Ligustilide protects PC12 cells from oxygen-glucose deprivation/reoxygenation-induced apoptosis via the LKB1-AMPK-mTOR signaling pathway

Autophagy has been shown to have a protective effect against brain damage.Ligustilide(LIG)is a bioactive substance isolated from Ligusticum chuanxiong,a traditional Chinese medicine.LIG has a neuroprotective effect;however,it is unclear whether this neuroprotective effect involves autophagy.In this study,PC12 cells were treated with 1×10^-5–1×10^-9 M LIG for 0,3,12 or 24 hours,and cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)assay.Treatment with 1×10^-6 M LIG for 3 hours had the greatest effect on cell proliferation,and was therefore used for subsequent experiments.PC12 cells were pre-treated with 1×10^-6 M LIG for 3 hours,cultured in 95%N2/5%CO2 in Dulbecco’s modified Eagle’s medium without glucose or serum for 4 hours,and then cultured normally for 16 hours,to simulate oxygen-glucose deprivation/reoxygenation(OGD/R).Cell proliferation was assessed with the MTS assay.Apoptosis was detected by flow cytometry.The expression levels of apoptosis-related proteins,Bcl-2 and Bax,autophagy-related proteins,Beclin 1 and microtubule-associated protein l light chain 3B(LC3-II),and liver kinase B1(LKB1)-5′-adenosine monophosphate-activated protein kinase(AMPK)-mammalian target of rapamycin(mTOR)signaling pathway-related proteins were assessed by western blot assay.Immunofluorescence staining was used to detect LC3-II expression.Autophagosome formation was observed by electron microscopy.LIG significantly decreased apoptosis,increased Bcl-2,Beclin 1 and LC3-II expression,decreased Bax expression,increased LC3-II immunoreactivity and the number of autophagosomes,and activated the LKB1-AMPK-mTOR signaling pathway in PC12 cells exposed to OGD/R.The addition of the autophagy inhibitor 3-methyladenine or dorsomorphin before OGD/R attenuated the activation of the LKB1-AMPK-mTOR signaling pathway in cells treated with LIG.Taken together,our findings show that LIG promotes autophagy and protects PC12 cells from apoptosis induced by OGD/R via the LKB1-AMPK-mTOR signaling pathway.

Chemical proteomics reveals ligustilide targets SMAD3,inhibiting collagen synthesis in aortic endothelial cells

Atherosclerosis is a persistent inflammatory state,while vascular endothelial fibrosis is one of the primary causes of atherosclerosis development.Although ligustilide(Lig) was shown to exert obvious antiatherogenic effects in previous studies,its precise mechanism has not been deeply discussed.In this paper,we designed a Lig-derived photoaffinity labelling(PAL) probe to identify potential therapeutic targets of Lig via chemical proteomics approach.Mothers against decapentaplegic homologue 3(SMAD3),a signal transmitter of transforming growth factor-β(TGF-β) which promotes the development of vascular fibrosis,was identified as a potential target of Lig.Lig suppressed the phosphorylation and nuclear translocation of SMAD3 by blocking the interaction between SMAD3 and TGF-β receptor 1,thereby inhibiting the collagen synthesis process.Hence,developing a novel SMAD3 inhibitor may present a promising therapeutic option for preventing vascular fibrosis.

Advances in Studies on Pharmacological Functions of Ligustilide and their Mechanisms

The article reviewed the research progress of ligustilide in recent years and elaborated its pharmacological functions and mechanisms in detail, especially in ischemic brain injury. Its mechanism includes reducing cerebral infarct volumes and improving neurobehavioral deficits, anti-oxidant and anti-apoptosis, antithrombotic activity, calcium channel blockers function, and effect on erythropoietin. Other pharmacological effects of ligustilide including inhibiting vascular smooth muscle cell proliferation, anti-inflammatory and analgesic effects, effects on LPS-induced endotoxic shock, inhibiting constriction effect, suppression of the central nervous system, and ameliorating the memory impairment induced by scopolamine and so on, are also introduced. Ligustilide has potential pharmacological value, which provides a reference for its further research and development.

Study on Rheology and Release Kinetics of Chuanqi Ophthalmic Microemulsion in situ Gel

[Objectives] To study the rheology and release kinetics of Chuanqi ophthalmic microemulsion in situ gel,so as to provide references for its future study and development. [Methods]The fluid properties and linear viscoelastic regions of this preparation were investigated by MCR 102 rheometer. The release kinetics of Chuanqi ophthalmic microemulsion in situ gel was evaluated by modified Franz diffusion cell method,the ligustrazine and ligustilide were selected as the indictors,and semi-permeable membrane was used as a barrier,sampling time point was 0. 5,1,2,4,6,and 8 h respectively. [Results]Chuanqi ophthalmic microemulsion in situ gel was a pseudoplastic fluid and it had a linear viscoelastic region. Taking the shear stress as the indicator,the linear viscoelastic region was 0-302. 74 Pa; taking the strain as the indicator,the linear viscoelastic region was 0-7. 45%. At the critical point,the storage modulus( G') = the loss modulus( G″) =2 976. 60 Pa,critical shear stress was 302. 74 Pa and critical strain was 7. 45%. The average cumulative release of ligustrazine of 6 samples within 8 h was 33. 71 μg,the average cumulative release rate reached 90. 08%,and the release kinetics followed Higuchi equation. The average cumulative release of ligustilide of 6 samples within 8 h was 68. 46 μg,the average cumulative release rate reached 84. 32%,and the release kinetics followed the zero-order kinetics equation. [Conclusions] Chuanqi ophthalmic microemulsion in situ gel has excellent viscoelasticity and its strain is reversible in a certain range. The release kinetics of ligustrazine is the result from synergistic effect of its physicochemical properties and matrix skeleton,while the release kinetics of ligustilide is mainly affected by its physicochemical properties.

Simultaneous Quantitative Analysis of Five Components in Angelica sinensis and Angelica acutiloba Acclimatized Growing in Vietnam by High-Performance Liquid Chromatography with Photodiode Array Detector

Background:Chinese Danggui(Angelica sinensis)and Japanese Danggui(Angelica acutiloba)are evaluated as the same in using and critical of quality control in Vietnamese Pharmacopoeia.In Vietnam,Japanese Danggui were acclimatized and cultivated in several regions in recent years.Despite the huge climatic difference between Vietnam and Japan,Japanese Danggui grows very well in Vietnam.However,there are no studies to assess the overall quality of this medicinal herbs.Aims and Objectives:The aim of this study is to develop a method to identify and assay simultaneously 5 component compounds of these crude herbs:chlorogenic acid,ferulic acid,scopoletin,xanthotoxin and ligustilide by high-performance liquid chromatography with photodiode array detector(HPLC-PDA).Materials and Methods:The procedure was optimized by using column Phenomenex Gemini 5μm PR C18,15 x 4,6 mm with the detection wavelength set at 321 nm for detector PDA and mobile phase was composed of(A)ACN and(B)aqueous solution 1%of acid acetic using a gradient elution.Analytes were performed at 30°C with a flow rate of 1.0 mL/min.Results:The developped method were fully validated for linearity,accuracy,recovery and met the validation requirements,proved practical applicability for the quality control of these herbs and related products.Conclusion:The method was successfully applied to the quantification of five markers simultaneously from collected samples.