| Part1The effect of Fluorofenidone on lipid peoxidation in UUO rats renal interstitial fibrosisObjective:To investigate whether Fluorofenidone (AKF-PD) can ameliorate the lipid peoxidation after ureteral obstruction; to investigate the effect of AKF-PD on renal oxidative stress, in order to get the better understandings of the mechanism underlying the anti-fibrogenic effect of AKF-PD.Methods:SD rats were submitted to unilateral ureteral obstruction (UUO) and studied after14days. Renal tissue after ureteral obstruction at day14was dyed by HE and Masson staining for general histology and collagen detection.Renal tissue after ureteral obstruction at day14, Western blot was used to assess the fibronectin(FN) expression.Immunostaining was used to assess the infiltration of collagen Ⅰ and Ⅲ. ELISA was done to assess the expression of8-iso-PGF2a and TRASB was done to assess the expression of MDA.Result:(1) HE and MASSON staining revealed that there is significant interstitial pathological change in rat injured kidney, and huge amount of collagen fiber deposited in renal tissue was seen. The Tubulointerstitial damage index and relative area of renal interstitial collagen significantly increased in UUO model14days after operation. All of above observations indicate that the establishing of the UUO model is successful. Compared with UUO group, treatment of AKF-PD and losartan significantly ameliorated renal interstitial fibrosis, and significantly reduced the deposit of collagen fiber, renal interstitial injury index, as well as the relative area of renal interstitial collagen (P<0.05) The tubulointerstitial injury scores and collagen I expression in the AKF-PD (500mg/kg) group were significantly lower than that in Losartan(20mg/kg) group (p<0.05).(2) The increase in oxidase activity in kidneys from UUO rats was also supported by evidence of lipid peroxidation given increased levels of8-iso-PGF2a and MDA as compared to those in sham rats. Both AKF-PD and Losartan treatment attenuated the8-iso-PGF2a increase, but only the former decreased (p<0.05) MDA generation.Conclusion:(1) AKF-PD ameliorats renal fibrosis. AKF-PD(500mg/kg) exerted a stronger inhibition than Losartan(20mg/kg) on protein expression of collagen III and tubulointerstitial injury in UUO rats.(2) AKF-PD significantly attenuated lipid peroxidation in UUO kidney, indicating that the beneficial effect of AKF-PD in renal interstitial fibrosis is attributable, at least in part, to an anti-lipid peroxidation action.Part2The effect of Fluorofenidone on NADPH oxidative and phosphorylated Akt in UUO rats renal interstitial fibrosisObjective:To investigate whether Fluorofenidone (AKF-PD) can ameliorate the expression of oxidative stress and phosphorylated Akt after ureteral obstruction; to investigate the effect of AKF-PD on renal oxidative stress, in order to get the explorations of the mechanism underlying the anti-oxidative stress effect of AKF-PD.Methods: SD rats were submitted to unilateral ureteral obstruction (UUO) and studied after14days. Renal tissue after ureteral obstruction at day14, Western blot was used to assess the Expression of Nox enzyme subunits and Akt. Cytochrome C reduction assay was done to assess the activity of Nox Enzyme.Result:(1)Kidneys from UUO rats underwent oxidative stress as evidenced by significantly increased NADPH oxidase activity, as compared to that in sham rats.Treatment with AKF-PD and Losartan ameliorated the increase oxidase activity(p<0.05) as compared to that UUO vehicle-treated rats. Supporting these findings, the kidney from UUO rats displayed increased (p<0.01) protein levels of four Nox subunits, namely Nox1and Nox2; conversely, While AKF-PD and Losartan treatment attenuated (p<0.01) Nox2expression, it did not affect (p>0.05) the expression of Noxl.There was no statistical difference between AKF-PD and Losartan treatment.(2) Western blot analysis for Akt was consistent with UUO rats developing kidney injury as evidenced by a significant appearance of phosphorylated Akt as compared to that in sham rats. Treatment with AKF-PD or Losartan did counteract (p<0.05) the increase in phosphorylated Akt expression as compared to untreated UUO rats.Conclusion:(1) AKF-PD ameliorats renal oxidative stress.