantly after injection with cisplatin alone (decreased 1.56 times) or in combination with CTPG(Cistanche Tubulosa Phenylethanoid Glycosides) (decreased 0.62 times). The numbers of CD11b+Gr-1− macrophages also decreased significantly although there was no significant change in their frequencies (Figure 9C). In addition, CTPG(Cistanche Tubulosa Phenylethanoid Glycosides) alone and CTPG+cisplatin treatment significantly decreased the frequencies of induced Tregs (decreased 0.33 and 0.37 times, respectively) (Figure 9D). These results suggested that the antitumor effect of CTPG+cisplatin might be correlated with the enhanced immunity characterized by the increased numbers of T cells and the decreased numbers of MDSCs and Tregs.
