Identification of antidepressant constituents from Xiangfu-chuanxiong herbal medicine pair via spectrum-effect relationship analyses, molecular docking and corticosterone-induced PC12 cells.

Herbal medicine pair, composed of two single herbs, is a relatively fixed minimum prescription unit in the traditional Chinese medicine's formula and has special significance in clinic. The combination of Xiangfu (the rhizoma of Cyperus rotundus L, XF) and Chuanxiong (the rhizoma of Ligusticum chuanxiong Hort, CX) has been recoded as an herbal medicine pair XF-CX in the Yuan Dynasty (1347 CE) of China and widely used in traditional Chinese medicine formula, including Chaihu Shugan San, which has been clinically used for treatment of depression. However, the optimal ratio of the XF-CX herbal medicine pair and its antidepressant constituents are still unclear. Herein, the antidepressive-like effects of XF-CX herbal medicine pairs with different ratios of XF and CX (2:1, 1:1, 1:2) were evaluated using behavioral despair animal models in mice, and then its potential antidepressant constituents were recognized by spectrum-effect relationship analyses. Finally, the potential antidepressant constituents of the XF-CX herbal medicine pair were validated by molecular docking with glucocorticoid receptor and corticosterone (CORT)-induced PC12 cell injury model. The results indicated that different ratios of XF-CX pairs had antidepressive-like effects, and the XF-CX (2:1) exhibited a more significant effect. Thirty-two potential antidepressant constituents in the XF-CX herbal medicine pair were screened out from the spectrum-effect relationship combined molecular docking analyses. Among them, senkyunolide A, cyperotundone, Z-ligustilide, and levistilide A were validated to have protective effects against CORT-induced injury in PC12 cells. Our findings not only obtained the optimal ratio of XF-CX in the herbal medicine pair for the treatment of depression but also its potential antidepressant constituents, which will benefit in elucidating the mechanism of action and promoting the application of the herbal medicine pair in the clinic.

[Research progress on mechanisms of ligustilide in treatment of nervous system diseases].

Ligustilide is the main active component of the volatile oil from Angelica sinensis and Ligusticum chuanxiong in the Umbelliferae family. It is a phthalein compound with anti-inflammatory, analgesic, antioxidant, anti-tumor, anti-atherosclerosis, neuroprotective, and other pharmacological effects. It can improve the permeability of the blood-brain barrier and has important potential in the treatment of neurodegenerative diseases and other nervous system diseases, such as Alzheimer's disease, ischemic stroke, Parkinson's disease, vascular dementia, and depression. Therefore, the mechanism of ligustilide in the treatment of nervous system diseases was summarized to provide a reference for drug development and clinical application.

Ligustilide covalently binds to Cys703 in the pre-S1 helix of TRPA1, blocking the opening of channel and relieving pain in rats with acute soft tissue injury.

ETHNOPHARMACOLOGICAL RELEVANCE: The natural anodyne Ligustilide (Lig), derived from Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort., has been traditionally employed for its analgesic properties in the treatment of dysmenorrhea and migraine, and rheumatoid arthritis pain. Despite the existing reports on the correlation between TRP channels and the analgesic effects of Lig, a comprehensive understanding of their underlying mechanisms of action remains elusive. AIM OF THE STUDY: The objective of this study is to elucidate the mechanism of action of Lig on the analgesic target TRPA1 channel. METHODS: The therapeutic effect of Lig was evaluated in a rat acute soft tissue injury model. The analgesic target was identified through competitive inhibition of TRP channel agonists at the animal level, followed by Fluo-4/Ca2+ imaging on live cells overexpressing TRP proteins. The potential target was verified through in-gel imaging, colocalization using a Lig-derived molecular probe, and a drug affinity response target stability assay. The binding site of Lig was identified through protein spectrometry and further analyzed using molecular docking, site-specific mutation, and multidisciplinary approaches. RESULTS: The administration of Lig effectively ameliorated pain and attenuated oxidative stress and inflammatory responses in rats with soft tissue injuries. Moreover, the analgesic effects of Lig were specifically attributed to TRPA1. Mechanistic studies have revealed that Lig directly activates TRPA1 by interacting with the linker domain in the pre-S1 region of TRPA1. Through metabolic transformation, 6,7-epoxyligustilide (EM-Lig) forms a covalent bond with Cys703 of TRPA1 at high concentrations and prolonged exposure time. This irreversible binding prevents endogenous electrophilic products from entering the cysteine active center of ligand-binding pocket of TRPA1, thereby inhibiting Ca2+ influx through the channel opening and ultimately relieving pain. CONCLUSIONS: Lig selectively modulates the TRPA1 channel in a bimodal manner via non-electrophilic/electrophilic metabolic conversion. The epoxidized metabolic intermediate EM-Lig exerts analgesic effects by irreversibly inhibiting the activation of TRPA1 on sensory neurons. These findings not only highlight the analgesic mechanism of Lig but also offer a novel nucleophilic attack site for the development of TRPA1 antagonists in the pre-S1 region.

