Ligustroflavone reduces necroptosis in rat brain after ischemic stroke through targeting RIPK1/RIPK3/MLKL

OBJECTIVE To explore the effect of ligustroflavone on ischemic brain injury in stroke rat and the under⁃lying mechanisms.METHODS A rat model of ischemic stroke was established by middle cerebral artery occlusion(MCAO).Administration of ligustroflavone(10,30,60 mg·kg-1,ig)15 min before ischemia,after which neurological deficit score and infarct volume were detected by longa score and TTC stain.The cell viability and necrosis rate of hypoxia-cultured PC12 cells(O2/N2/CO2,1:94:5,8 h)were evaluated by MTS and LDH release rate.Flow cytometry further verified the mortality rate of PC12 cells.Necroptosis-associated proteins(RIPK1,RIPK3 and MLKL/p-MLKL)were detected by Western blotting.The interaction between RIPK3 and RIPK1 or MLKL were confirmed by immunoprecipitation.Operating Environ⁃ment(MOE)program demonstrated the possible combination of ligustroflavone with RIPK1,RIPK3 and MLKL.RESULTS Ischemic injury(increase in neurological deficit score and infarct volume)and upregulation of necroptosis-associated proteins were showed in rat MCAO model.Administration of ligustroflavone(30 mg·kg^-1,ig)evidently improved neurological func⁃tion,reduced infarct volume,and decreased the levels of necroptosis-associated proteins except the RIPK1.Consistently,hypoxia-cultured PC12 cells caused cellular injury(LDH release and necroposis)concomitant with up-regulation of necroptosis-associated proteins,and these phenomena were blocked in the presence of ligustroflavone(25μmol·L^-1)except the elevated RIPK1 levels.Using the Molecular Operating Environment(MOE)program,we identified RIPK1,RIPK3,and MLKL as potential targets of ligustroflavone.Further studies showed that the interaction between RIPK3 and RIPK1 or MLKL was significantly enhanced,which was blocked in the presence of ligustroflavone.CONCLUSION Ligus⁃troflavone protects rat brain from ischemic injury,and its beneficial effect is related to the prevention of necroptosis through a mechanism involving targeting RIPK1,RIPK3,and/or MLKL.

Ligustroflavone ameliorates CCl_(4)-induced liver fibrosis through down-regulating the TGF-β/Smad signaling pathway

Liver fibrosis is a pathological process characterized by excess deposition of extracellular matrix(ECM)that are mainly derived from activated hepatic stellate cells.Previous studies suggested that ligustroflavone(LF)was an ingredient of Ligustrum lucidum Ait.with activities of anti-inflammation and anti-oxidation.In this study,we investigated whether LF had any effect on liver fibrosis.In our study,we established a mouse model of carbon tetrachloride(CCl4)-induced liver fibrosis and used TGF-β1-stimulated human hepatic stellate cell line(LX-2)to explore the effect of LF and associated underlying mechanism.LF was used in vivo with low dose(L-LF,5 mg·kg^(-1),i.p.,3 times each week)and high dose(H-LF,20 mg·kg^(-1),i.p.,3 times each week)and in vitro(25μmol·L^(-1)).Histopathological and biochemical assays investigations showed that LF delayed the formation of liver fibrosis;decreased AST,ALT activities and increased Alb activity in serum;decreased MDA level,Hyp content and increased GSH-Px concentration,SOD activity in liver tissues.Moreover,immunohistochemical,immunofluorescent and Western blot results showed that LF reduced the expressions of hepatic stellate cells specific marker proteins,including collagen I andα-SMA in vivo and in vitro.In addition,LF markedly suppressed TGF-β1-upregulated protein expressions of TβR I,TβR II,P-Smad2,P-Smad3 and Smad4 in LX-2 cells.Taken together,these findings demonstrated LF could decrease histopathological lesions,ameliorate oxidative injury,attenuate CCl4-induced liver fibrosis,which may be associated with down-regulating the TGF-β/Smad signaling pathway.

基于UPLC-Q/TOF-MS的中药水蜜丸制剂二至丸多成分稳定性研究

目的在遵循人用药品注册技术要求国际协调会(International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use,ICH)制剂稳定性指导原则的指导下,在采用超高效液相色谱-飞行时间质谱(UPLC-Q/TOF-MS)联用技术对中药传统水蜜丸制剂二至丸进行多成分定性定量研究的基础上,进行二至丸长期稳定性和加速稳定性研究,并考察了制剂影响因素对制剂稳定性的影响。方法采用Acquity UPLC BEH C18色谱柱(100 mm×2.1mm,1.7μm),流动相为0.1%甲酸水溶液-乙腈,梯度洗脱,体积流量0.2 m L/min,采用电喷雾电离源(ESI),正、负离子模式扫描。在遵循ICH制剂稳定性指导原则的指导下,进行了18个月的长期稳定性试验[温度(25±2)℃,相对湿度(RH)(60±5)%],加速稳定性试验[温度(40±2)℃,RH(75±5)%]和制剂影响因素试验(包装材料类型和密封条件)。结果采用以上方法共鉴定出二至丸中20个化学成分(红景天苷、蟛蜞菊内酯、10-羟基木犀榄苷二甲酯、木犀榄苷-11甲酯、马钱苷酸、松果菊苷、女贞苦苷、橄榄苦苷酸、毛蕊花苷/异毛蕊花苷、女贞苷、特女贞苷、ligustroflavone、特女贞苷异构体、阿富汗丁香苷F、异女贞苷、女贞酸、橄榄苦苷、女贞苷-G13、青藤碱、3-O-顺式-对-香豆酰委陵菜酸),并对其进行定量及归一化半定量研究。在长期稳定性研究中,20个成分均保持稳定;然而在加速稳定性试验条件下,11种成分(10-羟基木犀榄苷二甲酯、松果菊苷、女贞苦苷、橄榄苦苷酸、毛蕊花苷、女贞苷、特女贞苷、ligustroflavone、女贞酸、女贞苷-G13、青藤碱)的量明显下降。制剂影响因素试验结果显示采用钠钙玻璃包装材料替代现有市售聚酯包装,在密封或敞口条件下,制剂中各成分稳定性均显著提高。在采用相同包材时密封条件对制剂稳定性也存在显著性影响。结论通过较为系统的中药传统口服制剂类型水蜜丸的制剂稳定性研究,为中药制剂质量控制水平和临床用药安全提供技术支持。