Studies On The Effects And Mechanisms Of Active Compounds From Pericarpium Citri Reticulatae In Combination On Inflammation And Cancer Cells

The aim of the present study was to investigate the in vitro and in vivo anti-inflammatory effects of eriocitrin combined with resveratrol.In addition,this study focused on the mechanistic study of inhibitory effects of neohesperidin and docetaxel in combination on prostate cancer.Part I: Firstly,an in vitro inflammation model was established using LPS induced RAW264.7 macrophages.The effect of the combination on the growth of RAW264.7 cells was determined,and the appropriate doses was selected.The effect of the combination on the expression of NO was detected by Griess reagent,and the synergistic effect of eriocitrin and resveratrol was analyzed by CompuSyn software.Using tumor necrosis factor-alpha(TNF-?)and interleukin-1?(IL-1?)inflammatory mediators as indicators,ELISA was used to detect the inhibitory effect of the combination drugs on inflammatory mediators.Western blotting assay was used to detect the effect of combination therapy on the phosphorylation activation of mitogen-activated protein kinase(MAPK)signaling pathway and the expression of nuclear transcription factor NF-?B.Finally,the TPA-induced mouse ear swelling model was used to evaluate the effect of combination therapy on inflammation in vivo.The results showed that eriocitrin combined with resveratrol strongly inhibited LPS-induced NO,TNF-?,and IL-1?.Moreover,Eriocitrin combined with resveratrol potently inhibited NF-?B,phosphor-STAT3,and phosphor-AKT,which was accompanied by inhibition of phosphorylation in mitogen-activated protein kinase(MAPK)signaling pathways.Treatment with eriocitrin combined with resveratrol alleviated edema and subcutaneous tissue inflammation caused by TPA in vivo.This study also demonstrated that treatment with eriocitrin and resveratrol decreased the levels of the pro-inflammatory cytokines TNF-? and IL-1?.Part II : MTT assay was used to detect the effect of neohesperidin and docetaxel alone or in combination on the growth of prostate cancer cells LNCap,PC3,DU145 and prostate epithelial cells RWPE-1,and the appropriate dose was selected.Apoptosis was detected by propidium iodide single staining.The expression of nuclear factor NF-?B was detected byluciferase reporter gene assay.The effect of combined drug on prostate cancer cell migration was detected by scratch wound-healing assay.Western blotting was used to detect the effect of combination therapy on the phosphorylation activation of MAPK signaling pathway and the expression of Bcl-2,p-AKT and p-STAT3.Finally,the prostate cancer xenograft model was used to evaluate the effect of combination therapy on tumors in vivo.The results showed that a combination of neohesperidin and docetaxel produced synergistic effects inhibiting the growth of prostate cancer cells and inducing apoptosis in these cells.The combination of neohesperidin and docetaxel also strongly inhibited cell migration,colony formation and tumorsphere formation in PC-3 cells.Western blot analysis showed that the combined effects of neohesperidin and docetaxel on growth inhibition were associated with suppression of survivin and decreased levels of Bcl-2,phospho-Akt,phospho-JNK,and phospho-ERK.In addition,neohesperidin and docetaxel combination caused stronger suppression of NF-?B activity than either agent alone.The in vivo anti-cancer activity was investigated using immunodeficient(SCID)mice model.Neohesperidin or docetaxel alone resulted in slight inhibition of tumor growth while the combination of neohesperidin and docetaxel strongly inhibited the growth of the tumors.In summary,this is the first report for the combined anticancer and anti-inflammatory effects of active constituents from Pericarpium Citri Reticulatae and their mechanisms of actions.Our results indicate that neohesperidin had anticancer activity in vitro and had inhibitory efficacy on the development of PC-3 xenograft tumours in vivo,eriocitrin also showed anti-inflammatory activity in vitro and in vivo.Especially important,The current study demonstrated for the first time that the combination of neohesperidin and docetaxel synergistically inhibits the growth of prostate cancer.This study also suggests that eriocitrin combined with resveratrol is more effective than either of these compounds alone in relieving inflammation both in vitro and in vivo.