Sarsasapogenin regulates the immune microenvironment through MAPK/NF-kB signaling pathway and promotes functional recovery after spinal cord injury.

Spinal cord injury (SCI) occurs as a result of traumatic events that damage the spinal cord, leading to motor, sensory, or autonomic function impairment. Sarsasapogenin (SA), a natural steroidal compound, has been reported to have various pharmacological applications, including the treatment of inflammation, diabetic nephropathy, and neuroprotection. However, the therapeutic efficacy and underlying mechanisms of SA in the context of SCI are still unclear. This research aimed to investigate the therapeutic effects and mechanisms of SA against SCI by integrating network pharmacology analysis and experimental verification. Network pharmacology results suggested that SA may effectively treat SCI by targeting key targets such as TNF, RELA, JUN, MAPK14, and MAPK8. The underlying mechanism of this treatment may involve the MAPK (JNK) signaling pathway and inflammation-related signaling pathways such as TNF and Toll-like receptor signaling pathways. These findings highlight the therapeutic potential of SA in SCI treatment and provide valuable insights into its molecular mechanisms of action. In vivo experiments confirmed the reparative effect of SA on SCI in rats and suggested that SA could repair SCI by modulating the immune microenvironment. In vitro experiments further investigated how SA regulates the immune microenvironment by inhibiting the MAPK/NF-kB pathways. Overall, this study successfully utilized a combination of network pharmacology and experimental verification to establish that SA can regulate the immune microenvironment via the MAPK/NF-kB signaling pathway, ultimately facilitating functional recovery from SCI. Furthermore, these findings emphasize the potential of natural compounds from traditional Chinese medicine as a viable therapy for SCI treatment.

Integrated Network Pharmacology and UPLC/Q-TOF-MS Screen System to Exploring Anti-Inflammatory Active Components and Mechanism of Shunaoxin Pills.

Background: Chronic cerebral ischemia (CCI) is a pathological condition associated with a variety of cerebrovascular diseases. Shunaoxin pills (SNX) are a traditional Chinese medicine (TCM) used to improve blood circulation. However, its multicomponent and multitarget features make it difficult to decipher the molecular mechanisms. Objective: Thus, in this study, we aimed to identify the key anti-inflammatory components of SNX as markers for standardization and quality control and the potential pharmacological mechanisms of SNX in the treatment of CCI by network pharmacology to provide scientific evidence of its clinical efficacy. Methods: We evaluated the anti-inflammatory effect of SNX using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectroscopy (UPLC/Q-TOF-MS) combined with a dual-luciferase reporter assay for nuclear factor kappa B (NF-κB) inhibition to identify the active components in SNX. In addition, key pathways involved in the anti-inflammatory effect of SNX were predicted using a network pharmacology approach, and some crucial proteins and pathways were further validated by Western blotting. Results: Shunaoxin pills inhibited NF-κB through tumor necrosis factor-α (TNF-α) stimulation in 293T cells. The therapeutic effect may be related to 10 pathways regulated by ligustilide, ferulic acid, ligustrazine, and senkyunolide I. It was further confirmed that ligustilide could reduce the inflammatory response by inhibiting the phosphorylation of p38 and 3-phosphoinositide-dependent kinase 1 (PDK1). Conclusions: Ligustilide, senkyunolide I, ferulic acid, and ligustrazine could be used as anti-inflammatory Q-markers to control the quality of SNX, and p38 and PDK1 might be potential targets of SNX in the treatment of CCI.

Integrated artificial neural network analysis and functional cell based affinity mass spectrometry for screening a bifunctional activator of Ca2+ and ß2AR in aconite.

Arrhythmia, a common heart disease, is an abnormal frequency or rhythm of heartbeat caused by the origin or conduction obstacle of the heart. Aconite (Fuzi) has been regarded as an effective cardiotonic agent in traditional Chinese medicine (TCM), but it sometimes induces cardiac toxicity, such as arrhythmia, in the clinic. It is still a challenge to identify the active ingredients related to the therapeutic effect or toxicity in the application of aconite. To clarify which ingredient or derivative plays a key role of reducing toxicity and improving efficiency in the use of aconite, a serum pharmacology approach integrated with metabolomics and artificial neural network (ANN) analysis was used to in vivo screen Ca2+ and ß2AR regulators from the extracts of Heishunpian (HSP), which a processed lateral root of aconite. In addition, ß2AR transfected CHO and myocardium H9C2 functional cells-based affinity mass spectrometry (AMS) screening tests were carried out for evaluating the active ingredients in vitro. The results demonstrated that the monoester diterpenoid alkaloids (MDAs) represented by fuziline were the key bifunctional activators that could activate Ca2+ and ß2AR simultaneously. The effective compatibility of the calcium antagonists could regulate the heart rhythm to alleviate arrhythmia while maintain their cardiotonic effect, which was induced by aconite. In this study, ANN analysis based on serum pharmacology combined with AMS screen, which utilized with functional cells presented a powerful analytical strategy to the discovery and evaluation of active ingredients from a complex system.

