Background: Acute lung injury (ALI) is characterized by dysfunction of the alveolar epithelial membrane caused by acute inflammation and tissue injury. Qingwenzhike (QWZK) prescription has been demonstrated to be effective against respiratory viral infections in clinical practices, including coronavirus disease 2019 (COVID-19) infection. So far, the chemical compositions, protective effects on ALI, and possible anti-inflammatory mechanisms remain unknown.Methods: In this study, the compositions of QWZK were determined via the linear ion trap/electrostatic field orbital trap tandem high-resolution mass spectrometry (UHPLC-LTQ-Orbitrap MS). To test the protective effects of QWZK on ALI, an ALI model induced by lipopolysaccharide (LPS) in rats was used. The effects of QWZK on the LPS-induced ALI were evaluated by pathological changes and the number and classification of white blood cell (WBC) in bronchoalveolar lavage fluid (BALF). To investigate the possible underlying mechanisms, the contents of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein (MCP-1), interleukin-1β (IL-1β), interleukin-18 (IL-18), and immunoregulatory-related factors interferon-γ (IFN-γ) were detected by ELISA. Furthermore, the expression of Toll-like receptor 4 (TLR4), p-IKKα/β, IKKα, IKKβ, p-IκBα, IκBα, p-NF-κB, nuclear factor-κB (NF-κB), NOD-like receptor family pyrin domain containing 3 (NLRP3), cleaved caspase-1, pro-caspase-1, apoptosis-associated speck-like protein containing CARD (ASC), and β-actin were tested by Western blot.Results: A total of 99 compounds were identified in QWZK, including 33 flavonoids, 23 phenolic acids, 3 alkaloids, 3 coumarins, 20 triterpenoids, 5 anthraquinones, and 12 others. ALI rats induced by LPS exhibited significant increase in neutrophile, significant decrease in lymphocyte, and evidently thicker alveolar wall than control animals. QWZK reversed the changes in WBC count and alveolar wall to normal level on the model of ALI induced by LPS. ELISA results revealed that QWZK significantly reduced the overexpression of proinflammatory factors IL-6, TNF-α, MCP-1, IL-1β, IL-18, and IFN-γ induced by LPS. Western blot results demonstrated that QWZK significantly downregulated the overexpression of TLR4, p-IKKα/β, p-IκBα, p-NF-κB, NLRP3, cleaved caspase-1, and ASC induced by LPS, which suggested that QWZK inhibited TLR4/NF-κB signaling pathway and NLRP3 inflammasomes.Conclusions: The chemical compositions of QWZK were first identified. It was demonstrated that QWZK showed protective effects on ALI induced by LPS. The possible underlying mechanisms of QWZK on ALI induced by LPS was via inhibiting TLR4/NF-kB signaling pathway and NLRP3 inflammasome activation. This work suggested that QWZK is a potential therapeutic candidate for the treatments of ALI and pulmonary inflammation.

Backgrounds: Psoraleae Fructus (PF)-induced hepatotoxicity has been reported in clinical and animal experiments. However, the hepatotoxic constituents and mechanisms underlying PF-induced toxicity have remained unclear. Therefore, this study explored the potentially toxic PF components and revealed their relative mechanisms.Methods: The hepatotoxicity of PF water (PFW) and ethanol (PFE) extracts was compared using Kunming mice. The different compositions between PFW and PFE, which were considered toxic compositions, were identified using the UHPLC-Q-Exactive MS method. Then, L02 and HepG2 cell lines were used to evaluate the toxicity of these compositions. Cell viability and apoptosis were determined through the Cell Counting Kit-8 (CCK-8) assay and flow cytometry, respectively. An automatic biochemical analyzer detected the aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP). Lastly, we used high-content screening (HCS) to determine the levels of reactive oxygen species (ROS), lipid, and mitochondrial membrane potential (MMP).Results: The ethanol extraction process aggravated the hepatotoxicity of PF, causing more severe injuries. The content of psoralen, isopsoralen, bavachin, psoralidin, bavachinin, neobavaisoflavone, and bakuchiol was higher in the PFE than PFW. Bavachin, psoralidin, bavachinin, neobavaisoflavone, and bakuchiol induced cell apoptosis and the AST, ALT, and ALP leakages. Furthermore, these five constituents increased intracellular lipid accumulation and ROS levels but decreased the MMP level.Conclusion: The ethanol extraction process could induce severe PF hepatotoxicity. Bavachin, psoralidin, bavachinin, neobavaisoflavone, and bakuchiol are the main hepatotoxic ingredients. This mechanism could be associated with oxidative stress and mitochondrial damage-mediated apoptosis. Taken together, this study provides a basis for the clinical application of PF that formulates and improves its herbal standards.

