Z-Ligustilide purified from Ligusticum chuanxiong Hort. promotes motor ability in Caenorhabditis elegans by alleviating oxidative stress via the intestinal microvilli

ETHNOPHARMACOLOGICAL RELEVANCE: Based on years of clinical practice in China, Ligusticum chuanxiong Hort. (LCX) is known for promoting blood circulation, dispelling wind, and relieving pain. It is traditionally prescribed to relieve fatigue, muscle soreness, and weakness resulting from physical overexertion, reflecting its role in restoring vitality and alleviating movement-related discomfort. In modern pharmacology, inhibited oxidative stress leads to enhanced motor ability, and LCX possesses antioxidant, anti-inflammatory, and vasodilatory properties that may contribute to its traditional efficacy. However, the precise molecular mechanisms underlying these effects remain unclear. AIM OF THE STUDY: In this study, we extracted and identified the main active components of LCX using HPLC-MS/MS and response surface methodology, and analyzed the therapeutic effects and mechanisms of Z-ligustilide (LIG) on motor ability and oxidative stress imbalance using the Caenorhabditis elegans model. These actions were aimed at obtaining scientific evidence for the traditional use of LCX in fatigue-related disorders. MATERIALS AND METHODS: The components of LCX volatile oil were identified using liquid chromatography-triple quadrupole mass spectrometry, and the extraction process was optimized using Box-Behnken design-response surface methodology, which considered three variables (petroleum ether to ethanol ratio, extraction time, and solid-to-liquid ratio) to isolate high-purity LIG from the oil. Subsequently, LIG was administered to C. elegans, and its effects on physiological properties, such as mobility, lifespan, growth, reproductive ability, defecation, and feeding frequency, were evaluated. To further explore the regulatory mechanisms of LIG, we used an oxidative stress/aging-related genetic mutant model of C. elegans and combined western blotting and fluorescence imaging to evaluate the anti-inflammatory and antioxidative effects of LIG on the organism. RESULTS: The optimal conditions for extracting LIG were petroleum ether-to-ethanol ratio of 6:4, extraction time of 3 h, and solid-to-liquid ratio of 250 mg/mL, which resulted in a purity of 98.73 % after column chromatography. Compared with traditional methods, this optimized extraction process avoids the structural isomerization or degradation of LIG caused by high temperatures during steam distillation while also reducing the excessive impurities and low extraction efficiency associated with single-solvent extraction. The activity results indicated that LIG could improve motor ability in C. elegans caused by peroxidation and also enhance the phosphorylation level of PMK-1. Inhibition of either PMK-1 or GST-4 blocked the antioxidant activity of LIG. Furthermore, the loss of function in CDH-8 in cdh-8(cas1109) or cdh-8(ok628) mutants lacking intestinal microvilli completely inhibited the effect of LIG on the improvement of movement and oxidative stress response in C. elegans. These findings indicated that LIG was primarily detected in the intestine; signals were transmitted throughout the body via the intestine, which then regulate the CDH-8/NSY-1/SEK-1/PMK-1/SKN-1/GST-4 signaling axis, thus alleviating oxidative stress and ultimately promoting motor movement in organisms. CONCLUSION: Our research showed that LIG, a major component of LCX volatile oil, is sensed by microvilli, which triggers the PMK-1/p38 pathway and activates SKN-1, thereby stimulating the expression of GST-4, an oxidative stress effector, and regulating ROS production and improving mobility. Our findings provide new insights into therapeutic strategies involving LIG for treating diseases related to oxidative stress.