| ObjectiveCyclosporine A (CsA), a potent immunosuppressant, is the most widely used drug in organ transplantation and in the management of several autoimmune disorders. Despite being so very potent, its use for therapeutic purpose remains challenging due to the occurrence of disastrous side effects, namely, acute and chronic nephrotoxicity, severe hypertension, and neurotoxicity. Schisandra chinensis extracts (SCE) from mature fruit of Schisandra chinesis, has been one of the most widly used in traditional herbal medicines in traditional Chinese medicine for thousands of years in People’s Republic of China. Additionally, SCE have been revealed to possess multipharmaceutical bioactivities, including antioxidant, anti-inflammatory, anti-microbial, cardioprotective, hepatoprotective, which are extremely useful for the treatment of hepatitis, renal insufficiency and neuroasthenia. Clinically, more recently coadministered with SCE and its preparations for the treatment of CsA induced side effects in China, While the potential renoprotective and molecular mechanism underlying the benefits of SCE on CsA induced nephrotoxicity remains to be explored. Therefore, this study aimed to investigate the possible effect of SCE on the pharmacokinetics of CsA in rats by LC/MS/MS, and further to elucidate the potential mechanisms by which it hinders the development of CsA induced nephrotoxicity. Meanwhile, we can further interpret the renal protective mechanism of SCE or SchB.Methods1. Quantification of CsA by liquid chromatography tandem mass spectrometry in biological samplesChromatographic analysis was performed using the Agilent 1260 series HPLC system, and the separation was performed using a C18 column (2.1×50mm,3.5μm particle size, Waters) at 60℃. The mobile phase consisted of methanol-water (80:20, v/v, containing lOmM ammonium acetate), and it was pumped at flow rate of 0.2mL/min,10μL injection volume, FK520 as the internal standard. The detection was performed by multiple reation monitoring mode via electrospray ionization source operating in the positive ionization mode. Then, the selectivity, precision and accuracy, matrix effect, extraction recovery, linearity and stability were investigated.2. Effect of different doses of SCE on the pharmacokinetics of CsAMale Sprague Dawley rats were randomly divided into four groups, each group consisting of six rats:(1) CsA only group:rats were administered tap water simultaneously with CsA in a span of 10 minutes. (2) SCE administered at a dose of 54mg/kg. (3) SCE administered at a dose of 108mg/kg. (4) SCE administered at a dose of 216mg/kg. In the latter three groups:SCE was administered by gavage to the animals at relevant doses, and 10 minutes post-SCE administration, all rats were given CsA at a dose of 25mg/kg. Both SCE and CsA were given in an administration volume of 10 mL/kg body weight. Blood sample (400μL) was collected using heparinized tubes through the abdominal aorta at 0, 0.083,0.25,0.50,0.75,1.0,2.0,3.0,4.0,6.0,8.0,12.0,24.0,36.0 and 48.0 hours. Blood sample were removed protein, sample concentration and further to analynzed with LC/MS/MS. Pharmacokinetic parameters were calculated using a non-compartment model in a noncompartmental analysis performed by a pharmacokinetic program (Data Access Service, Version 2.1).3. SCE protects against CsA induced nephrotoxicity study in ratsForty-two rats were randomized into six subgroups of seven rats each and treated daily for 28 days as follows:(1) Control group:gavaged with tap water and fed normal salt diet for 28 days. (2) Vehicle control group:gavaged with olive oil and fed low salt diet for 28 days. (3) CsA group:gavaged with 25mg/kg CsA and fed low salt diet for 28days in order to establish a nephrotoxic effect. (4) SCE group:gavaged with 108mg/kg SCE and fed low salt diet for 28 days. (5) 54mg/kg SCE+CsA group:gavaged with 54mg/kg SCE+25mg/kg CsA and fed low salt diet for 28 days. (6) 108mg/kg SCE+25mg/kg CsA group:gavaged with 108mg/kg SCE+25mg/kg CsA and fed low salt diet for 28 days. Blood samples were collected from aorta abdominalis, and these samples were then separated by the process of centrifugation at 4000rpm within a span of 10 minutes. The serum was aspirated and stored at-80℃ until it was needed for Cr and BUN analyses. Thereafter, their abdomens were opened and longtitudinal section of the right kidney was takern and fixed in 4% paraformaldehyde at 4℃ for 48 hours. These longitudinal sections from each animal were then embedded in paraffin blocks for histopathological and immunohistochemical analyses. The renal cortex of the left kidney was frozen in liquid nitrogen and stored at-80℃ for Western blot (4-HNE, HO-1, Nrf2, P-gp, Cleaved caspase 3, Bax, LC3A/B) and various biochemical analyses (MDA, CAT, GSH-Px).4. Effect of Schisandrin B protects against nephrotoxicity induced by CsA in HK-2 cells via oxidative stress, apoptosis and autophagyHK-2 cells model were conventionally cultured, cell viability and toxicity were evaluated by MTT assay for discussing the effect of co-administered with SchB. SchB and CsA co-treatment for HK-2 cells, Lacate dehydrogenase (LDH), reduced glutathione (GSH), reactive oxygen species (ROS), mitochondrial membrane potential disruption (MMP) assays was detected. Apoptotic cells were quantitated using the annexin V:PI apoptosis detection kit. For autophagy detection, used the Cyto-ID (?) green stain solution and confocal fluorescence microscopy.4-HNE, HO-1, NQO1, GCLM, Nrf2, Bax, Bcl-2, Beclinl expression levels were detected by Western blot for illuminating SchB protects against nephrotoxicity induced by CsA from perspective of oxidative stress, apoptosis, autophagy mechanism and further to provide experiment basis for the clinical application.Results1. It is established determination of CsA in whole