Inhibitory Effect Of Qinggan Huayu Granule On Primary Hepatic Carcinoma Induced By Diethylnitrosamine In Rats And Its Mechanism

ObjectiveQinggan Huayu decoction is professor Shukun Yao’s empirical formula on the basis of his clinical experience in treating primary liver cancer (PLC) more than thirty years. According to traditional Chinese medicine theory, Qinggan Huayu decoction has the following effects: clearing heat and detoxifying, invigorating spleen and eliminating dampness, promoting blood circulation and removing blood stasis, softening hardness and dissipating nodulation, etc. Our previous studies showed that Qinggan Huayu decoction can enhance patients’ immune function, prolong living time and improve quality of life, thereby may be a potential product which can be used for the treatment of PLC. Qinggan Huayu Granule is an optimized product on the basis of Qinggan Huayu decoction. The study aims to assess the effects of QGHYG in rat with PLC induced by diethylinitrosamine, and explored relevant mechanisms of action via relevant signal pathways.Methods94 Specific pathogen-free healthy male wistar rats (weight 190 to 210g) were included in this study. All of the rats were adaputiely fed for one week, then randomly divided into the following six groups:blank (no treatment) group (n=10), model group (n=20), Qinggan Huayu Granule low-dose group (n=16), Qinggan Huayu Granule medial-dose group (n=16), Qinggan Huayu Granule high-dose group (n=16), and norcantharidin group (n=16). All groups, except blank group, were made model of PLC by intraperitoneal injection of diethylinitrosamine. Dosing schedules in three Qinggan Huayu Granule groups (from low-dose group to high-dose group) were 0.6g/kg,1.2g/kg and 2.4g/kg per day, respectively. Dosing schedules of norcantharidin was 3mg/kg per day. All of the drugs were assigned through intragastric administration with a same volume of 15 ml/kg. Meanwhile, rats in blank group and model group were given equal volumes of normal saline. Intragastric administration was conducted for 16 weeks. After 12 hours’fasting, all rats were given anesthesia and drew blood through abdominal aortas. After that, all rats were executed in order to get liver and spleen. Indexes tested in this study included mortality of rat, incidence of liver cancer, wet weight of liver, liver index, wet weight of spleen, spleen index, alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), serum a-L-fucosidase (AFU), total bile acid (TBA), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-17 (IL-17), T lymphocyte subsets in peripheral blood, the expression of p-Akt, p-MEK, p-ERK and VEGF in tumor tissue by immunohistochemical method and western blot, the expression level of Akt mRNA, RKIP mRNA and PTEN mRNA in tumor tissue by RT-PCR.Results1 Effects of Qinggan Huayu Granule in rats with PLC induced by diethylinitrosamine1.1 Effects on survival rate of rat, incidence of liver cancer, weight of rat, liver index and spleen index.① Survival rate of rat:survival rate of rat in model group was 65%, and in Qinggan Huayu Granule medial-dose group was 100%. There was statistical difference between the two groups (p<0.05).②Incidence of liver cancer:incidence of liver cancer in model group was 100%, in three Qinggan Huayu Granule groups (low-medial-high dose) was 100%,87.5%,100%, respectively, and in norcantharidin group was 93.8%. There was no statistical difference among the five groups (p>0.05).③Weight of rat:compared with model group, rats in three Qinggan Huayu Granule groups (low-medial-high dose) and norcantharidin group had higher weight (p<0.01).④liver index and spleen index:compared with model group, rats in norcantharidin group had lower liver index (p<0.05), meanwhile, rats in Qinggan Huayu Granule medial-dose group and Qinggan Huayu Granule high-dose group had lower liver index and spleen index (p<0.01). 1.2 Improvement of liver functionCompared with blank group, rats in model group had higher level of ALT, AST, GGT, ALP, AFU and TBA (p<0.01). Meanwhile, compared with model group, rats in three Qinggan Huayu Granule groups as well as in norcantharidin group had lower level of ALT, AST, GGT, ALP and AFU (p<0.01). Compared with model group, rats in Qinggan Huayu Granule medial-dose group, Qinggan Huayu Granule high-dose group and norcantharidin group also had lower level of TBA (p<0.01 or p<0.05). Moreover, rats in Qinggan Huayu Granule medial-dose group had lower level of ALT, AST, GGT, ALP, AFU and TBA, compared with norcantharidin group (p<0.01 or p<0.05). 1.3 Effects on T lymphocyte subsets and serum IL-6, IL-10, IL-17Compared with model group, rats in three Qinggan Huayu Granule groups (low-medial-high dose) had more CD4+cells in peripheral blood (p<0.01 or p<0.05). Moreover, rats in Qinggan Huayu Granule medial-dose group and Qinggan Huayu Granule high-dose group also had high ratio of CD4+/CD8+(p<0.01 orp<0.05).Compared with blank group, rats in model group had lower level of IL-6 and IL-17 (P<0.01). Compared with model group, rats in three Qinggan Huayu Granule groups (low- medial-high dose) as well as in norcantharidin group had higher level of IL-6 and IL-10 (P<0.01). Compared with model group, rats in Qinggan Huayu Granule medial-dose group, Qinggan Huayu Granule high-dose group and norcantharidin group also had higher level of IL-17 (P<0.01). What’s more, compared with norcantharidin group, rats in Qinggan Huayu Granule medial-dose group and Qinggan Huayu Granule high-dose group still had higher level of IL-6 and IL-17 (P<0.05).2 Molecular mechanisms of the antitumor effect of Qinggan Huayu Granule2.1 Effect on Ra#MEK/ERK signaling pathwayThe immunohistochemical analysis and Western blot measurement showed that:compared with model group, the expression of p-MEK and p-ERK in medial-dose and high-dose group markedly decreased(P<0.01, P<0.05). RT-PCR show that the expression level of RKIP mRNA in medial-dose group was significant higher than model group(P<0.05).2.2 Effect on PI3K/Akt signaling pathwayThe immunohistochemical analysis and Western blot measurement showed that:compared with model group, the expression of p-Akt in low-dose, medial-dose and high-dose group markedly decreased(P<0.01, P<0.05). RT-PCR show that the expression level of PTEN mRNA in medial-dose group and high-dose were significant higher than model group(P<0.01).Concision1 Qinggan Huayu Granule has a significant inhibitory action on DEN-induced PLC rats.2 Qinggan Huayu Granule may boost immune system in DEN-induced PLC rats.3 The antitumor effect of Qinggan Huayu Granule may be associated with the inhibition of PI3K/Akt signaling pathway and RaflMEK/ERK signaling pathway.4 Qinggan Huayu Granule has a potential value for clinical application in foture, and deserves to be researched further.