Ligustilide inhibits Purkinje cell ferritinophagy via the ULK1/NCOA4 pathway to attenuate valproic acid-induced autistic features.

BACKGROUND: Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder in which social impairment is the core symptom. Presently, there are no definitive medications to cure core symptoms of ASD, and most therapeutic strategies ameliorate ASD symptoms. Treatments with proven efficacy in autism are imminent. Ligustilide (LIG), an herbal monomer extracted from Angelica Sinensis and Chuanxiong, is mainly distributed in the cerebellum and widely used in treating neurological disorders. However, there are no studies on its effect on autistic-like phenotypes and its mechanism of action. PURPOSE: Investigate the efficacy and mechanism of LIG in treating ASD using two Valproic acid(VPA)-exposed and BTBR T + Itpr3tf/J (BTBR) mouse models of autism. METHODS: VPA-exposed mice and BTBR mice were given LIG for treatment, and its effect on autistic-like phenotype was detected by behavioral experiments, which included a three-chamber social test. Subsequently, RNA-Sequence(RNA-Seq) of the cerebellum was performed to observe the biological changes to search target pathways. The autophagy and ferroptosis pathways screened were verified by WB(Western Blot) assay, and the cerebellum was stained by immunofluorescence and examined by electron microscopy. To further explore the therapeutic mechanism, ULK1 agonist BL-918 was used to block the therapeutic effect of LIG to verify its target effect. RESULTS: Our work demonstrates that LIG administration from P12-P14 improved autism-related behaviors and motor dysfunction in VPA-exposed mice. Similarly, BTBR mice showed the same improvement. RNA-Seq data identified ULK1 as the target of LIG in regulating ferritinophagy in the cerebellum of VPA-exposed mice, as evidenced by activated autophagy, increased ferritin degradation, iron overload, and lipid peroxidation. We found that VPA exposure-induced ferritinophagy occurred in the Purkinje cells, with enhanced NCOA4 and Lc3B expressions. Notably, the therapeutic effect of LIG disappeared when ULK1 was activated. CONCLUSION: LIG treatment inhibits ferritinophagy in Purkinje cells via the ULK1/NCOA4-dependent pathway. Our study reveals for the first time that LIG treatment ameliorates autism symptoms in VPA-exposed mice by reducing aberrant Purkinje ferritinophagy. At the same time, our study complements the pathogenic mechanisms of autism and introduces new possibilities for its therapeutic options.

Dual role of Nrf2/HO-1 pathway in Z-ligustilide-induced ferroptosis against AML cells.

BACKGROUND: The scarcity of drugs targeting AML cells poses a significant challenge in AML management. Z-Ligustilide (Z-LIG), a phthalide compound, shows promising pharmacological potential as a candidate for AML therapy. However, its precise selective mechanism remains unclear. PURPOSE: In order to assess the selective inducement effects of Z-LIG on ferroptosis in AML cells and explore the possible involvement of the Nrf2/HO-1 pathway in the regulation of ferroptosis. METHODS: Through in vitro cell proliferation and in vivo tumor growth tests, the evaluation of Z-LIG's anticancer activity was conducted. Ferroptosis was determined by the measurement of ROS and lipid peroxide levels using flow cytometry, as well as the observation of mitochondrial morphology. To analyze the iron-related factors, western blot analysis was employed. The up-regulation of the Nrf2/HO-1 axis was confirmed through various experimental techniques, including CRISPR/Cas9 gene knockout, fluorescent probe staining, and flow cytometry. The efficacy of Z-LIG in inducing ferroptosis was further validated in a xenograft nude mouse model. RESULTS: Our study revealed that Z-LIG specifically triggered lipid peroxidation-driven cell death in AML cells. Z-LIG downregulated the total protein and nuclear entrance levels of IRP2, resulting in upregulation of FTH1 and downregulation of TFR1. Z-LIG significantly increased the susceptibility to ferroptosis by upregulating ACSL4 levels and simultaneously suppressing the activity of GPX4. Notably, the Nrf2/HO-1 pathway displayed a twofold impact in the ferroptosis induced by Z-LIG. Mild activation suppressed ferroptosis, while excessive activation promoted it, mainly driven by ROS-induced labile iron pool (LIP) accumulation in AML cells, which was not observed in normal human cells. Additionally, Nrf2 knockout and HO-1 knockdown reversed iron imbalance and mitochondrial damage induced by Z-LIG in HL-60 cells. Z-LIG effectively inhibited the growth of AML xenografts in mice, and Nrf2 knockout partially weakened its antitumor effect by inhibiting ferroptosis. CONCLUSION: Our study presents biological proof indicating that the selective initiation of ferroptosis in leukemia cells is credited to the excessive activation of the Nrf2/HO-1 pathway triggered by Z-LIG.

