Autophagy inhibition mediated by trillin promotes apoptosis in hepatocellular carcinoma cells via activation of mTOR/STAT3 signaling.

Apoptosis and autophagy have been shown to act cooperatively and antagonistically in self-elimination process. On the one side, apoptosis and autophagy can act as partners to induce cell death in a coordinated or cooperative manner; on the flip side, autophagy acts as an antagonist to block apoptotic cell death by promoting cell survival. Our previous research indicated that trillin could induce apoptosis of PLC/PRF/5 cells, but the effects of trillin on autophagy as well as its functional relationship to apoptosis have not been elucidated. Here, the running study aims to investigate the function and molecular mechanism of trillin on autophagy with hepatocellular carcinoma (HCC) cells. The objective of this study is to investigate the molecular mechanism of trillin on autophagy in HCC cells. Protein levels of autophagy markers beclin1, LC3B, and p62 were detected by western blotting. 6-Hydroxyflavone and stattic were used to test the role of trillin regulation of autophagy via serine threonine kinase (AKT)/extracellular-regulated protein kinases (ERK) 1/2/mammalian target of rapamycin (mTOR)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Flow cytometry was used to detect caspase 3 activity and apoptosis in PLC/PRF/5 cells treated with trillin for 24 h with or without rapamycin, stattic, and 6-hydroxyflavone. The protein level of autophagy marker beclin1 was decreased, whilst the protein level of p62 was significantly increased by trillin treatment, indicating trillin treatment led to inhibition of autophagy in HCC cells. Trillin treatment could reduce the protein levels of p-AKT and p-ERK1/2, but enhance the protein levels of mTOR and p-mTOR, suggesting that trillin could inhibit AKT/ERK rather than mTOR. The AKT/ERK activator 6-hydroxyflavone could reverse the loss of AKT and ERK1/2 phosphorylation induced by trillin, implying that trillin impairs autophagy through activated mTOR rather than AKT/ERK. STAT3 and p-STAT3 were significantly upregulated by the trillin treatment with an increase in dose from 0 to 50 µM, suggesting that autophagy inhibition is mediated by trillin via activation of STAT3 signaling. The STAT3 inhibitor stattic significantly reversed the increased STAT3 phosphorylation at tyrosine 705 induced by trillin. The mTOR signaling inhibitor rapamycin reversed the trillin-induced mTOR phosphorylation enhancement but exerted no effects on total mTOR levels, suggesting trillin treatment led to inhibition of autophagy in HCC cells through activating mTOR/STAT3 pathway. Furthermore, caspase 3 activities and the total rate of apoptosis were increased by trillin treatment, which was reversed by rapamycin, stattic, and 6-hydroxyflavone, proving that trillin promotes apoptosis via activation of mTOR/STAT3 signaling. Trillin induced autophagy inhibition and promoted apoptosis in PLC/PRF/5 cells via the activation of mTOR/STAT3 signaling. Trillin has the potential to be a viable therapeutic option for HCC treatment.

Game dynamics of emotion evolution based on the Moran process.

In this paper, emotions are classified into four types, namely, respect for the strong, envying the strong, sympathy for the weak, and bullying the weak. The corresponding relationship between the four emotion types and the two behaviors of competition and cooperation is then defined. The payoff matrices of the game based on emotions are obtained and the evolutionary dynamics of the four emotion types in a finite population based on the Moran process are studied. Next, we derive the absorption probabilities of a 4×4 symmetric evolutionary game of the population. The influence of the payoff parameters and the natural selection intensity on the result of the group evolution are then analyzed. The calculations indicate that there are differences in the absorption probabilities of the four absorption states of the system. At a steady state, individuals of the types envying the strong and bullying the weak have the highest probability of occupying the entire population, and individuals of the type respect for the strong and sympathy for the weak have the lowest one. By comparing the level of cooperation and average payoffs at a steady state, we observe that the level of cooperation and average payoffs based on the proposed model are better than those of the prisoner's dilemma game with two behaviors. Therefore, emotional evolution can promote cooperation and achieve better group fitness.

Trillin prevents proliferation and induces apoptosis through inhibiting STAT3 nuclear translocation in hepatoma carcinoma cells.

Trillin is a constituent of total Trillium Tschonoskii Maxim (TTM), which is extracted from TTM and displayed anti-tumor effect in many tumor cell lines. However, the anti-tumor mechanism of trillin is still unclear. This study demonstrated that trillin could dramatically inhibit hepatoma carcinoma cell proliferation, induce apoptosis and decrease migration and invasion through suppressing phosphorylated STAT3 translocated to nucleus. Trillin could down-regulate Bcl-2 and Survivin, up-regulate cleaved PRAP, leading to dramatically apoptosis; trillin could also down-regulate MMP1, MMP2, MucI and VEGF, which displayed an inhibition effect on hepatocellular tumor cells invasion and development. The results of this study indicated the potential utility of trillin as a STAT3 inhibitor for the treatment of cancers.