Sinigrin Enhanced Antiasthmatic Effects of Beta Adrenergic Receptors Agonists by Regulating cAMP-Mediated Pathways.

Millions of patients suffer from asthma worldwide. However, the first-line drugs used to treat asthma, namely, the beta-adrenergic receptors agonists (ß-agonists), are not recommended for use as monotherapy because of their severe dose-related side effects. This limitation has prompted the search for new therapies, which can be used in conjunction with ß--agonists so that lower doses can be administered. Sinigrin is a major compound found in many antiasthmatic medicinal plants. In this study, we explored the antiasthmatic activity of sinigrin when used in combination with ß-agonists and its underlying mechanism. Sinigrin enhanced the asthma-relieving effects of isoproterenol and reduced the effective isoproterenol dose in an acute-asthma model in guinea pigs. Mechanistically, sinigrin enhanced the cAMP levels induced by ß-agonists by inhibiting PDE4. The resulting increase in cAMP levels stimulated the activity of the downstream effector protein kinase A, which would be expected to ultimately induce the relaxation of airway smooth muscle. In conclusion, sinigrin enhances the asthma-relieving effects of ß-agonists by regulating the cAMP signaling pathway and represents a potential add-on drug to ß-agonists for the treatment of asthma.

Analyze and Identify Peiminine Target EGFR Improve Lung Function and Alleviate Pulmonary Fibrosis to Prevent Exacerbation of Chronic Obstructive Pulmonary Disease by Phosphoproteomics Analysis.

Chronic obstructive pulmonary disease (COPD) has been a major public health problem and is still a formidable challenge for clinicians. It is urgent to find new compounds for minimizing the risk of disease progression and exacerbation especially in the early phase of COPD. A traditional Chinese medicine (TCM) formula, Chuan Bei Pi Pa dropping pills (CBPP), was tested in this study to investigate its potential mechanisms in preventing the exacerbation of COPD. Phosphoproteomics analysis for a smog stimulated early stage COPD mice model was employed to detect the underlying molecular mechanisms of CBPP. In addition, protein-protein interaction (PPI) and bioinformatics analyses were included to analyze the key proteins and predict the key bioactive compounds. The results indicated that peiminine (PEI) target epidermal growth factor receptor (EGFR) prevented the exacerbation of COPD by inhibiting the EGFR signaling pathway, and ursolic acid (UA) can alleviate inflammation disorders via inhibition of CASP3 on mitogen-activated protein kinase (MAPK) signaling pathway. After in vivo and in vitro evaluations, we revealed that PEI from CBPP, as a lead compound, can improve lung function and alleviate pulmonary fibrosis by acting on the EGFR and MLC2 signaling pathways. Furthermore, the approach described here is an effective way to analyze and identify the bioactive ingredients from a mixture by functional proteomics analysis.

Effect of N-methyltyramine on the regulation of adrenergic receptors via enzymatic epinephrine synthesis for the treatment of gastrointestinal disorders.

BACKGROUND: Citri Reticulatae Pericarpium (CRP), Aurantii Fructus Immaturus (AFI) and Aurantii Fructus (AF) are all important Citrus species used in traditional Chinese medicines (TCMs) for the treatment of gastrointestinal disorders. Although they have been used since ancient times and are still in use today, the mechanistic basis for their regulation of adrenergic receptors (ARs) is still not clear. PURPOSE: In this study, we aimed to determine the active components and mechanisms of action of CRP, AFI and AF in treating gastrointestinal disorders related to ARs. METHODS: First, the phenethylamine alkaloid components of CRP, AFI and AF were identified and compared across 30 samples of three Citrus species by UPLC-Q/TOF-MS in combination with content difference analysis. Second, the effect of the main active alkaloid component on AR-based gastrointestinal disorders was investigated by an in vivo small intestinal propulsive test and an in vitro relaxing small intestinal smooth muscle activity test. The mechanism of AR regulation of the active alkaloid was further studied by evaluating its effect on relaxing small intestinal smooth muscle in the presence of an inhibitor. Lastly, the enzymes, which played an important role in epinephrine synthesis and AR regulation, were detected by immunohistochemistry. RESULTS: Three phenethylamine AR regulators (N-methyltyramine, synephrine and hordenine) in CRP, AFI and AF were characterized. It was found that N-methyltyramine could relax mouse small intestinal smooth muscle and inhibit small intestinal propulsion. The effect of N-methyltyramine on relaxing small intestinal smooth muscle could be inhibited by a-methyl-l-tyrosine. The enzymes related epinephrine synthesis and AR function were found in the mouse small intestine. The biotransformation process that converts N-methyltyramine to epinephrine was determined. CONCLUSION: The treatment of gastrointestinal disorders of CRP, AFI and AF is associated with their alkaloid component N-methyltyramine via the regulation of ARs, and the mechanism is considered to be the biotransformation of N-methyltyramine to epinephrine by serial synthase, which takes place at the nerves cells in small intestine.