Ligustilide, A Novel Senolytic Compound Isolated from the Roots of Angelica Acutiloba.

Senescent cells accumulate with age and contribute to age-related diseases and organ dysfunctions. Early evidence suggests that removal of senescent cells using senolytic drugs improves the aging phenotype in mice and may improve the health of individuals with chronic diseases. Signs of skin aging, including wrinkles, and sagging, occur largely due to the accumulation of senescent fibroblasts within the dermis; However, there is currently no skin treatment that eliminates senescent cells. In this study, human fibroblasts subjected to replicative aging and ionizing radiation exposure are used to screen plant extracts for potential senescent cell-destructive and/or senescent cell-forming activities. Angelica acutiloba-a traditional Chinese herbal medicine-selectively kills senescent cells without affecting the proliferating cells. Among the major components of this herb, ligustilide shows promising senescent cell-destructive properties, and selectively eliminates senescent cells by inducing an apoptosis. Moreover, ligustilide markedly inhibits senescence-associated secretory phenotypes. Administration of ligustilide to mouse skin eliminates senescent cells and increases dermal collagen density and subcutaneous adipose tissue content; it selectively promotes death of senescent cells without affecting non-senescent cells. These results provide evidence that a natural compound-ligustilide-may exhibit therapeutic effects on the skin aging phenotype by specifically inducing apoptosis in senescent cells.

Ligustilide enhances pregnancy outcomes via improvement of endometrial receptivity and promotion of endometrial angiogenesis in rats.

Ligustilide (LIG) is the main active ingredient of Angelica sinensis (Oliv.) Diels, which could promote focal angiogenesis to exert neuroprotection. However, there was no report that verified the exact effects of LIG on endometrial angiogenesis and the pregnancy outcomes. To explore the effects of LIG on low endometrial receptivity (LER) and angiogenesis, pregnancy rats were assigned into Control (saline treatment), LER (hydroxyurea-adrenaline treatment), LIG 20 mg/kg and LIG 40 mg/kg groups. Hematoxylin and eosin (H&E) staining was performed to evaluate endometrial morphology. Quantitative real-time PCR, immunofluorescence staining, western blot and immunohistochemistry staining were employed to assess the expression of endometrial receptivity factors and angiogenesis-related gene/protein, respectively. RNA sequencing was used to analyze the effects of LIG on LER caused by Kidney deficiency and blood stasis. We found that endometrial thickness and the implanted embryo number were substantially reduced in the hydroxyurea-adrenaline-treated pregnancy rats. At the same time, the gene and protein expressions of ERα, LIF, VEGFA and CD31 in the endometrium were markedly reduced, while the expressions of MUC1, E-cadherin were increased in the LER group. Administration of LIG raised the endometrial thickness and implanted embryos, as well as reversed the expressions of these factors. Collectively, our findings revealed that LIG could facilitate embryo implantation via recovery of the endometrium receptivity and promotion of endometrial angiogenesis.

Ligustilide prevents thymic immune senescence by regulating Thymosin ß15-dependent spatial distribution of thymic epithelial cells.

