Preparation And Preliminary Pharmaceutical Research Of S-allylmercaptocysteine Nanosuspension For The Treatment Of Acute Lung Injury

Acute lung injury(ALI),as a common syndrome with high morbidity and mortality up to 40%caused by non-cardiogenic stimuli such as pneumonia,sepsis and trauma,is characterized by diffuse alveolar damage,refractory hypoxemia,and non-cardiogenic pulmonary edema.The present study shows that oxidative stress and inflammation are important pathogenesis of acute lung injury.Unfortunately,despite a series of treatments such as inhaled nitric oxide and glucocorticoids have been applied in the ALI treatment,none of these available therapies have been proven to effectively reduce the mortality or improve the life quality of survivors.Therefore,exploring complementary and alternative medicines for ALI treatment is indispensable.Garlic(Allium sativum),a bulbous perennial plant,has been proved owning a wide range of pharmacological effects,which are mostly attributed to its organosulfur compounds.S-allylmercaptocysteine(SAMC),one of the remarkable aqueous soluble sulfur-containing compounds from garlic,has pharmacological activities of anti-cancer,liver protection,anti-inflammation,anti-oxidation and so on.Although the research on the pharmacological effects of SAMC is constantly developing,its solubility also limits its further research.To date,there is no research on preparing SAMC new formulation to improve its physic-chemical properties;in addition,most of the current researches on the pharmacological activities of SAMC are limited to the evaluation of anti-cancer activity,and there are few reports on the application of SAMC in the treatment of ALI.Therefore,a new SAMC nanosuspension was prepared in this study to improve the solubility and dissolution rate of SAMC.Meanwhile,a preliminary study on the efficacy of SAMC on the treatment of ALI and its related mechanisms were conducted in our study.The main contents in this paper include four parts:(1)preparation and characterization of SAMC nanosuspension(SAMC-NS);(2)preparation of lyophilized SAMC nanosuspension;(3)study on the anti-inflammatory and anti-oxidative effects and mechanisms of SAMC in vitro;(4)study of SAMC-NS on the pharmacological activities and molecular mechanism against ALI in vivo.1.Preparation and characterization of SAMC nanosuspension:The main contents of this part included the methodology establishment of SAMC content determination,prescription screening of SAMC-NS,preparation process optimization and characterization of SAMC-NS.The microfluidization method was used to prepare SAMC-NS in this study.Particle size,particle size distribution and short-term physical stability were selected as indicators for prescription screening and optimizing process to examine the effects of variables including stabilizers,the ratio of drug to stabilizer,the homogenization pressure and the number of homogenization cycles on the particle size and PDI of SAMC-NS.Then,the physic-chemical characterization of the optimum nanosuspension formulation were evaluated including particle size,PDI,Zeta potential,appearance and transmission electron microscope(TEM)morphology.The results show that the appearance of SAMC-NS was clear and transparent and the morphology of the particles under the TEM was spherical.The mean particle size was 275.93±4.66 nm with a narrow particle size distribution(PDI:0.124± 0.025)and the Zeta potential value was-18.97±1.05 mV.2.Preparation of lyophilized SAMC nanosuspension:The main contents of this part included the investigation of the freeze-drying process of SAMC-NS,the quality evaluation of lyophilized product,the determination of saturation solubility and in vitro dissolution.To increase the stability,freeze-drying method was employed to lyophilize SAMC-NS.Taking the appearance,color and redispersibility of the lyophilized product as indicators,the commonly used lyoprotectants and the amount of addition were screened and finally,5%(w/v)mannitol was selected as the lyoprotectant of SAMC-NS.The quality evaluation of the lyophilized product included appearance,redispersibility,particle size,PDI and Zeta potential.The results show that the lyophilized SAMC-NS appeared as white and loose powder with good redispersion.After re-dissolution,the particle size,PDI and Zeta potential of the lyophilized SAMC-NS were 394.60±9.51 nm,0.45±0.09 and-18.43±5.61 mV,respectively.The saturation solubility was examined in comparison with coarse powder,physical mixture and lyophilized SAMC-NS.Our results show that the saturation