(2) AKF-PD reduced Akt phosphorylation in fibrotic kidneys.Part3The effect of Fluorofenidone on oxidative stress and phosphorylated Akt and ERK in Ang Ⅱ-Induced Rat Proximal Tubular Epithelial (NRK-52E) Cells Objective:To investigate whether Fluorofenidone (AKF-PD) can ameliorate the expression of oxidative stress and phosphorylated Akt、ERK by Ang II stimulated rat NRK-52E cells; to investigate the effect of AKF-PD on renal oxidative stress, Further discussion the mechanism underlying the anti-oxidative stress effect of AKF-PD.Methods:NRK-52E cells assigned to four groups:control、AKF-PD、Losartan and inhibitor of the PI3K inhibitor of LY294002, MEK inhibitor of U0126and NADPH oxidase inhibitor DPI. To examine the effects of AKF-PD and Losartan on Ang Ⅱ induced expression of p-Akt, p-ERK, Noxl and Nox2by Western blot, and were subsequently induced by Ang Ⅱ for15minutes (p-Akt,p-ERK) and24h (Nox1, Nox2, and Collagen Ⅰ (1α) before cellular protein extraction. ROS levels were determined by measuring the oxidative conversion of cell-permeable2’,7’-ichlorofluorescein diacetate (DCFH-DA) to fluorescent dichlorofluorescein (DCF) using a Microplate Reader.Result:(1) Groups of24h serum-starved cells were treated with Ang Ⅱ plus AKF-PD, Losartan or DPI and DCF fluorescence was determined60min later. All AKF-PD, Losartan and DPI attenuated (p<0.01) Ang Ⅱ induced ROS generation by48,43and79%, respectively.(2) Cell pretreatment (24h) with AngⅡ alone increased (P＜0.01) the protein expression of Noxl and Nox2. While, co-treatment with the Nox inhibitor DPI blocked the Ang Ⅱ-induced expression of both Noxl and Nox2(P<0.01), co-treatment with AKF-PD only blocked (P＜0.05) Ang Ⅱ-induced Nox2expression.(3) Increased levels of p-Akt and p-ERK were detected as early as15 min after AngⅡ treatment.Co-treatment of AngⅡ with AKF-PD and Losartan dramatically decreased levels of p-Akt and p-ERK at15min. Moreover, addition of the PI3K inhibitor, LY294002, and the MEK inhibitor, U0126, exerted stronger inhibition effect on AngⅡ-induced p-Akt and p-ERK than AKF-PD.Conclusion:(1) AKF-PD inhibited expression of the ROS and Nox2subunit in rat NRK-52E Cells.(2) AKF-PD inhibited Ang Ⅱ-Induced phosphorylation of Akt and ERK in NRK-52E cells.Part4The effect of Fluorofenidone on PI3K/Akt and MEK/ERK Signaling Pathways in rat proximal tubular epithelial cellsObjective:To investigate whether Fluorofenidone (AKF-PD) can ameliorate the expression of oxidative stress and phosphorylated Akt、ERK by Overexpressing PI3K P110α-CAAX and MEK1Q56P inNRK-52E cells; to investigate the effect of AKF-PD on renal oxidative stress, go deep into the mechanism underlying the anti-oxidative stress effect of AKF-PD.Methods:Plasmids constitutively expressing the active form of MEK (MEK1Q56P) or PI3K (PI3K P110α-CAAX), respectively. Transient transfection of cells with one or the other plasmid was conducted using Lipofectamine2000reagent following the protocol provided by the manufacturer. The production of ROS was then measured in these groups of cells (see below) following treatment with AKF-PD and Losartan for24h. Each experiment was carried in triplicate. The oxidative conversion of cell-permeable DCFH-DA to fluorescent DCF was assessed by flow cytometry analysis using FACS calibur.Result:(1) To elucidate the molecular mechanism by which AKF-PD blocked Nox activationMand interstitial matrix accumulation induced by oxidative stress, we inhibited the PI3K/Akt signaling pathway. Hence, NRK-52E cells were transfected with a plasmid constitutively expressing the active form of PI3K (PI3K P110α-CAAX). Transiently transfected NRK-52E cells displayed an increased expression of p-Akt, p47phox, Nox2and collagen I (al) as well as increased production of ROS. The addition of AKF-PD reduced ROS formation (p<0.01), and expression of all proteins but Nox2.(2) To elucidate the potential role of the MEK/ERK pathway on AKF-PD inhibition of ROS generation NRK-52E, transfection was performed using a plasmid (MEK1Q56P) constitutively expressing MEK. Transiently transfected NRK-52E cells overexpressing MEK displayed an increased expression of p-ERK, Nox2and collagen type I (α1) as well as increased ROS production, all of which were counteracted by the addition of AKF-PD.Conclusion:(1) AKF-PD inhibited the PI3K/Akt signaling pathway in rat proximal tubular epithelial cells overexpressing PI3K.(2) AKF-PD inhibited the MEK/ERK signaling pathway in rat proximal tubular epithelial cells overexpressing MEK.(3)AKF-PD inhibits the expression of the oxidative stress was at least partly via PI3K/Akt and MEK/ERK signaling pathways. |
PDF Full Text Request