BACKGROUND: Thymus is the most crucial organ connecting immunity and aging. The progressive senescence of thymic epithelial cells (TECs) leads to the involution of thymus under aging, chronic stress and other factors. Ligustilide (LIG) is a major active component of the anti-aging Chinese herbal medicine Angelica sinensis (Oliv.) Diels, but its role in preventing TEC-based thymic aging remains elusive. PURPOSE: This study explored the protective role of Ligustilide in alleviating ADM (adriamycin) -induced thymic immune senescence and its underlying molecular mechanisms. METHOD: The protective effect of Ligustilide on ADM-induced thymic atrophy was examined by mouse and organotypic models, and conformed by SA-ß-gal staining in TECs. The abnormal spatial distribution of TECs in the senescent thymus was analyzed using H&E, immunofluorescence and flow cytometry. The possible mechanisms of Ligustilide in ADM-induced thymic aging were elucidated by qPCR, fluorescence labeling and Western blot. The mechanism of Ligustilide was subsequently validated through actin polymerization inhibitor, genetic engineering to regulate Thymosin ß15 (Tß15) and Tß4 expression, molecular docking and ß Thymosin-G-actin cross-linking assay. RESULTS: At a 5 mg/kg dose, Ligustilide markedly ameliorated ADM-induced weight loss and limb grip weakness in mice. It also reversed thymic damage and restored positive selection impaired by ADM. In vitro, ADM disrupted thymic structure, reduced TECs number and hindered double negative (DN) T cell differentiation. Ligustilide counteracted these effects, promoted TEC proliferation and reticular differentiation, leading to an increase in CD4+ single positive (CD4SP) T cell proportion. Mechanistically, ADM diminished the microfilament quantity in immortalized TECs (iTECs), and lowered the expression of cytoskeletal marker proteins. Molecular docking and cross-linking assay revealed that Ligustilide inhibited the protein binding between G-actin and Tß15 by inhibiting the formation of the Tß15-G-actin complex, thus enhancing the microfilament assembly capacity in TECs. CONCLUSION: This study, for the first time, reveals that Ligustilide can attenuate actin depolymerization, protects TECs from ADM-induced acute aging by inhibiting the binding of Tß15 to G-actin, thereby improving thymic immune function. Moreover, it underscores the interesting role of Ligustilide in maintaining cytoskeletal assembly and network structure of TECs, offering a novel perspective for deeper understanding of anti thymic aging.

Ligustilide-loaded liposome ameliorates mitochondrial impairments and improves cognitive function via the PKA/AKAP1 signaling pathway in a mouse model of Alzheimer's disease.

BACKGROUND: Oxidative stress is an early event in the development of Alzheimer's disease (AD) and maybe a pivotal point of interaction governing AD pathogenesis; oxidative stress contributes to metabolism imbalance, protein misfolding, neuroinflammation and apoptosis. Excess reactive oxygen species (ROS) are a major contributor to oxidative stress. As vital sources of ROS, mitochondria are also the primary targets of ROS attack. Seeking effective avenues to reduce oxidative stress has attracted increasing attention for AD intervention. METHODS: We developed liposome-packaged Ligustilide (LIG) and investigated its effects on mitochondrial function and AD-like pathology in the APPswe/PS1dE9 (APP/PS1) mouse model of AD, and analyzed possible mechanisms. RESULTS: We observed that LIG-loaded liposome (LIG-LPs) treatment reduced oxidative stress and ß-amyloid (Aß) deposition and mitigated cognitive impairment in APP/PS1 mice. LIG management alleviated the destruction of the inner structure in the hippocampal mitochondria and ameliorated the imbalance between mitochondrial fission and fusion in the APP/PS1 mouse brain. We showed that the decline in cAMP-dependent protein kinase A (PKA) and A-kinase anchor protein 1 for PKA (AKAP1) was associated with oxidative stress and AD-like pathology. We confirmed that LIG-mediated antioxidant properties and neuroprotection were involved in upregulating the PKA/AKAP1 signaling in APPswe cells in vitro. CONCLUSION: Liposome packaging for LIG is relatively biosafe and can overcome the instability of LIG. LIG alleviates mitochondrial dysfunctions and cognitive impairment via the PKA/AKAP1 signaling pathway. Our results provide experimental evidence that LIG-LPs may be a promising agent for AD therapy.

Ligustilide delays senescence of auditory cortex in mice by inhibiting ferritinophagy / 中国药理学通报

Aim To investigate the mechanism of ligu aged 2 months of the same strain were used as the constilide (LIG) in delaying the senescence of auditory trol (Ctrl) group. Auditory brainstem response test was cortex and treating central presbycusis. Methods used to detect the auditory threshold of mice before and Forty C57BL/6J mice aged 13 months were randomly di after treatment. Levels of serum MDA and activity of vided into ligustilide low-dose(L-LIG) group, ligustil serum SOD were detected to display the level of oxidative ide medium-dose (M-LIG) group, ligustilide high-dose stress. The pathological changes of auditory cortex were (H-LIG) group and aging (Age) group, and 10 mice observed by HE staining. Ferroptosis was observed by

Targeting RXFP1 by Ligustilide: A novel therapeutic approach for alcoholic hepatic steatosis.

