| Background/AimsCurrently, chronic kidney disease (CKD) has become a primary public healthy problem in the world. It has been demonstrated that tubular damage is one of the pathological features of CKD progressing to end-stage renal disease. Tubular damage mainly refers to tubular epithelial cell death. The ways of cell death include necrosis, apoptosis and autophagy. Among them, apoptosis and autophagy are important mechanisms in regulating tubular epithelial cell death. Apoptosis has a close relationship with the TGF-β1/JNK signaling pathway. Autophagy has a close relationship with the AMPK/mTOR/p70S6K and MAPKs signaling pathways. Dahuang Fuzi Decoction (DFD) (from Jin Kui Yao Lue), a classical prescription, with the effect of warming yang-deficiency and eliminating turbidity, is widely used in treating CKD patients in clinic and has a significant effect. Previous studies have been proved that DFD and its bioactive component rhein have anti-apoptotic or anti-fibrotic effects both in vivo and in vitro. However, the underlying pharmacological mechanisms are still obscure. Thus, a hypothesis is proposed as follows:DFD and its bioactive component rhein may protect against tubular injury by regulating apoptosis or autophagy. Based on the above hypothesis, firstly, the effect and mechanism of DFD in protecting against tubular damage by regulating apoptosis are studied in this thesis; secondly, the effect and mechanism of DFD and rhubarb (the monarch drug in DFD) in attenuating tubular injury by intervening autophagy are demonstrated; finally, the molecular mechanism of rhein, a bioactive component of DFD, in regulating tubular injury by intervening autophagy is elucidated.Methods1. In experiment 1, twenty-seven Sprague-Dawley (SD) rats were randomly divided into four groups, a normal group (A), a vehicle group (B), a Dahuangfuzi Decoction group (C), and a allopurinol group (D). Rats in B, C, and D groups were orally given adenine suspension at the dose of 150 mg/kg·d from day 1 to day 14 to establish a tubular injury (or renal failure) model. Rats in C group were administered Dahuangfuzi Decoction at the dose of 2.5 g/kg·d and the ones in D group were administered allopurinol solution at the dose of 0.03 g/kg·d, while the ones in A and B groups were given distilled water respectively from day 15 to day 35. During the drug administration, rats were administered adenine suspension (150 mg/kg) in B, C and D groups every 3 days to stabilize the renal function of model rats. The rats’ body weights were weighed every week. Blood urea nitrogen (BUN), serum creatinine (Scr), serum uric acid (SUA),24 h urinary protein excretion (Upro) and urinary N-acetyl-D-glucosaminidase (UNAG) were detected on days 0,14 and 35. All rats were sacrificed at the end of the 3rd week after drug administration. Then we withdrew blood, removed and weighed kidneys, examined renal macroscopic morphology, tubulo-interstitial morphological changes and the characters of tubular epithelial cells apoptosis (including TUNEL staining, and the protein expressions of Bcl-2, Bax and Cleaved caspase-3), and detected the blood biochemical and urinary parameters, as well as the protein expressions of TGF-β1, JNK and p-JNK, which are the related molecules in TGF-β1/JNK signaling pathway. This experiment suggested that DFD protects against tubular damage by reducing apoptosis in vivo.2. In experiment 2, thirty-three SD rats were randomly divided into five groups, a normal group (A), a vehicle group (B), a Dahuangfuzi Decoction group (C), a rhubarb group (D) and a valsartan group (E). Rats in B, C, D and E groups were orally given adenine suspension at the dose of 150 mg/kg·d from day 1 to day 14 to establish a tubular injury (or renal failure) model. Rats in C group were administered Dahuangfuzi Decoction at the dose of 2.5 g/kg·d, the ones in D group were administered rhubarb solution at the dose of 1 g/kg·d and the ones in E group were administered vehicle solution at the dose of 8 mg/kg·d, while the ones in A and B groups were given distilled water from day 15 to day 35. During the drug administration, rats were given adenine suspension (150 mg/kg) in B, C, D and E groups every 3 days to stabilize the renal function of model rats. All rats were sacrificed at the end of the 3rd week after drug administration. Then we extracted kidneys to examine the immunohistochemical stainings and protein expressions of autophagic marker LC3, as well as renal fibrotic markers Collagen Ⅰ and Fibronectin. This experiment proposed that DFD and rhubarb attenuates tubular injury by intervening autophagy in vivo.3. In experiment 3, NRK-52E cells were in starvation-induced autophagy by exposing to hank’s balanced salt solution (HBSS), with or without rhein. The protein expressions of LC-3 Ⅰ/Ⅱ, the related molecules in the Akt/AMPK/mTOR/p70S6K signaling pathway (p-Akt Ser473, Akt, p-AMPK, AMPK, mTOR, p-mTOR Ser2448 and p-p70S6K), and the related molecules in MAPKs signaling pathway (p-p38, p-Erk and p-JNK) were detected. The protein expression of LC-3 Ⅰ/Ⅱ was detected in NRK-52E cells treated by HBSS and