ABSTRACT
Objectives:To investigate the therapeutic effect and mechanism of modified Fuzi Lizhongtang on ulcerative colitis （UC） model rats. Method:The 72 male SD rats were randomly divided into normal group，model group，sulfasalazine group（0.5 g·kg-1），modified Fuzi Lizhongtang high，medium and low-dose group （23.62，11.81，5.91 g·kg-1）. These rats were used to replicate the UC rat model by 2，4，6-trinitrobenzene sulfonic acid （TNBS）-ethanol composite modeling and treated by gavage for 2 weeks. The general condition of rats in each group was observed. After anesthesia，blood was collected from abdominal aorta and colonic tissue was taken. Semi quantitative evaluation by the colon mucosa damage index （CMDI），the pathological changes of colonic tissue were observed by the hematoxylin and eosin （HE） staining. The contents of serum interleukin-4 （IL-4），IL-6，IL-10 and tumor necrosis factor-α （TNF-α） were detected by enzyme-linked immunosorbent assay （ELISA）. The expressions of mammalian target of rapamycin（mTOR） and phosphorylated ribosomal protein S6 kinase 1 （p-S6K1） in colonic mucosa were detected by immunohistochemistry （IHC） and Western blot. Result:Compared with normal group，the CMDI score of the model group rats was significantly increased （P<0.01）. The contents of IL-4 and IL-10 in serum were significantly decreased，the contents of IL-6 and TNF-α were significantly increased （P<0.01）. The expression levels of mTOR and p-S6K1 in colonic mucosa were up-regulated （P<0.01）. Compared with model group，the CMDI score of the modified Fuzi Lizhongtang high dose group was significantly decreased （P<0.05）. In modified Fuzi Lizhongtang high and medium dose group，the contents of IL-6 and TNF-α were significantly decreased （P<0.01） and the contents of IL-4 and IL-10 in serum were significantly increased （P<0.05，P<0.01）. In the modified Fuzi Lizhongtang high dose group，the expression level of mTOR and p-S6K1 protein was down-regulated significantly （P<0.05，P<0.01）. Conclusion:Modified Fuzi Lizhongtang high dose group can significantly reduce the congestion and edema，inflammatory cell infiltration，gland distortion，disorder of arrangement and other pathological manifestations of UC colon mucosa，and its mechanism may be related to its down-regulation of mTOR/p-S6K1 signal and the regulation of inflammatory factors secretion.
ABSTRACT
@#Objective: To investigate the effect of sulforaphane (SFN) on CD8+ T cells differentiation, phenotype and the secretion of intracellular cytokines, as well as to study the underlying molecular mechanism. Methods: In the in vitro culture experiment, the cells were categorized into control group, SNF 10 mmol/L group and SNF 20 mmol/L group according to the SNF concentration. The effect of SFN treatment on CD8+ T cells differentiation, phenotype and cytokine secretion were detected by flow cytometry, and the effect of mTOR siRNA on the expression of CD127 and LKRG1 in CD8+T cells was also detected by flow cytometry. Expression of Bcl-2 and Bcl-6 were analyzed by qRT-PCR. The effect of SFN on apoptosis of CD8+T cells was examined byAnnexin-V/PI staining. The protein expressions of p-mTOR, p-S6 and b-actin were detected by western blotting. Results: SFN significantly promoted the formation of memory precursor CD8+ T cells and decreased the expression level of PD-1 and Tim-3 in CD8+T cells(P<0.01); meanwhile, after the treatment of SFN, the expressions of anti-apoptosis genes Bcl-2 and Bcl-6 were significantly increased while the apoptosis of CD8+ T cells was significantly inhibited and the protein expressions of p-mTOR and p-S6 were also significantly inhibited(P<0.05 or P<0.01). Moreover, mTOR siRNA could significantly increasethe expression of CD127 and decrease the expression of LKRG1 (all P<0.01). Conclusion: Sulforaphone promotes the formation of memory precursor CD8+T cells possibly by inhibiting the p-mTOR signaling pathway, and this could obtain more T cells to provide new thoughts for clinical immunotherapy.
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Purpose To investigate the expression and significance of type L amino acid transporter 1 (LAT1 ) and phosphorylated s6 ribosomal protein (p-s6) in breast cancer tissues and their correlation. Methods LAT1 protein and p-s6 protein were detected by immunohistochemical EnVision two step method in 178 cases of breast cancer and 78 cases of benign breast lesion, and the relationship between the expression and clinicopathological parameters was analyzed. Results The positive rate of LAT1 in breast cancer was 36.5％, which was significantly higher than that of breast benign lesion tissues (23.1 ％, P＜ 0.05 ), the positive rate of p-s6 in breast cancer tissues was33.2％, which was significantly higher than that of breast benign lesion tissues (12.8％, P＜0.05). There was a positive correlation between the expression of LAT1 protein and p-s6 protein in breast cancer tissues (r = 0.345, P＜ 0.05). The expression of LAT1 protein in breast cancer was correlated with tumor diameter, axillary lymph node metastasis, TNM staging and HER-2 level (P＜ 0.05), but not associated with the patient's age, histological grade, ER, and PR levels (P＞ 0.05). The expression of p-s6 protein was related to axillary lymph node metastasis, TNM staging, age and ER level (P＜ 0.05), but not associated with tumor diameter, histological grade, PR and HER-2 levels (P＞ 0.05 ). Multivariate analysis showed that the expres sion of LAT1 protein was related to tumor diameter and expression level of HER-2. The expression of p-s6 protein was related to axillary lymph node metastasis. Conclusion The expression of LAT1 protein and p-s6 protein in breast cancer is up-regulated, and the expression of these two proteins is positively related, which implying that LAT1 and p-s6 might play a synergistic role in the development and progression of breast cancer.