BACKGROUND: Ligustilide (Lig) is the main active ingredient of Umbelliferae Angelicae Sinensis Radix (Chinese Angelica) and Chuanxiong Rhizoma (Sichuan lovase rhizome). Lig possesses various pharmacological properties and could treat obesity by regulating energy metabolism. However, the impact and regulatory mechanism of Lig on alcoholic hepatic steatosis remains unclear. PURPOSE: This study aimed to explore the therapeutic effect of Lig on alcoholic hepatic steatosis and its related pharmacological mechanism. RESULTS: With chronic and binge ethanol feeding, liver tissue damage and lipid accumulation in mice suffering alcoholic hepatic steatosis were significantly improved after Lig treatment. Lig effectively regulated the expression levels of lipid metabolism-related proteins in alcoholic hepatic steatosis. In addition, Lig reduced RXFP1 expression, inhibited the activation of NLRP3 inflammasome, and blocked NET formation. Lig reduced the infiltration of immune cells to the liver and the further prevented the occurrence of alcohol-stimulated inflammatory response in liver. Lig significantly regulated lipid accumulation in alcohol exposed AML12 cells via modulating PPARα and SREBP1. In MPMs, Lig decreased the expression of RXFP1, inhibited the activation of NLRP3 in macrophages stimulated by LPS/ATP, and slowed down the occurrence of inflammatory response. CONCLUSION: Lig sustained lipid metabolism homeostasis in alcoholic hepatic steatosis, through inhibiting the activation of NLRP3 inflammasomes and the formation of NETs, especially targeting RXFP1 in macrophages.

Z-Ligustilide Combined with Cisplatin Reduces PLPP1-Mediated Phospholipid Synthesis to Impair Cisplatin Resistance in Lung Cancer.

Lung cancer is a malignant tumor with one of the highest morbidity and mortality rates in the world. Approximately 80-85% of lung cancer is diagnosed as non-small lung cancer (NSCLC), and its 5-year survival rate is only 21%. Cisplatin is a commonly used chemotherapy drug for the treatment of NSCLC. Its efficacy is often limited by the development of drug resistance after long-term treatment. Therefore, determining how to overcome cisplatin resistance, enhancing the sensitivity of cancer cells to cisplatin, and developing new therapeutic strategies are urgent clinical problems. Z-ligustilide is the main active ingredient of the Chinese medicine Angelica sinensis, and has anti-tumor activity. In the present study, we investigated the effect of the combination of Z-ligustilide and cisplatin (Z-ligustilide+cisplatin) on the resistance of cisplatin-resistant lung cancer cells and its mechanism of action. We found that Z-ligustilide+cisplatin decreased the cell viability, induced cell cycle arrest, and promoted the cell apoptosis of cisplatin-resistant lung cancer cells. Metabolomics combined with transcriptomics revealed that Z-ligustilide+cisplatin inhibited phospholipid synthesis by upregulating the expression of phospholipid phosphatase 1 (PLPP1). A further study showed that PLPP1 expression was positively correlated with good prognosis, whereas the knockdown of PLPP1 abolished the effects of Z-ligustilide+cisplatin on cell cycle and apoptosis. Specifically, Z-ligustilide+cisplatin inhibited the activation of protein kinase B (AKT) by reducing the levels of phosphatidylinositol 3,4,5-trisphosphate (PIP3). Z-ligustilide+cisplatin induced cell cycle arrest and promoted the cell apoptosis of cisplatin-resistant lung cancer cells by inhibiting PLPP1-mediated phospholipid synthesis. Our findings demonstrate that the combination of Z-Ligustilide and cisplatin is a promising approach to the chemotherapy of malignant tumors that are resistant to cisplatin.

Anti-Glioma Effects of Ligustilide or n-Butylphthalide on Their Own and the Synergistic Effects with Temozolomide via PI3K/Akt Signaling Pathway.