blood samples by LC/MS/MS method, that was quantitated patent drug of CsA by multiple reaction monitoring (MRM) mode. The result showed that CsA metabolites have no impact on this method. The methodological evaluation showed that specificity, matrix effect, extraction recovery, precision and accuracy, linearity and stability were within the FDA acceptance criteria of bioanalytical method validation. All in all, it is indicated that the method apply to monitor blood concentration of CsA and guide for dosage in clinical practice. It can further to reduce the side effect happened.2. A LC/MS/MS method for the determination of CsA in whole blood sample was used to determine the pharmacokinetics of CsA in rats following a CsA alone dose or co-treatment with different dose of SCE (54,108,216mg/kg). The result showed as follow:(1) 25mg/kg CsA alone:The results showed pharmacokinetic parameters of CsA (Cmax, 2608±585.89ng/mL, Tax,6.33±1.50h, t1/2,19.92±6.43h, AUC0â†'∞,82851.36±16615.30ng h/mL).(2) 54mg/kg SCE±25mg/kg CsA:The results showed pharmacokinetic parameters of CsA (Cmax,3456.674±251.89ng/mL, Tmax,6.8±1.10h, t1/2,29.66±14.27h, AUC0â†'∞, 132564.2±50934.74ng h/mL).(3) 108 mg/kg SCE±25mg/kg CsA:The results showed pharmacokinetic parameters of CsA (Cmax,3026.806±409.61ng/mL, Tmax,8.67±1.63h, t1/2,28.44±9.00h,AUC0â†'∞ 114030.90±21573.65ng h/mL).(4) 216 mg/kg SCE±25mg/kg CsA:The results showed pharmacokinetic parameters of CsA (Cmax,3286.45±1418.41ng/mL, Tmax,13.5±7.55h, t1/2,34.94±12.67h, AU0â†'∞, 173777.70±95727.64).The above results showed that after the oral administration of different dose of SCE to rats, the pharmacokinetic parameters of CsA were found to be increased or extended, that it could be illustrate SCE change the pharmacokinetic behavior in vivo when CsA was administered in combination with different doses of SCE. In other words, with the help of reducing the dose of CsA in clinical practice, it has reduced the possibility of side effects and financial burden.3. After the aforementioned 28 days, compared with 25mg/kg CsA group, the results showed that the CsA induced increase in Cr, BUN and MDA levels was abolished when rats were treated concurrently with different doses of SCE. One hand, treatment with SCE showed significant improvement in CAT and GSH-Px activity. On the other hand, it was revealed that the coadministered with SCE showed less renal damage with fewer tubular cells affected, inflammation and renal tubularinterstitial fibrosis, which can explain SCE against CsA induced nephrotoxicity effectively. Meanwhile, When exposed to concomitant administration of SCE along with CsA, the increase in HO-1, Nrf2 and P-gp expression levels, the decrease in 4-HNE, Bcl-2, Cleaved caspase 3 and LC3A/B, it can be deduced that the renoprotective mechanism of SCE is associated with attenuation of oxidative stress, apoptosis, autophagy.4. The experimental results in HK-2 cell model as follow:(1) The viability of HK-2 cells was decreased by CsA in concentration-and time-dependent manners; when the cells were co-treated with various concentration of SchB (2.5,5.0, 10.0μM) for 24h and proliferation increased in a dose-dependent manner, meanwhile, the LDH leakage from the cell to culture medium and ROS levels gradually decreased; pretreatment with SchB at increasing concentration of 2.5,5.0, 10.0μM for 24h reversed the decreased GSH and 4-HNE expression levels of HK-2 cells induced by CsA. This finding indicates that the renoprotective effect of SchB is achieved with anti-oxidative stress to protect cells from toxicity.(2) Using the Annexin V/PI double staining detected showed that CsA increased HK-2 cell apoptosis not only in a dose dependent manner, but also in a time dependent way. Pretreatment with 2.5,5.0, 10.0μM for 24h showed the apoptosis rate reduce to 18.12%, 16.80%,15.25%, which apparently protective action compared with 10μM CsA alone. On the other hand, a pronounced increase was observed in the mitochondrial membrane potential and Bcl-2/Bax expression levels following SchB pretreatment, which suggest that exposure to SchB increase the mitochondrial membrane depolarized through the intrinsic mitochondrial apoptotic pathway.(3) According to instructions of Cyto-ID (?) green stain solution using flow cytometric analysis and confocal microscopic examination. The result showed that CsA exhibited a concentration-and time- dependent increase autophagy activity. The increased autophagy activity mediated by CsA was blocked by pretreatment with SchB. We further comfirmed the autophagy inhibiting effects of SchB on HK-2 cell mediated by CsA using confocal microscopic examination to observe lysosome specific fluorescence dye. SchB treatment caused a significant decrease the lysosome specific fluorescence dye, meanwhile, the marker of autophagy Beclinl expression was down regulated. These data suggest that SchB reduces CsA induced autophagy.(4) The study also found that, pretreatment with SchB could cause nuclear accumulation of Nrf2 in association with downstream activation of Nrf2 mediated â…¡ phase detoxifying enzyme and oxidative response expression such as HO-1, NQO1, GCLM, these suggest that SchB could protect against the oxidative damage of HK-2 cells induced by CsA via the activation of Nrf2/ARE signal pathway. ConclusionWhole blood concentrations of CsA in rats were significantly increased after cotreatment with lignans of Schisandra chinesis and were found to be protective against CsA induced renal nephrotoxicity, the mechanism of which may be related with the decreased of ROS levels, stabilizd mitochondria membrane potential, regulated Nrf2 signaling pathway for anti oxidative stress, attenuated apoptosis by means of mitochondrial apoptotic pathway and decreased cell autophagy. |
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