Anti-inflammatory effects of Shufengjiedu capsule for upper respiratory infection via the ERK pathway.

BACKGROUND: Shufengjiedu Capsule (SFJD) is a type of Chinese traditional medicine compound for the treatment of acute upper respiratory tract infection. The present work aims to decipher the mechanism of SFJD. METHODS: In this study, we used target prediction and RNA sequence (RNA-Seq) based on transcriptome analysis to clarify the inflammation-eliminating mechanism of SFJD. Firstly, Pseudomonas aeruginosa (PAK) was used to induce acute lung injury in KM mice. After being treated by SFJD, the differently expressed genes were analyzed by RNA-Seq. Secondly, the chemical constituents of SFJD were identified by ultra-performance liquid chromatography quadrupole/time of flight mass spectrometry (UPLC/Q-TOF-MS) and submitted to PharmMapper to predict targets. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and String 9.1 websites were employed to establish the interaction network of inflammation of these targets. RESULTS: The results indicated that SFJD alleviated PAK induced lung injury in KM mice. We infer that the mechanism is a complex network containing 15 pathways related to inflammation regulated by 16 types of components from six types of herbs via 29 proteins. The ERK signaling pathway was a key pathway among them, which was predicted to be regulated by 14 types of components in SFJD. Phillyrin, emodin, and verbenalin were screened out by binding capacity, and the synergistic effect of them was further confirmed. CONCLUSIONS: Various components of SFJD ameliorated PAK induced upper respiratory tract infection via multiple targets, of which ERK phosphorylation might be the key event regulated specifically by verbenalin, phillyrin and emodin.

The hemostatic effect study of Cirsium setosum on regulating α1-ARs via mediating norepinephrine synthesis by enzyme catalysis.

BACKGROUND: Cirsium setosum (CS) is the aboveground part of Cephalanoplos segetum Kitam. Although it has been used as a hemostatic treatment for thousands of years and is still in use today, the mechanism of CS on regulating ARs is still not clear. PURPOSE: In this study, we aimed to clarify the mechanism of CS on regulating ARs. METHODS: We developed a simple method based on UPLC/Q-TOF MS combined adrenergic receptor dual-luciferase reporter assay systems for the rapid determination of active constituents in CS. The mechanism of tyramine, the main active component for regulating ARs, was further investigated by an in vitro norepinephrine biotransformation test and in vivo vaso activity tests. RESULTS: Two phenethylamine ARs regulators (tyramine and N-methyltyramine) in CS were characterized, and it was found that tyramine could induce vasoconstriction via regulation of α1-ARs by mediating norepinephrine synthesis. CONCLUSION: The hemostatic effect of CS is associated with tyramine and N-methyltyramine, via regulation of α1-ARs, and the mechanism of tyramine is related to mediating norepinephrine synthesis by enzyme catalysis.

Identification of NF-κB inhibitors following Shenfu injection and bioactivity-integrated UPLC/Q-TOF-MS and screening for related anti-inflammatory targets in vitro and in silico.

ETHNOPHARMACOLOGICAL RELEVANCE: Shenfu injection (SFI) is a commercial medicinal product approved by the China Food and Drug Administration that is widely used in the treatment of stroke and coronary heart disease. However, the material basis and the mechanism of SFI are not fully understood. AIM OF THE STUDY: With network pharmacology analysis, our research committed to identify the anti-inflammatory ingredients and mechanism of SFI by combining high-throughput screening. MATERIALS AND METHODS: We developed a bioactivity-based UPLC/Q-TOF-MS method followed by network pharmacology and identified the anti-inflammatory active ingredients of SFI from two different perspectives of network computing and high throughput screening. Then we verified the anti-inflammatory effect of SFI in vitro with endothelial cells. After detecting the cell viability, the expression of interleukin-6 (IL-6), inhibitor of nuclear factor kappa-B kinase (IKK), phosphorylated IKK, phosphorylated NF-κB and phosphorylated IκB-α from the supernatant were determined. RESULTS: SFI could significantly suppress inflammatory responses, and the mechanism may be via an NF-κB-dependent pathway. The results of high throughput screening (HTS) revealed that protopanaxadiol glycosides (ginsenosides Rb1, Rb2, Rb3, Rc and Rd), protopanaxatriol glycosides (ginsenosides Rg1, Rg2, Re, Rf and F1), diester-type alkaloids (fuziline and neoline) and aconine derivatives (mesaconine and benzoyl-mesaconine) have anti-NF-κB activity. The three compounds (including benzoyl-mesaconine, fuziline and neoline) are the first reported SFI compounds to have NF-κB inhibitor activity. CONCLUSIONS: SFI may play a critical role in counteracting inflammation through the NF-κB signaling pathway. The active ingredients are protopanaxadiol glycosides, protopanaxatriol glycosides, diester-type alkaloids and aconine derivatives.