Background: Ligustilide (LIG) and n-butylphthalide (NBP) have neuroprotective effects in cerebral ischemia; however, their roles in gliomas are not well-known.This study aimed to explore the anti-glioma effects of LIG and NBP individually and the synergistic effects of temozolomide (TMZ) via the PI3K/Akt Signaling Pathway. Materials and Methods: Cytotoxicity of LIG and NBP alone and in combination with TMZ in U251 cells was determined using the CCk-8. The effect of compounds alone or in combination on cell migration was detected using the wound healing assay, and the invasion was evaluated by transwell assays, respectively. Cell apoptosis was quantified by flow cytometry and the changed expressions of proteins were detected by Western blotting. Results: The results showed that LIG and NBP significantly inhibited the growth of U251 cells at concentrations of 4-10 µg/mL and 1.5-6 µg/mL in a dose-dependent manner (p<0.05, p<0.01). The combination of 20 µg/mL TMZ with LIG in the concentration range of 4-10 µg/mL or with NBP of 0.5-6 µg/mlachieved synergistic effect towardsU251 cells. LIG and NBP, alone or in combination with TMZ, markedly inhibited cell invasion (p< 0.001) and enhanced apoptosis (p< 0.05). The combination of TMZ with LIG or NBP markedly inhibited cell migration (p< 0.001). Western blot analysis showed that LIG, NBP, and TMZ, alone and in combination, significantly decreased the expression of Bcl-2, p-PI3K, and p-Akt, and increased the expression of Bax. Conclusion: Both LIG and NBP exert anti-glioma effects on their own through the PI3K/Akt pathway and enhance TMZ-mediated anti-glioma efficiency via the same pathway.

Z-ligustilide preferentially caused mitochondrial dysfunction in AML HL-60 cells by activating nuclear receptors NUR77 and NOR1.

BACKGROUND: Nuclear receptors NUR77 and NOR1 were identified as critical targets in acute myeloid leukemia (AML) therapy. Previously, we showed that Z-ligustilide (Z-LIG) selectively targeted AML by restoring NUR77 and NOR1. However, its downstream mechanisms are yet to be elucidated. METHODS: SRB staining assay was used to measure cell viability. Cell apoptosis, mitochondrial membrane potential and mitochondrial reactive oxygen species were analyzed using flow cytometry. The potential targets of Z-LIG in AML HL-60 cells were evaluated by RNA sequencing. Changes in RNA levels were measured using quantitative RT-qPCR and western blot analysis was used to detect the expression of proteins. RESULTS: Z-LIG preferentially induced mitochondrial dysfunction in HL-60 cells compared with 293T cells. Furthermore, RNA sequencing revealed that mitochondrial transcription and translation might be potential Z-LIG targets inhibiting HL-60 cells. NUR77/NOR1 overexpression significantly reduced the mitochondrial ATP and mitochondrial membrane potential and increased mitochondrial reactive oxygen species in HL-60 cells but not in 293T cells. Moreover, Z-LIG induced mitochondrial dysfunction by restoring NUR77 and NOR1 in HL-60 cells. Compared with HL-60 cells, the apoptosis-inducing activities of NUR77/NOR1 and Z-LIG were significantly reduced in HL-60 ρ0 cells depleted in mitochondrial DNA (mt-DNA). Moreover, NUR77/NOR1 and Z-LIG downregulated mitochondrial transcription and translation related proteins in HL-60 cells. Notably, Z-LIG remarkably reduced mitochondrial ATP in primary AML cells and showed anti-AML activity in mouse models of human AML. CONCLUSIONS: Collectively, our findings suggested that Z-LIG selectively induces mitochondrial dysfunction in AML HL-60 cells by restoring NUR77 and NOR1, a process associated with interference in mtDNA transcription.

Exploring the Mechanism of Chuanxiong Rhizoma against Thrombosis Based on Network Pharmacology, Molecular Docking and Experimental Verification.

Chuanxiong rhizoma (CX) has been utilized for centuries as a traditional herb to treat blood stasis syndromes. However, the pharmacological mechanisms are still not completely revealed. This research was aimed at exploring the molecular mechanisms of CX treatment for thrombosis. Network pharmacology was used to predict the potential anti-thrombosis mechanism after correlating the targets of active components with targets of thrombosis. Furthermore, we verified the mechanism of using CX to treat thrombosis via molecular docking and in vitro experiments. Network pharmacology results showed that a total of 18 active ingredients and 65 targets of CX treatment for thrombosis were collected, including 8 core compounds and 6 core targets. We revealed for the first time that tissue factor (TF) had a close relationship with most core targets of CX in the treatment of thrombosis. TF is a primary coagulation factor in physiological hemostasis and pathological thrombosis. Furthermore, core components of CX have strong affinity for core targets and TF according to molecular docking analysis. The in vitro experiments indicated that Ligustilide (LIG), the representative component of CX, could inhibit TF procoagulant activity, TF mRNA and protein over-expression in a dose-dependent manner in EA.hy926 cells through the PI3K/Akt/NF-κB signaling pathway. This work demonstrated that hemostasis or blood coagulation was one of the important biological processes in the treatment of thrombosis with CX, and TF also might be a central target of CX when used for treating thrombosis. The inhibition of TF might be a novel mechanism of CX in the treatment of thrombosis.

