1.Network pharmacology predicts the mechanism and related experimental research on the effective components of Salvia miltiorrhiza and Carthamus tinctorius against cerebral ischemic stroke
Huiyuan ZHU ; Qi MIAO ; Jiang WANG ; Bin LUO ; Haitong WAN ; Wenxuan WANG ; Bingyao DONG ; Shengbin XIAO ; Shan DANG
Journal of Xi'an Jiaotong University(Medical Sciences) 2021;42(3):474-483
【Objective】 To explore the effect mechanism of Salvia miltiorrhiza and safflower on combined anti-ischemic stroke and verify relevant action targets in middle cerebral artery occlusion (MCAO) rat model based on network pharmacology. 【Methods】 ①Traditional Chinese Medicine Systems Pharmacology (TCMSP) and GeneCards databases were used to screen the active components, component targets and ischemic stroke targets of Salvia miltiorrhiza and safflower respectively. The above data were imported into STRING database for protein interaction network analysis, and Cytoscape3.8.0 software was used to construct protein interaction network (PPI) and component target interaction network. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation analysis of target genes were performed using David online analysis tool. ② In this experiment, a rat model of ischemic stroke was prepared by using improved MCAO method, and immunohistochemical method and Real-time quantitative polymerase chain reaction (REAL-TIME PCR) to detect the positive expressions of NLRP3 inflammatory body and NF P65 protein in the brain tissue of rats in each group so as to explore the functional mechanism of anti-inflammation reaction against cerebral ischemia injury. 【Results】 ① A total of 87 effective components, corresponding to 253 targets, 1448 targets for ischemic stroke and 161 targets related to drugs and diseases, were screened from the Salvia milticorrhiza and safflower drug pairs. We obtained 730 biological processes, 81 cell components and 128 molecular functions through GO analysis, and 127 signal pathways through KEGG analysis. ②Immunohistochemical method and Real-time PCR determination results showed that compared with control group rats, model group rats had significantly increased tissue NLRP3 inflammatory body and NFkBp65 protein expressions (P<0.01). Compared with those in the model group, NLRP3 inflammatory body and NFkBp65 protein expressions significantly decreased in Dan red compatibility groups and nim horizon groups (P<0.01). 【Conclusion】 Compatibility of effective components in salvia miltiorrhiza, and carthamus tinctorius can further downregulate the release of inflammatory corpuscle NLRP3 through NFkB signaling pathway by blocking inflammatory lesions and thus plays the role of fighting against inflammatory damage.
2.Effects of Qingguang’an Granules on mitochondrial autophagy of retinal ganglion cells in rats with chronic ocular hypertension
TANG Yu ; ZHU Bingyao ; SHI Jian ; LIU Qianhong ; CHEN Lihao ; PENG Qinghua ; PENG Jun ; YAO Xiaolei
Digital Chinese Medicine 2022;5(3):295-304
Objective To investigate the effect and underlying mechanism of Qingguang’an Granules (青光安颗粒剂, QGAG) on mitochondrial autophagy (mitophagy) of retinal ganglion cells (RGCs) in rats with chronic ocular hypertension (COH). Methods Sixty Sprague Dawley (SD) rats, half males and half females, were randomly assigned to three groups: the control, model, and QGAG (2.5 g/kg) groups, with 20 rats in each group. Rats’ model of COH was established by cauterizing episcleral veins in the model group and QGAG group. Three weeks after successful modeling, rats in the QGAG group were intragastrically administered with QGAG, while rats in the control group and the model group received an equal dose of normal saline. After three months of intragastric administration, intraocular pressure (IOP) of all rats was measured. The mitophagy was monitored by the immunofluorescence method, the mitochondrial membrane potential was measured using the JC-1 method, and the morphological changes of mitophagy in RGCs were observed by transmission electron microscopy. Meanwhile, rat RGCs were labeled using the fluorescent gold method, and RGCs density in each group was calculated. Moreover, RGCs apoptosis was observed by TdT-mediated dUTP Nick-End Labeling (TUNEL) assay. Finally, the expression levels of Parkin, optineurin, microtubule-associated protein 1 light chain 3-II/microtubule-associated protein 1 light chain 3-I (LC3-II/LC3-I), recombinant lysosomal associated membrane protein 1 (LAMP1), and B-cell lymphoma-2 (Bcl-2) in RGCs were determined by Western blot assay. The corresponding mRNAs were detected through quantitative real-time polymerase chain reaction (qRT-PCR). Results The QGAG reduced IOP in COH rats, and inhibited mitophagy and apoptosis of RGCs (P < 0.05). Besides, the QGAG significantly increased the expression levels of Parkin and Bcl-2 (P < 0.05), and inhibited the expression levels of optineurin, LAMP1, and LC3-II/LC3-I (P < 0.05) in RGCs of COH rats. Conclusion The QGAG can inhibit mitophagy in RGCs of COH rats and show a protective effect against optic nerve damage caused by glaucoma, which may be mediated through the mitophagy ubiquitination via the Parkin/PINK1-related pathway.