Z-Ligustilide Induces c-Myc-Dependent Apoptosis via Activation of ER-Stress Signaling in Hypoxic Oral Cancer Cells.

Z-ligustilide (or ligustilide) is found in Angelica sinensis (Oliv.) Diels and may exert potential benefits in cancer treatment. Previous research has reported that ligustilide has anti-cancer effects on several types of cancer cells. However, studies of ligustilide on oral cancer cells have not been reported, especially under hypoxic conditions. This study focuses on the molecular mechanism of ligustilide-induced apoptosis in hypoxic oral cancer cells. We found that in hypoxic TW2.6 cells, ligustilide inhibited cell migration and induced caspase-dependent apoptosis. Accumulation of c-Myc accompanied by BH3-only members suggests that ligustilide may induce c-Myc-dependent apoptosis. In addition, we reported that ligustilide has an effect on ER-stress signaling. By using inhibitors of c-Myc, IRE1α, and ER-stress inhibitors, we found that cell morphologies or cell viability were rescued to some degree. Moreover, ligustilide is able to increase the expression of γ-H2AX and enhance the occurrence of DNA damage in oral cancer cells after radiation treatment. This result suggests that ligustilide has potential as a radiation sensitizer. Altogether, we propose that ligustilide may induce c-Myc-dependent apoptosis via ER-stress signaling in hypoxic oral cancer cells.

Ligustilide alleviates podocyte injury via suppressing the SIRT1/NF-κB signaling pathways in rats with diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is one of the common chronic microvascular complications of diabetes, and podocyte injury and dysfunction are strictly related to the pathogenesis of DN. Studies have shown that ligustilide (LIG) has anti-inflammatory, antioxidant, and anti-apoptotic activities. This study was designed to investigate the therapeutic effect of LIG in DN rats and their mechanisms. METHODS: DN rat models (n=10) were induced by streptozotocin (STZ) combined with a high-fat diet. Rats in the LIG group were intragastrically administered with LIG daily for eight weeks, and animals in the positive control group were treated with Losartan potassium. The body weight and blood glucose were checked weekly during the treatment. The pathological changes of kidney tissue were observed with hematoxylin and eosin (HE) staining. Blood lipid profiles and renal function-related markers, including total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), blood urea nitrogen (BUN), and serum creatinine (Scr) were monitored using a biochemical analyzer. The protein expression of nephrin was determined by immunohistochemistry and Western blotting. Finally, Western blot was used to determine the protein expression of Sirtuin 1 (SIRT1) and nuclear factor-kappa B (NF-κB). RESULTS: Compared with the healthy control group, rats in the DN group have slower weight gain, increased blood sugar level, renal lesions, and impaired renal function, along with decreased nephrin expression, abnormally activated NF-κB, and inhibited SIRT1 protein expression. All the above conditions were improved after intervention with either losartan potassium or LIG. CONCLUSIONS: LIG attenuates podocyte injury by regulating the SIRT1/NF-κB signaling pathway and thereby exerts its protective effect on renal function in DN rats.

Ligustilide modulates oxidative stress, apoptosis, and immunity to avoid pathological damages in bleomycin induced pulmonary fibrosis rats via inactivating TLR4/MyD88/NF-KB P65.