Migraine is a major cause of disability worldwide, particularly in young adults and middle-aged women. Xiongshao Zhitong Recipe (XZR) is a traditional Chinese medicine prescription used for treating migraine, but its bioactive components and therapeutic mechanisms remain unclear. We aimed to confirm the therapeutic effect of XZR on migraine and to determine the possible mechanism and bioactive components of XZR. Here, a sensitive UHPLC-LTQ-Orbitrap MS assay was carried out to analyze the ingredients of XZR, and a total of 62 components were identified, including coumarins, phenolic acids, phthalides, flavonoids, and terpenoids; among them, 15 components were identified in the serum samples after XZR treatment. We established a rat model of migraine via nitroglycerin (NTG) injection. The in vivo experiments demonstrated that XZR attenuated allodynia and photophobia in rats with NTG-induced migraine, and XZR also demonstrated analgesic effects. XZR reversed the abnormal levels of nitric oxide, 5-hydroxytryptamine (5-HT), calcitonin gene-related peptide (CGRP), and substance P (SP) to normal levels. XZR also downregulated inflammatory reactions, including mast cell degranulation and serum IL-1β, IL-6, and TNF-α levels. In terms of mechanism, we revealed that XZR treated NTG-induced migraine through the inhibition of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) expression in both the trigeminal nucleus caudalis (TNC) and periaqueductal gray matter (PAG), as well as the total NOS enzyme activity, which regulated the NF-κB signaling pathway. Additionally, imperatorin and xanthotoxin, two major ingredients of XZR, showed a high binding affinity to nNOS (Gly468-Leu616). In vitro, XZR, imperatorin, and xanthotoxin inhibited the nNOS expression and the NF-κB signaling pathway in lipopolysaccharide (LPS)-stimulated PC12 cells. In conclusion, we demonstrated the therapeutic effects of XZR and provided evidence that XZR played a critical anti-inflammatory role by suppressing NOS and NF-κB signaling pathway activation. Imperatorin and xanthotoxin were potential bioactive components of XZR. The findings from this study supported that XZR was a candidate herbal drug for migraine therapy.

Migraine is a prevalent neurological disorder accompanied by a considerable economic burden. Xiongshao Zhitong granules (XSZT) have anti-inflammatory and analgesic functions in the clinic and are used for migraine therapy. However, the mechanisms by which XSZT treats migraine remain unclear. To discover the underlying mechanism and active ingredients of XSZT in the treatment of migraine. The nitroglycerin (NTG)-induced chronic migraine (CM) model was established and used to detect the therapeutic effect of XSZT on migraine. To elucidate the mechanism, we detected transient receptor potential vanilloid 1 (TRPV1) -mediated NOD-like receptor protein 3 (NLRP3) inflammasome activation in the CM rat model and the LPS-induced inflammatory BV-2 cell model using Western blotting, immunofluorescence and ELISA techniques. The potentially active ingredients of XSZT were determined by UHPLC-LTQ-Orbitrap MS, molecular docking, and surface plasmon resonance. Our findings revealed that XSZT reduced the number of head scratching, increased the periorbital pain threshold and shortened the time spent in the dark box, decreased c-Fos expression in the CM rat model, suggesting an analgesic effect of XSZT on migraine. XSZT inhibited neurogenic inflammation, including downregulating CGRP, TNF-α, IL-1β and IL-18 levels and decreasing the degranulation rate of mast cells. Additionally, XSZT suppressed the expression and activation of TRPV1 and the NLRP3 inflammasome in the trigeminal nucleus caudalis. In vitro experiments confirmed that activated TRPV1 increased the level of the NLRP3 inflammasome by increasing intracellular calcium levels. Galloylpaeoniflorin, isogastrin, ellagic acid and salvianolic acid A interacted with TRPV1 and inhibited IL-1β secretion. XSZT plays a therapeutic role in migraine through regulating TRPV1-mediated NLRP3 inflammatory activation and galloylpaeoniflorin, isogastrin, ellagic acid and salvianolic acid A might be the active ingredients of XSZT, which provides an experimental basis for the clinical treatment of migraine.

