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ORIGINAL ARTICLE
Year : 2023 | Volume
: 9
| Issue : 2 | Page : 201-211
Network pharmacology-based and pharmacological evaluation of the effects of Curcumae Radix on cerebral ischemia–Reperfusion injury
Shang-Xia Zhang1, Yu-Hong Wang1, Hong-Ping Long2, Jian Liu2, Hong-Qing Zhaoa1, Jian Yi2, Jia Ling1
1 Science and Technology Innovation Center/State Key Laboratory Breeding Base of Chinese Medicine Powder and Innovative Medicine, Hunan University of Chinese Medicine Changsha 410208, China
2 Central Laboratory, The First Affiliated Hospital of Hunan University of Chinese Medicine, Changsha 410007, China
Correspondence Address:
Dr. Hong-Ping Long
Central Laboratory, The First Affiliated Hospital of Hunan University of Chinese Medicine Changsha 410007
China
Dr. Yu-Hong Wang
Science and Technology Innovation Center/State Key Laboratory Breeding Base of Chinese Medicine Powder and Innovative Medicine, Hunan University of Chinese Medicine Changsha 410208
China
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/2311-8571.370154
Objective: This study aimed to investigate the network pharmacology of curcumae radix (CR, Yujin) and explore the mechanism of CR in the treatment of cerebral ischemia–reperfusion injury (CIRI). Materials and Methods: Network analysis and pharmacological evaluation were performed to explore the protective role of CR to treat CIRI. The potential target genes of the active components and CIRI were identified using SwissTarget Prediction, Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine, GeneCards, and Online Mendelian Inheritance in Man. Furthermore, network analysis was performed using Cytoscape software. Gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were performed using the R software. In vivo experiments were performed using the water extract of CR (WECR) on PC12 cells induced by hypoxia/reoxygenation (H/R) to simulate ischemia/reperfusion injury. Results: The results exhibited that 21 active compounds identified in CR were associated with 73 targets of CIRI. Functional analysis showed that multiple pathways, including response to stress, regulation of apoptotic process, and hypoxia-inducible factor 1 signaling pathway, were significantly enriched. In addition, STAT3, IL4, HIFIA, and CTNNB1 were predicted to be the most important genes among the 36 hub genes. Furthermore, WECR treatment significantly improved PC12 cell injury and decreased apoptosis levels in cells induced by H/R, with malondialdehyde contents reduced and superoxide dismutase or glutathione peroxidase levels increased. Conclusions: Network analysis and pharmacological evaluation of CR could provide valuable directions for further research on CR and improve comprehension of CIRI.
Keywords: Cerebral ischemia–reperfusion injury, curcumae radix, network pharmacology, pharmacological evaluation
Cerebral ischemia–reperfusion injury (CIRI) is a complicated and heterogeneous disease that seriously endangers human health.[1] Several studies have shown that it may lead to multiple complications, such as ischemia, collateral circulation, distal microthrombosis, systemic blood pressure changes, and comorbidities.[2],[3] Although current treatment strategies, such as recovery of cerebral blood flow by thrombolysis and revascularization, are safe and effective to cure CIRI, these methods are only applied in the very early stages when the patient has suffered few strokes. Therefore, it is important to identify potential therapeutic drugs to treat CIRI. Recently, many studies have demonstrated that natural products, an alternative source for the treatment of CIRI, have attracted increasingly more attention, owing to traditional and multitarget characteristics.[4],[5]
Curcumae radix (CR, Yujin), the dried root tuber of Curcuma longa L., is frequently used in traditional medicine in South and Southeast Asia.[6] Several reports have demonstrated its diverse pharmacological effects, including anti-inflammatory, anticancer, antiviral, and antimicrobial.[7],[8] For example, CR and its active compounds, such as curcuminoids, can exert anticancer effects by regulating multiple cell-signaling pathways involving proliferation and apoptosis.[9] