Macamides are very important secondary metabolites produced by Lepidium meyenii Walp, which possess multiple bioactivities, especially in the neuronal system. In a previous study, we observed that macamides exhibited excellent effects in the recovery of injured nerves after 1-methyl-4-phenylpyridinium (MPP⁺)-induced dopaminergic neuronal damage in zebrafish. However, the mechanism underlying this effect remains unclear. In the present study, we observed that N-benzylhexadecanamide (XA), which is a typical constituent of macamides, improved the survival rate of neurons in vitro. We determined the concentration of neurotransmitters in MN9D cells and used it in conjunction with an integrated proteomics and lipidomics approach to investigate the mechanism underlying the neuroprotective effects of XA in an MPP⁺-induced neurodegeneration cell model using QqQ MS, Q-TOF MS, and Orbitrap MS. The statistical analysis of the results led to the identification of differentially-expressed biomarkers, including 11 proteins and 22 lipids, which may be responsible for the neuron-related activities of XA. All these potential biomarkers were closely related to the pathogenesis of neurodegenerative diseases, and their levels approached those in the normal group after treatment with XA. Furthermore, seven lipids, including five phosphatidylcholines, one lysophosphatidylcholine, and one phosphatidylethanolamine, were verified by a relative quantitative approach. Moreover, four proteins (Scarb2, Csnk2a2, Vti1b, and Bnip2) were validated by ELISA. The neurotransmitters taurine and norepinephrine, and the cholinergic constituents, correlated closely with the neuroprotective effects of XA. Finally, the protein–lipid interaction network was analyzed. Based on our results, the regulation of sphingolipid metabolism and mitochondrial function were determined to be the main mechanisms underlying the neuroprotective effect of XA. The present study should help us to better understand the multiple effects of macamides and their use in neurodegenerative diseases.

To evaluate whether Xiaoerfupi (XEFP), a traditional Chinese medicine formula, can ameliorate functional dyspepsia (FD) through regulation of the HTR3A and c-FOS. The FD rat model was established through administration of iodoacetamide (IA) and interval fasting. XEFP group rats received XEFP for 3 weeks. Detection of gastric emptying and gastrin were performed to assess the interventional effect of XEFP. The constituents of XEFP were submitted to BATMAN-TCM, an online bioinformatics analysis tool, to predict the targets related to dyspepsia. Furthermore, the prediction was validated via Western blot assay. XEFP enhanced gastric emptying of rats (XEFP middle dose vs. FD model: 71.87 ± 15.21% vs. 30.07 ± 12.76%, P < 0.01) and simultaneously increased gastrin in FD rats (XEFP middle dose vs. FD model: 63.61 ± 17.90 vs. 26.14 ± 7.78 pg/ml, P < 0.01). KEGG enrichment analysis revealed that the neuroactive ligand-receptor interaction was successfully enriched (P-value = 2.2E-13, Benjamini = 2.0E-11). Combining different Bioinformatics analysis implied that XEFP regulates HTR3A and c-FOS. Subsequently molecular biological studies confirmed that the expression of HTR3A and c-FOS in the model group was upregulated in rats in comparison with the control group. Furthermore, the expression of HTR3A and c-FOS in the XEFP group (middle dose) compared with the model group was significantly reduced (P < 0.01). XEFP may ameliorate FD through regulation of the HTR3A and c-FOS.

Natural products from traditional medicine inherit bioactivity from their source herbs. However, the pharmacological mechanism of natural products is often unclear and studied insufficiently. Pathway fingerprint similarity based on “drug-target-pathway” heterogeneous network provides new insight into Mechanism of Action (MoA) for natural products compared with reference drugs, which are selected approved drugs with similar bioactivity. Natural products with similar pathway fingerprints may have similar MoA to approved drugs. In our study, XYPI, an andrographolide derivative, had similar anti-inflammatory activity to Glucocorticoids (GCs) and non-steroidal anti-inflammatory drugs (NSAIDs), and GCs and NSAIDs have completely different MoA. Based on similarity evaluation, XYPI has similar pathway fingerprints as NSAIDs, but has similar target profile with GCs. The expression pattern of genes in LPS-activated macrophages after XYPI treatment is similar to that after NSAID but not GC treatment, and this experimental result is consistent with the computational prediction based on pathway fingerprints. These results imply that the pathway fingerprints of drugs have potential for drug similarity evaluation. This study used XYPI as an example to propose a new approach for investigating the pharmacological mechanism of natural products using pathway fingerprint similarity based on a “drug-target-pathway” heterogeneous network.

Cerebral ischemia has led to a high rate of both disability and mortality with massive healthcare costs. Although transcriptional regulation is typically mediated by different combinations of TFs, a combined regulatory unit to synergistically activate transcription has remained unclear in cerebral ischemia, especially in different drug treatments. In this study, TFs alterations after 6 h cerebral ischemic injury and repair were performed by a concatenated tandem array of consensus transcription factor response elements (catTFREs), and vital TFs were obtained by TFs-target imbalanced network. Drug intervention used Danhong injection (DHI) and BNC (BuChang NaoXinTong Capsules), which has been widely prescribed in Chinese herb medicine for the treatment of cerebrovascular and cardiovascular diseases. There were 198 TFs identified after 6 h MCAO operation, and six TFs (Sox2, Smad3, FoxO1, Creb1, Egr,1 and Smad4) were considered as critical TFs in response to cerebral ischemia. Moreover, Smad3 was identified as a hub TF among six vital TFs, and the transcription activity of Smad3 was further verified. These 6 TFs were all reversed by DHI or BNC, indicating different medications may regulate different transcription factors through TF synergy. Moreover, validation results indicated that Smad3 was a putative target TF for DHI and BNC-mediated protection against cerebral ischemia. The observations of the present study provide a fresh understanding of biomolecules and possible new avenues for therapeutic interventions, in addition to the new intervention pattern for different treatments for ischemia stroke.

Retinal degenerative diseases are related to retinal injury because of the activation of the complement cascade, oxidative stress-induced cell death mechanisms, dysfunctional mitochondria, chronic neuroinflammation, and production of the vascular endothelial growth factor. Anti-VEGF therapy demonstrates remarkable clinical effects and benefits in retinal degenerative disease patients. Hence, new drug development is necessary to treat patients with severe visual loss. He xue ming mu (HXMM) tablet is a CFDA-approved traditional Chinese medicine (TCM) for retinal degenerative diseases, which can alleviate the symptoms of age-related macular degeneration (AMD) and diabetic retinopathy (DR) alone or in combination with anti-VEGF agents. To elucidate the mechanisms of HXMM, a quantitative evaluation algorithm for the prediction of the effect of multi-target drugs on the disturbance of the disease network has been used for exploring the specific pathology of HXMM and TCM precision positioning. Compared with anti-VEGF agents, the drug disturbance of HXMM on the functional subnetwork shows that HXMM reduces the network robustness on the oxidative stress subnetwork and inflammatory subnetwork to exhibit the anti-oxidation and anti-inflammation activity. HXMM provides better protection to ARPE-19 cells against retinal injury after H₂O₂ treatment. HXMM can elevate GSH and reduce LDH levels to exhibit antioxidant activity and suppress the expression of IL-6 and TNF-α for anti-inflammatory activity, which is different from the anti-VEGF agent with strong anti-VEGF activity. The experimental result confirmed the accuracy of the computational prediction. The combination of bioinformatics prediction based on the drug attack on network robustness and experimental validation provides a new strategy for precision application of TCM.