Ischemic stroke (IS) elicits intertwined metabolic derangements and neuroinflammatory responses that propagate pathogenic cascades. Rhubarb (RR) is widely applied to treat IS in the clinic. However, its underlying pharmacological mechanisms remain incompletely elucidated. To dissect the mechanism and pharmacological basis of RR's ability to ameliorate IS from the side of modulating tryptophan-kynurenine (TRP-KYN) metabolism and neuroinflammation. An embolic middle cerebral artery occlusion (MCAO) model was employed in rats to simulate human IS. Neurological deficit scores, hematoxylin-eosin, 2,3,5-triphenyltetrazolium chloride, alcian blue-periodic acid-Schiff staining, Western blotting, targeted metabolomics, immunofluorescence, and enzyme-linked immunosorbent assay were utilized to evaluate the efficacy of RR against cerebral ischemic injury, gut permeability, TRP-KYN metabolism levels, and neuroinflammation. Surface plasmon resonance and cell-based assays were further utilized to screen and validate potential bioactive ingredients of RR for the treatment of IS. In addition to mitigating cerebral ischemic damage, RR treatment substantially restored intestinal barrier function in IS rats by reducing leaky gut biomarkers, ameliorating histopathological alterations, and upregulating colonic tight junction proteins. Moreover, RR modulated TRP-KYN metabolism, concomitant with regulated expression and enzymatic activity of indoleamine 2,3-dioxygenase 1 (IDO-1) and the triggering receptor expressed on myeloid cells-1 (TREM-1) levels in both colon tissue and serum. Within the central nervous system, RR attenuated neuroinflammation triggered by MCAO through orchestrating microglial polarization and cytokine levels, accompanied by suppression of the TREM-1-mediated inflammatory signaling cascade. Chrysophanol 8-O-β-d-glucoside, rutinum, lindleyin, and (-)-catechin gallate from RR were identified as potential IDO-1 inhibitors, while rhein, lindleyin, methyl gallate, and chrysophanol 8-O-β-d-glucoside were identified as potential TREM-1 inhibitors, capable of attenuating the overexpression of IDO-1 and TREM-1 or TREM-1 and NOD-like receptor protein 3 (NLRP3). This study demonstrates that RR exerts therapeutic effects in IS by improving the intestinal barrier function, reprogramming the IDO-1-dependent TRP-KYN pathway, and attenuating neuroinflammation. These effects are attributed to its bioactive components, including rutinum, chrysophanol 8-O-β-d-glucoside, lindleyin, (-)-catechin gallate, rhein, and methyl gallate.

Exposure to 4-vinylcyclohexene diepoxide (VCD) has been associated to reproductive toxicity, However, the dynamic ovarian function alterations over time in rats and the underlying mechanisms remain unclear. This study investigated the effects of VCD on ovarian function in female Sprague-Dawley (SD) rats on the 25, 60 and 135 days after VCD injection and discovered related mechanisms. Our results showed that VCD had little ovotoxicity on the 25^th day, as no significant changes were observed except for the reduction of preantral follicles. However, on the 60^th day and 135^th day, rats exhibited irregular estrous cycles, abnormal serum sex hormone levels, ovarian morphological damage, decreased primordial and preantral follicles, and increased atretic follicles. We further found that on the 60^th and 135^th day, VCD might exert ovotoxicity by decreasing antioxidant enzyme levels, inhibiting of the Kelch-like ECH-associated protein 1 (KEAP1)/nuclear factor erythroid 2-related factor 2 (NRF2) and PTEN-induced putative kinase 1 (PINK1)/E3 ubiquitin ligase (Parkin) signaling pathway. Interestingly, on the 25^th day, KEAP1/NRF2 and PINK1/Parking signaling pathway were activated. This study provided a wider application in using laboratory rodents as a model of ovarian failure and highlighted that VCD-induced ovotoxicity was linked to the regulation of the KEAP1/NRF2 and PINK1/Parkin signaling pathways.