Regulation Effect Of Fuzheng Jiedu Decoction On The Tumor-associated Macrophage Remodeling Of Micro Vessel In Tumor-burdened Model

Background:Recurrence and metastasis of gastric cancer has plagued clinicians and limited the improvement of clinical effects for several years. Recently, studies have shown that immunosuppression microenvironment plays an important role for tumor cells in surviving, growing, escaping immunological surveillance and forming microvessels. The completed national natural science foundation (NO.81072802):regulation effect of Fuzheng Jiedu Decoction(FZJD) on the TEMs remodeling of lymphangiogenesis shows that FZJD can induce TEMs translation from M2 to Ml, reverse hypoxic and immunosuppressive of tumor microenvironment, restrain lymphangiogenesis by intervening PI3K/AKT/mTOR pathways, it also found that FZJD depress the production of capillaries. Based on that, this experiment further discusses the regulation effect of FZJD on the Tumor-associated macrophage remodeling of micro vessel in a tumor-burdened model.Methods:The main parts of this study include cell experiments and animal experiments. Firstly, we established a tumor-burdened model of gastric cancer (national natural science foundation of NO.81072802 reference method has been completed); and then the experimental animals were randomly divided into 4 groups:model control group (C), Fuzheng Jiedu group (FZJD-treated),5-Fu group (5-Fu-treated),5-Fu+FZJD group(5-Fu+FZJD). Each group intervened in 14 days with corresponding drugs respectively. Lately, we evaluated the regulating effects of FZJD on tumor growth and angiogenesis. Simultaneously, FACS was used to test the contents of immune cells of CD4+T cells, CD8+T cells. Treg T cells, for assessing the levels of immuno-suppression in microenvironment. RT-PCR was used to detect the expression levels of pro-angiogenic factors VEGFa、MMP-9、COX-2 and immune suppressor IL-6、IL-10、CCL-17、Arg-1. Western blot detected the expression of Angl、Ang2、 PI3K、Rheb、p70S6K1 in tumor and IHP described the expression of AKT、TSC1、 mTOR、PERK、ATF-6、IRE1α.Results:1. Tumor weight:Model control group had a maximum tumor weight(1.29±0.39g), while the tumor weight of 5-Fu+FZJD group(0.48±0.24g) was significantly smaller than control group (P<0.05), FZJD-treated and 5-Fu-treated were separately 0.93±0.37g, 0.62±0.30g; Tumor inhibition rate:inhibition rate of FZJD-treated,5-Fu-treated, 5-Fu+FZJD were separately 28.20%、52.09%、62.59%.2. Ultrasonic observation on each group of mice tumor blood vessel distribution and blood flow parameters detection:The control group had rich blood flow and density vascular of tumor tissue. Its vascular was distributed in tumor surrounding in chaos, featured with untypical, straight into the tumor center and thin pipe diameter of tumors blood vessels. However, the distribution of tumors blood vessels in FZJD were extremely smaller than control group, which featured with intensive, regulative and 1-2 bigger size vessels straight into the tumor center. The number and signal of blood vessels in 5-Fu and 5-Fu+FZJD was not obvious though both of them featured with partial blood vessels deeply inside. Blood flow parameters PSV,EDV, RI:FZJD-treated>C> 5-Fu-treated>5-Fu+FZJD. Verbal rating scale of blood alder was similar to the blood flow parameters. The degree of classification of FZJD was the maximum, the other groups were respectively control group,5-Fu,5-Fu+FZJD, with statistical significance (P<0.05)3. The blood perfusion and hypoxia of tumor tissues:Blood perfusion of tumor tissues were respectively C>FZJD-treated>5-Fu-treated>5-Fu+FZJD; state of hypoxia in tumor tissues was the same. Control group had the most serious hypoxia situation, however, when FZJD or 5-Fu intervene, the situation becoming better.4. The influence on tumor-associated macrophage immunosuppression m icroenviron ment:The ratio of CD4+/CD8+T cells in control group was 1.44±0.09, which in a lower level. The splenic tissues of control group was with higher Treg ratio (2.5±0.56), overexpression TAM2 associated immunosuppressive factors:IL-6, IL-10, CCL-17, Arg-1. It was supposed that the tumor microenvironment was in immunosuppressive state. When FZJD intervening, ratio of CD4+/CD8+T cells level up-regulated (1.72±0.14),5-Fu+FZJD group had more effective significant (1.96±0.31); ratio of Treg cells reduced, FZJD、5-Fu and 5-Fu+FZJD was separately 1.37±0.44, 1.67±0.20,1.15±0.55, and associated immunosurppressive factors of treatment group was also less expressed.5. The promote angiogenesis factors expression of tumor associated macrophages:Gene transcription of VEGFa\MMP-9\COX-2 was maximum in control group,after 5-Fu and FZJD treatment, the transcription level was reducing (P<0.05). The levels in 5-Fu+FZJD were most obvious. Gene expression of VEGFa:C> 5-Fu-treated>FZJD-treated>5-Fu+FZJD;MMP-9,COX-2:O5-Fu-treated>FZJD-treat ed>5-Fu+FZJD. In comparison with FZJD,5-Fu,5-Fu+FZJD had significantly lower expression of MMP-9(P<0.05). While 5-Fu,5-Fu+FZJD had significantly lower expression of COX-2 than control group and FZJD(P<0.05).Control group had higher expression of Ang-1,Ang-2, after 5-Fu and FZJD treatment, its expression were obviously suppressed(P<0.05). Expression level of Ang-1:C>FZJD-treated>5-Fu-treated>5-Fu+FZJD, in which control group had better effect than other groups with statistic significance. Ang-2 expression level in groups except control group was lower but with no statistic significance.6. Intervening PI3K/AKT/mTOR pathways:Control group had high expression of PI3K, Rheb, p70S6K1, after FZJD intervene, the level of PI3K, Rheb was lower.,5-Fu+FZJD group had obvious effect, compared with control group P<0.05. The expression of p70S6K1 both reduced in FZJD and 5-Fu+FZJD groups, but only 5-Fu+FZJD had statistic significance compared to control group.Control group had higher OD ratio of mTOR, AKT, TSC1, after treatment, the ratio reduced. With comparision of mTOR OD in control group, the FZJD and 5-Fu+FZJD group reduced significantly.5-Fu+FZJD had minimum AKT OD compared with control group and FZJD (P<0.05). FZJD and 5-Fu had lower expression of TSC1 OD, but 5-Fu+FZJD was the lowest compared with control group (P<0.05).7. The regulation of tumor microenvironment of china-africa folded protein response (UPR)Control group had higher expression of GRP-78, after FZJD and 5-Fu intervene.the level reduced with statistic significance.5-fu had the minimum expression of GRP-78. but each group had no statistic significance.Control group had highest expression of ATF-6, IRE 1a, PERK OD. after FZJD and 5-Fu intervene, the level reduced.5-Fu+FZJD had minimum expression of ATF-6, IRE1a. other groups had statistic significance compared with control group. FZJD could reduce the expression of PERK OD. but compared with 5-Fu+FZJD, the effect had no statistic significance.Conclution:FZJD decoction can effectively suppress the growth of MFC tumor cells, generation of tumor blood vessels and improve blood perfusion, hypoxia in tumor microenvironment. The probable mechanisms were related to FZJD that regulating TAMs activation and transcription, suppressing expression of PI3K/AKT/TSC1/Rheb/mTOR pathways and creation of china-africa folded protein response (UPR). FZJD could reverse the formation of tumor immunosuppressive microenvironment. reduce the expression of promoting angiogenesis factors, further regulate the tumor vascular remodeling.