[Effects of Shiquanyuzhentang on immunologic function of H22 tumor-bearing mouse].

OBJECTIVE: To investigate the effects of Shiquanyuzhentang (Traditional Chinese Medicine) on the immunologic function of tu-mor-bearing mice and its mechanism of antitumor. METHODS: Thirty SPF grade male Kunming mice transplanted with H22 hepatocellular carci-noma were divided into three groups randomly:model group,positive control group and Shiquanyuzhentang group(n=10). Another 10 mice were selected as normal control group. Normal control group and model group received normal saline and distilled water supplementation by 10 mL/kg everyday. Positive control group and Shiquanyuzhentang group received Shengyi(80 mg/ml)and Shiquanyuzhentang decoction at the doses of 8 g/kg and 18 g/kg respectively everyday. After 14 days of continuous administration, the mice were killed and the thymus, spleen index, tumor inhibition rate,peripheral blood leukocytes,lymphocyte content,cell percentage of T cell subsets CD3, CD4, CD8,interleukin 2 (IL-2)in serum,tumor necrosis factor-α(TNF-α),interferon ß(IFN-ß) content,lymphocyte proliferation ability and NK cell were measured. RESULTS: Compared with normal control group, the weight of mice and thymus and spleen index of the Shiquanyuzhentang group were increased significantly(P<0.05);leukocyte and lymphocyte CD3、CD4、CD8 and TNF-α were increased greatly(P<0. 05),IL-2 and IFN-ß were de-creased significantly(P<0.05). Compared with model group,thymus and spleen index of Shiquanyuzhentang group were increased significant-ly(P<0.05);leukocyte and lymphocyte CD3、CD4、IL-2、IFN-ß and TNF-α of Shiquanyuzhentang group were increased significantly(P<0. 05),CD8 content was decreased significantly(P<0.05);NK cell and lymphocyte proliferation were increased significantly(P<0.05). CONCLUSIONS: Shiquanyuzhentang can promote the growth of immune organs in mice bearing H22, enhance immune function and is beneficial to the recovery of tumor body.

[The protective effects and mechanisms of Trillium tschonoskii Maxim on rats' liver damage induced by lipopolysaccharide].

OBJECTIVE: To study the protective effects of Trillium tschonoskii maxim (TTM) on rats' oxidative stress and hepatotoxicity in-duced by lipopolysaccharide (LPS). METHODS: Sixty SD rats were randomly divided into TTM high, medium and low dose groups, model group, Dexamethasone (DEX) control group and blank control group with ten rats in each group. The TTM high, medium and low dose groups were treated with 8, 4, 2 g/(kg·d) TTM by intragastric administration and model group, DEX control group and blank control group were treated with the same amount of distilled water respectively. The TTM high, medium and low dose groups, model group, DEX control group were injected intraperitoneal with 1 mg/kg LPS and the DEX control group was injected intraperitoneal with 5 mg/kg DEX, the blank control group was injected with same amount of normal saline every five days. The indexes of rats' thymus and spleen were measured in 30 days. The activities of serum nitric oxide synthase (NOS), superoxide dismutase (SOD) and the contents of nitric oxide (NO), glutathione (GSH), glu-cosinolates barbituric acid reaction product(TBARS), white cells interleukin-6(IL-6), IL-10 and tumor necrosis factor-α(TNF-α), the activi-ties of liver SOD, GSH-Px and the contents of GSH and TBARS were measured. RESULTS: TTM high dose group was significantly different in body weight in 19~30 days(P<0.05); The index of thymus in TTM high, medium and low dose groups and the index of spleen in TTM high dose group were decreased significantly compared with those of the model group. The activity of serum NOS and the contents of TBARS and NO in TTM high, medium and low dose groups were decreased significantly(P<0.05). The activity of serum SOD in TTM high dose group and the contents of GSH in TTM medium and low dose groups were increased significantly(P<0.05). The contents of serum IL-6 and TNF-αin TTM high, medium dose groups were decreased significantly and the contents of serum IL-10 were increased significantly(P<0.05). The con-tents of liver TBARS in TTM high, medium dose groups were decreased significantly. The activity of liver SOD in TTM high, medium and low dose groups, the activity of GSH-Px in TTM high, medium dose groups and the contents of GSH in TTM high dose group were increased signifi-cantly(P<0.05). CONCLUSIONS: TTM has a certain effect to delay the rats' atrophy of thymus and spleen generated by LPS. It can effectively reduce the activity of NOS in serum, reduce the formation of NO, improve the activity of SOD, GSH-Px and the contents of GSH, reduce lipid peroxidation, decrease the excessive secretion of IL-6, TNF-α, increase the contents of IL-10, which can resist inflammation and protect the liver.