BACKGROUND: Pulmonary fibrosis (PF) is a fatal disease with increasing incidence. Ligustilide (LIG) has been shown to inhibit oxidative stress, apoptosis, and inflammation. Here we investigated the possible effect of LIG on bleomycin-induced PF in Sprague-Dawley rats. METHODS: PF rats were set up through a single endotracheal injection of bleomycin (5 mg/kg). Then rats were treated with 20, 40, and 80 mg/kg LIG for four weeks, and the effects were estimated. RESULTS: Overall, LIG significantly improved ventilation and reduced hyperplasia, and treatment of LIG reduced fibrosis as indicated by Masson staining and reduced expression of transforming growth factor-beta (TGF-ß), Fibronectin, and alpha-smooth muscle actin (α-SMA). Oxidative stress was induced with bleomycin while inhibited with LIG, as showed with rebalanced serum lactate dehydrogenase (LDH), and tissue superoxide dismutase (SOD), glutathione peroxidase (GSH) and malondialdehyde (MDA). Apoptosis was further inhibited with LIG, as shown with Terminal dUTP nick-end labeling (TUNEL) staining and expression of Caspase-3, Caspase-9, Bax, and Bcl-2. Th1/Th2 balance was also rebuilt as evaluated with CD4 and IFNγ/IL-4 labeled flow cytometry of peripheral blood mononuclear cells (PBMCs) and expression of inducible nitric oxide synthase (iNOS) and IL-10 in the serum and lung. Protein expression of Toll-like receptor 4 (TLR4), HSP60-TLR4-myeloid differentiation factor 88 (Myd88) and nuclear factor-kappa B (NF-κB) p-P65/P65 was significantly reduced with LIG treatment. All the effects of LIG exhibited in a dose-dependent way. CONCLUSIONS: LIG improved bleomycin-induced PF with improved ventilation, reduced fibroblast, reduced oxidative stress and apoptosis, and rebalanced Th1/Th2 immunity, through TLR4/MyD88/NF-κB P65 signaling.

Neuroprotective Effect of Ligustilide through Induction of α-Secretase Processing of Both APP and Klotho in a Mouse Model of Alzheimer's Disease.

Emerging evidence suggests that alpha-processing single transmembrane proteins, amyloid precursor protein (APP) and anti-aging protein Klotho, are likely to be involved in the progression of Alzheimer's disease (AD). The natural phthalide Ligustilide (LIG) has been demonstrated to protect against aging- and amyloid-ß (Aß)-induced brain dysfunction in animal models. The present study is to investigate the effects of LIG on cognitive deficits and metabolism of both APP and Klotho and its underlying mechanism in AD double-transgenic (APP/PS1) mice and cultured human cells. Our results show that treatment with LIG significantly ameliorated memory impairment and Aß levels and plaques burden. Specifically, LIG might act as a potent enhancer of α-secretase, disintegrin, and metalloprotease 10 (ADAM10), leading to upregulation of alpha-processing of both APP and Klotho and subsequent increases in the levels of both soluble APP fragment (sAPPα) and soluble Klotho (sKL) with inhibition of IGF-1/Akt/mTOR signaling in AD mice and cultured cells. Moreover, the specific ADAM10 inhibitor (G1254023X) effectively reversed LIG-induced alpha-processing of both APP and Klotho in vitro, while Klotho gene knockdown by small interfering RNA significantly blunted LIG-mediated inhibition of IGF-1/Akt/mTOR signaling in vitro. Taken together with the reported neuroprotective effects of both sAPPα and sKL as well as autophagy induction by Akt/mTOR pathway inhibition, our findings suggest that neuroprotection of LIG against AD is associated with induction alpha-processing of APP and Klotho and potential Aß clearance. Whether LIG might induce Aß autophagic clearance and the underlying mechanisms need to be further studied.

(Z)-ligustilide increases ferroportin1 expression and ferritin content in ischemic SH-SY5Y cells.

The mechanisms involved in the antioxidant and anti-apoptotic properties of (Z)-ligustilide (LIG) are not fully elucidated. Based on the accumulated data, we hypothesized that LIG might be able to reduce ischemia/reperfusion-induced increase in brain iron by regulating expression of iron transport proteins. We therefore investigated the effects of LIG on iron uptake protein transferrin receptor 1, iron exporter protein ferroportin 1, iron storage protein ferritin light chain and also hypoxia inducible factor-1 alpha (HIF-1 alpha) in oxygen-glucose deprivation/reoxygenation (OGD/R)-treated SH-SY5Y cells, using Western blot analysis. We demonstrated that LIG completely reversed the OGD/R-induced reduction of ferroportin 1, increased ferritin light chain content, and also suppressed the OGD-induced increase in HIF-1 alpha in SH-SY5Y cells. These findings imply that LIG might reduce the OGD/R-induced increase in brain iron by promoting cell iron release and iron corporation into ferritin, and also by inhibiting the HIF-1 alpha-induced increase in transferrin-bound iron uptake and iron accumulation in the brain, consequently attenuating iron-mediated free radical formation, oxidative stress and apoptosis.