Bafilomycin A1 (an autophagic inhibitor) with or without rhein, in NRK-52E cells treated by HBSS and rhein with or without Rapamycin (an mTOR inhibitor), in NRK-52E cells or the cells transfected with deptor plasmid (an mTOR inhibitor) treated by HBSS with or without rhein, or in NRK-52E cells treated by HBSS with or without SB203580 (a p38 inhibitor) or PD098059 (an Erk inhibitor). The protein expressions of LC-3 Ⅰ/Ⅱ, p-AMPK, AMPK and p-p70S6K were detected in NRK-52E cells treated by HBSS and rhein with or without Metformin (an AMPK inducer). mRFP-LC3 fluorescent dots were examined in NRK-52E and hela cells transfected with mRFP-LC3 plasmid treated by HBSS and Bafilomycin A1 with or without rhein, SB203580 or PD098059, or treated by HBSS, Bafilomycin Al and rhein with or without Metformin. This experiment showed that, rhein, a bioactive component of DFD, regulates tubular injury by intervening autophagy in vitro.Results1. In model rats after the administration of adenine suspension for 14 days, Upro, UNAG, BUN, Scr and SUA were increased significantly, and the changes of tubular interstitial injury and tubular epithelial cells apoptosis were notable. After the 3-week drug administration, DFD and allopurinol solution reduced the levels of renal function, Upro and UNAG, ameliorated the apoptosis of tubular epithelial cells and interstitial fibrosis, down-regulated the protein expressions of Bax and Cleaved caspase-3, up-regulated the protein expression of Bcl-2, as well as inhibited the protein expressions of TGF-β1 and p-JNK. DFD was more effective than the allopurinol solution.2. In model rats after the administration of adenine suspension for 14 days, tubules were dilated, tubular epithelial cells were reduced, renal interstitial areas were increased, as well as the immunohistochemical stainings and protein expressions of LC3, Collagen Ⅰ and Fibronectin were increased significantly. After the 3-week drug administration of DFD, rhubarb and valsartan solutions, dilated tubules, reduced tubular epithelial cells and increased renal interstitial areas were slightly improved, and the immunohistochemical stainings and protein expressions of LC3, Collagen Ⅰ and Fibronectin were reduced significantly. The differences in DFD, rhubarb and valsartan groups were not significant.3. (1) Rhein suppressed LC3 conversion induced by HBSS in NRK-52E cells, as well as inhibited HBSS and Bafilomycin A1 induced LC3 conversion and mRFP-LC3 fluorescent dots. The results showed that rhein can inhibit HBSS induced autophagy in NRK-52E cells. (2) Rhein increased the protein expressions of p-mTOR Ser2448 and p-p70S6K inhibited by HBSS. Rapamycin increased LC3 conversion exposed to HBSS and rhein. HBSS-induced LC3 conversion cannot be inhibited by rhein in NRK-52E cells transfected with deptor plasmid. The results indicated that rhein inhibits HBSS induced autophagy in NRK-52E cells by activating the mTOR/p70S6K signaling pathway. (3) Rhein suppressed HBSS-induced p-AMPK protein expression in NRK-52E cells, not affecting p-Akt Ser473 protein expression. Metformin increased LC3 conversion and p-AMPK protein expression inhibited by the co-treatment of HBSS and rhein, as well as suppressed p-p70S6K protein expression. Further, Metformin increased mRFP-LC3 fluorescent dots exposed to HBSS, Bafilomycin A1 and rhein. The results demonstrated that rhein inhibits HBSS induced autophagy in NRK-52E cells by the AMPK signaling pathway without affecting the Akt signaling pathway. (4) Rhein inhibited HBSS induced p-p38 and p-Erk protein expressions in NRK-52E cells, not affecting p-JNK protein expression. SB203580 and PD098059 can reduce HBSS-induced LC3 conversion in NRK-52E cells, as well as mRFP-LC3 fluorescent dots exposed to HBSS and Bafilomycin A1. The results suggested that rhein inhibits HBSS induced autophagy in NRK-52E cells by inhibiting p38 and Erk MAPKs signaling pathways. Rhein may act as the analogue of Erk and p38 inhibitors.Conclusions1. DFD reduced tubular epithelial cells apoptosis and improved renal interstitial fibrosis in adenine-induced rats by reducing the protein expressions of TGF-β1 and p-JNK, the key signaling molecules in the TGF-β1/JNK signaling pathway. Thus, the TGF-β1/JNK signaling pathway is not only a vital pathway in regulating apoptosis, but also a target of DFD in protecting against adenine-induced tubular injury.2. In adenine-induced renal injury rats, DFD and rhubarb can not only improve dilated tubules, reduced tubular epithelial cells, and increased renal interstitial areas, but also reduce the protein expressions of LC3, Collagen Ⅰ and Fibronectin. Hence, autophagy is not only an important way to induce tubular injury, but also a target of DFD and rhubarb in improving tubular injury.3. Rhein can inhibit HBSS-induced autophagy in NRK-52E cells by regulating AMPK/mTOR/p70S6K and p38/Erk MAPKs signaling pathways. Therefore, AMPK/mTOR/p70S6K and p38/Erk MAPKs signaling pathways are not only vital pathways in modulating autophagy, but also the targets of rhein in regulating tubular injury. |
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