[Rich selenium-Banqiao-Codonopsis Pilosula mixture enhances immune function of aging mice].

OBJECTIVE: To investigate the effect of rich selenium-Banqiao-Codonopsis Pilosula (RSBCP) mixture on the immune functions of the aging mice. METHODS: Sixty Kunming mice (half male and half female) were randomly divided into high-, middle- and low-dose RSBCP mixture groups, model group and control group, with twelve mice in each group. The control group was injected intraperitoneally with 500 mg/(kg.d) normal saline and the other groups were injected with the same amount of D-galactose. The RSBCP mixture high-, middle- and low-dose groups were respectively gavaged with 100, 50, 25 g/(kg.d) RSBCP mixture at the moment when D-galactose was injected. The model group and control group were given the same amount of distilled water instead. The mice were killed at the 30th days and the indexes of thymus and spleen were measured. The serum levels of IgG, IgM, C3, C4, interleukine 2 (IL-2), IL-6 and tumor necrosis factor alpha (TNF-α) were detected by ELISA and the major T-cell subsets of splenocytes were analyzed by flow cytometry. RESULTS: Compared with the control group, the indexes of thymus and spleen in the model group decreased obviously; the serum levels of IgG, IgM, C3, C4, IL-2 and IL-6 were reduced significantly and the level of TNF-α increased significantly. Compared with the model group, the indexes of thymus and spleen in the RSBCP mixture groups increased obviously; the levels of IgG, IgM, C3, C4, IL-2 and IL-6 were raised significantly and the level of TNF-α decreased significantly. Compared with the control group, the indexes of thymus and spleen in the RSBCP mixture high-dose group increased obviously; the levels of IgG, IgM, C3, C4, IL-2 and IL-6 were lifted significantly and the level of TNF-α decreased significantly. The indexes of thymus and spleen and the levels of IgG, IgM, C3, C4, IL-2 and IL-6 in the RSBCP mixture middle- and low-dose groups had no obvious differences from those of the control group. Compared with the control group, the model group showed significantly decreased numbers of CD3⁺ T, CD4⁺ T, CD44⁺ T cells and significantly increased number of CD8⁺ T cells. Compared with the model group, the RSBCP mixture groups showed significantly increased numbers of CD3⁺ T, CD4⁺ T, both the high- and middle-dose groups showed significantly increased numbers of CD44⁺ T cells and significantly decreased number of CD8⁺ T cells. CONCLUSION: RSBCP mixture could delay the atrophy of thymus and spleen in the aging mice, dramatically elevate serum levels of IgG, IgM, C3, C4, IL-2, IL-6, lower the level of TNF-α, and influence the proportions of T cell subsets. The mixture plays a role in enhancing the immune function of aging mice.

[The effect of Wu-He Dipsacus asper on mice-aging model induced by D-galactose].

OBJECTIVE: To study the effect of Wu-He Dipsacus asper (WHDA), Traditional Chinese Medicine, injection on mice-aging model induced by D-galactose. METHODS: Forty-eight Kunming mice (24 male and 24 female) were randomly divided into control group, model group, positive control group, 7.2 g/kg WHDA group, 3.6 g/kg WHDA group and 1.8 g/kg WHDA group with eight in each group. The model was induced through injecting D-galactose into peritoneal cavity and Morris water maze was used to detect the learning and cognitive ability of mice. The skin hydroxyproline, brain tissue malondialdehyde (MDA), lipofuscin (LP), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels of mice were detected; the IL-2 and IL-6 levels in serum of mice were detected by using double antibody sandwich ELISA method. RESULTS: Each WHDA group was significantly reduced in latency period compared with the model group during Morris water maze test (P < 0.05) and the number of mice in model group through the platform was less than other mice in each group (P < 0.05). The levels of MAD and LP of the control group and each WHDA group were less than model group in the detection of heart, brain tissue oxidation index (SOD, MAD, LP and GSH-Px, P < 0.05). The activity of SOD and GSH-Px in the control group and each WHDA group was significantly higher than that in the model group (P < 0.05). The skin hydroxyproline content of mice which had been injected with D-galactose was significantly lower than that in the control group (P < 0.05) and the skin hydroxyproline content of mice of WHDA group was significantly higher than that in the model group (P < 0.05). The IL-2, IL-6 levels in serum of mice in WHDA group were significantly higher than those in the control group and the model group (P < 0.05) and the IL-2, IL-6 levels in serum of mice in the model group were lower than those in the control group (P < 0.05). CONCLUSION: The effective constituents of WHDA have a variety of biological activity which can have a good effect on anti-aging by different ways, improving learning and memory function, eliminating free radicals antioxidant, and enhancing the body immunity and other aspects.