solubility of lyophilized SAMC-NS was 1.87±0.05 mg/ml,which was significantly increased than that of coarse SAMC powder.Dissolution behavior of the lyophilized SAMC-NS in vitro was estimated using a dissolution apparatus with the paddle method.Only 21.0%and 17.9%had dissolved at 2 min for the coarse SAMC and the physical mixture,respectively,while the dissolution rate of lyophilized SAMC-NS was up to 93.9%.Thus,lyophilized SAMC-NS apparently improved dissolution rate of SAMC.3.Study on the anti-inflammatory and anti-oxidative effects and mechanisms of SAMC in vitro:The main contents of this part included the measurement of mRNA expression levels of inflammatory cytokines,the determination of oxidative stress markers,and the exploration of SAMC anti-inflammatory and anti-oxidant mechanism.We established a cell model of ALI by adding 1 ?g/ml LPS into NR8383 cell culture medium and then the cells were treated with SAMC.After 24 hours,the changes of inflammation and oxidative stress markers levels were measured,and the expression of related signaling pathway proteins was detected by Western blot.Briefly,we found that SAMC weakened the inflammatory response by reducing the amount of NO secretion and the mRNA expression levels of TNF-?,IL-6,and IL-1?.Besides,SAMC obviously reversed oxidative stress by decreasing the content of MDA and increasing the levels of SOD and GSH.Furthermore,Western blot experiments showed that SAMC exerted anti-inflammatory and anti-oxidative effects via inhibiting the NF-?B signaling pathway and activating Nrf2 signaling pathway.(4)Study of SAMC-NS on the pharmacological activities and molecular mechanism against ALI in vivo:The main contents of this part included the establishment of ALI mice model,the effect of SAMC-NS on lung edema in ALI mice,histopathological evaluation,the evaluation of anti-inflammatory and anti-oxidative effects of SAMC-NS,and the exploration of relevant signaling pathways.A mice ALI model was established by intratracheal instillation of LPS.BalB/c mice were treated with SAMC-NS(10,30 and 60 mg/kg)or positive control N-acetylcysteine(NAC,500 mg/kg)by gavage after intratracheal instillation of LPS for 30 min.These mice were sacrificed 24 h after LPS administration,and the bronchoalveolar lavage fluid(BALF)as well as lung tissues were collected for SAMC-NS pharmacodynamic evaluation and molecular mechanism study.Our results indicate that the treatment with SAMC-NS not only ameliorated the histological changes but also decreased LPS-triggered lung edema.Inflammatory cell counting in BALF and ELISA assay results proved that SAMC-NS displayed an anti-inflammatory effect through reducing inflammatory cells infiltration and inhibiting pro-inflammatory cytokines production including tumor necrosis factor alpha(TNF-?),interleukin-1?(IL-1?),interleukin-6(IL-6).Moreover,the measurement results of oxidative stress markers in lung tissue homogenates indicated that SAMC-NS attenuated oxidative stress evoked by LPS via diminishing malondialdehyde(MDA)formation and reversing glutathione(GSH)and superoxide dismutase(SOD)depletion.Western blot and immunohistochemistry were used to explore the mechanism of SAMC-NS in the treatment of ALI.SAMC-NS regulated inflammation by suppressing the activation of nuclear factor-kappaB(NF-?B)signaling pathway,which in turn inhibited the expression of the pro-inflammatory mediator iNOS and COX2 proteins.Meanwhile,SAMC-NS reversed oxidative stress by activating the Keap1/Nrf2 signaling pathway to up-regulate the expression of endogenous antioxidant/detoxifying proteins,including HO-1 and NQO1 proteins.In summary,our results demonstrate that SAMC-NS effectively attenuated LPS-induced ALI which was largely dependent upon the inhibition of inflammation and oxidative stress via NF-?B and Keap1/Nrf2 signaling pathways.In conclusion,this project first prepared SAMC nanouspension by microfluidization method to improve the solubility of SAMC and characterized them.In addition,in order to further improve the stability of SAMC-NS,freeze-drying technology was employed to prepare lyophilized SAMC-NS and its quality was evaluated.The results show that SAMC-NS significantly improved solubility and dissolution rate of SAMC.Then,the therapeutic effect of SAMC on acute lung injury was evaluated at both cellular and animal levels and the mechanism was explored.Our results show that SAMC had a good therapeutic effect on LPS-induced ALI and the mechanism was largely dependent upon the inhibition of inflammation and oxidative stress via NF-?B and Nrf2 signaling pathways.