Guideline on treating community-acquired pneumonia with Chinese patent medicines.

Community-acquired pneumonia (CAP) is one of the most common infectious diseases, and its morbidity and mortality increase with age. Resistance and mutations development make the use of anti-infective therapy challenging. Chinese patent medicines (CPMs) are often used to treat CAP in China and well tolerable. However, currently there are no evidence-based guideline for the treatment of CAP with CPMs, and the misuse of CPMs is common. Therefore, we established a guideline panel to develop this guideline. We identified six clinical questions through two rounds of survey, and we then systematically searched relevant evidence and performed meta-analyses, evidence summaries and GRADE decision tables to draft recommendations, which were then voted on by a consensus panel using the Delphi method. Finally, we developed ten recommendations based on evidence synthesis and expert consensus. For the treatment of severe CAP in adults, we recommend Tanreqing injection, Reduning injection, Xuebijing injection, Shenfu injection, and Shenmai injection respectively. For the treatment of non-severe CAP in adults, we recommend Tanreqing injection, Reduning injection, Lianhua Qingwen capsule/granule, Qingfei Xiaoyan Pill and Shufeng Jiedu capsule respectively. CPMs have great potential to help in the fight against CAP worldwide, but more high-quality studies are still needed to strengthen the evidence.

Ligustilide covalently binds to Cys129 of HMGCS1 to ameliorate dyslipidemia.

Dyslipidemia is characterized by elevated levels of total cholesterol and triglycerides in serum, and has become the primary human health killer because of the major risk factors for cardiovascular diseases. Although there exist plenty of drugs for dyslipidemia, the number of patients who could benefit from lipid-lowering drugs still remains a concern. Ligustilide (Lig), a natural phthalide derivative, was reported to regulate lipid metabolic disorders. However, its specific targets and underlying molecular mechanism are still unclear. In this study, we found that Lig alleviated high fat diet-induced dyslipidemia by inhibiting cholesterol biosynthesis. Furthermore, a series of chemical biological analysis methods were used to identify its target protein for regulating lipid metabolism. Collectively, 3-hydroxy-3-methylglutaryl coenzyme A synthetase 1 (HMGCS1) of hepatic cells was identified as a target for Lig to regulate lipid metabolism. The mechanistic study confirmed that Lig irreversibly binds to Cys129 of HMGCS1 via its metabolic intermediate 6,7-epoxyligustilide, thereby reducing cholesterol synthesis and improving lipid metabolism disorders. These findings not only systematically elucidated the lipid-lowering mechanism of Lig, but also provided a new structural compound for the treatment of dyslipidemia.

Antiproliferative and Anti-Inflammatory Activities of Deprungsith Formulation and Its Bioactive Compounds Against Mild Psoriasis and Potential of Metabolic Herb-Drug Interactions.

Psoriasis is an incurable, chronic and auto-immune skin disorder with a global prevalence rate of approximately 2-3%. The study investigated the antipsoriasis activities of Deprungsith formulation and its bioactive components and their potential for inhibitory activities on human cytochrome P450 (CYP450). HaCaT and peripheral blood mononuclear cells (PBMCs) from healthy volunteers (n = 9) and psoriasis patients (n = 10) were exposed to Deprungsith formulation (Thai traditional medicine for psoriasis consisting of 16 plants), ethyl p-methoxycinnamate (EPMC), ligustilide and cyclosporin for 24 and 48 h. The antiproliferative, cell apoptosis and cell cycle arrest activities were evaluated using MTT assay and flow cytometry, respectively. The pro-inflammatory cytokine mRNA expression levels were measured using real-time polymerase chain reaction (RT-PCR). The CYP450 inhibitory effect was investigated using a bioluminescent-based CYP450 assay. Deprungsith formulation and the bioactive compounds inhibited HaCaT cells and PBMCs with weak to moderate potencies. EPMC and ligustilide combination produced an additive effect. Most substances arrested cell transition at sub-G1 and S phases, leading to early and late apoptosis induction. With prolonged exposure (48 h), all test substances decreased PBMCs necrosis. The mRNA expression of all pro-inflammatory cytokines was downregulated. Deprungsith formulation, EPMC, ligustilide and ferulic acid inhibited CYP1A2, CYP2C9, CYP2D6 and CYP3A4 activities with weak to moderate potencies. Deprungsith formulation and bioactive components induced cell apoptosis by inhibiting cell transition at specific cell cycle phases, which was correlated with the mRNA downregulation of interleukin (IL-6, IL-12p19, IL-23) and tumor necrosis factor (TNF-α). There is a low risk of potential adverse drug reactions and toxicity due to CYP450 interaction when Deprungsith formulation is concurrently administered with modern medicines.

[Molecular mechanism of ligustilide attenuating OGD/R injury in PC12 cells by inhibiting ferroptosis].

The aim of this study is to explore the mechanism of ligustilide, the main active constituent of essential oils of traditional Chinese medicine Angelicae Sinensis Radix, on alleviating oxygen-glucose deprivation/reperfusion(OGD/R) injury in PC12 cells from the perspective of ferroptosis. OGD/R was induced in vitro, and 12 h after ligustilide addition during reperfusion, cell viability was detected by cell counting kit-8(CCK-8) assay. DCFH-DA staining was used to detect the level of intracellular reactive oxygen species(ROS). Western blot was employed to detect the expression of ferroptosis-related proteins, glutathione peroxidase 4(GPX4), transferrin receptor 1(TFR1), and solute carrier family 7 member 11(SLC7A11), and ferritinophagy-related proteins, nuclear receptor coactivator 4(NCOA4), ferritin heavy chain 1(FTH1), and microtubule-associated protein 1 light chain 3(LC3). The fluorescence intensity of LC3 protein was analyzed by immunofluorescence staining. The content of glutathione(GSH), malondialdehyde(MDA), and Fe was detected by chemiluminescent immunoassay. The effect of ligustilide on ferroptosis was observed by overexpression of NCOA4 gene. The results showed that ligustilide increased the viability of PC12 cells damaged by OGD/R, inhibited the release of ROS, reduced the content of Fe and MDA and the expression of TFR1, NCOA4, and LC3, and improved the content of GSH and the expression of GPX4, SLC7A11, and FTH1 compared with OGD/R group. After overexpression of the key protein NCOA4 in ferritinophagy, the inhibitory effect of ligustilide on ferroptosis was partially reversed, indicating that ligustilide may alleviate OGD/R injury of PC12 cells by blocking ferritinophagy and then inhibiting ferroptosis. The mechanism by which ligustilide reduced OGD/R injury in PC12 cells is that it suppressed the ferroptosis involved in ferritinophagy.

Ligustilide, a novel SIRT1 agonist, alleviates lipopolysaccharide-induced acute lung injury through deacetylation of NICD.

Development and progression of sepsis-induced acute lung injury (ALI) involve apoptosis and oxidative stress in lung epithelial cells. Ligustilide (LIG) is one of the main bioactive constituents derived from the Angelica sinensis. As a novel SIRT1 agonist, LIG owns powerful anti-inflammatory and antioxidative properties, exerting remarkable therapeutic effects on cancers, neurological disorders, and diabetes mellitus. However, whether LIG could protect against lipopolysaccharide (LPS)-induced ALI by activating SIRT1 remains unclear. Mice underwent intratracheal LPS injection to mimic sepsis-induced ALI while MLE-12 cells were treated with LPS for 6 h to establish an in vitro ALI model. At the same time, mice or MLE-12 cells were treated with different doses of LIG to access its pharmacological effect. The results demonstrated that LIG pretreatment could improve LPS-induced pulmonary dysfunction and pathological injury, apart from increasing 7-day survival rate. In addition, LIG pretreatment also decreased inflammation, oxidative stress and apoptosis during LPS-induced ALI. Mechanically, LPS stimulation decreased the expression and activity of SIRT1 but increased the expression of Notch1 and NICD. And LIG could also enhance the interaction between SIRT1 and NICD, thus deacetylating NICD. In vitro experiments also unveiled that EX-527, a selective SIRT1 inhibitor, could abolish LIG-elicited protection in LPS-treated MLE-12 cells. And in SIRT1 knockout mice with ALI, LIG pretreatment also lost its effects on inflammation, apoptosis, and oxidative stress during ALI.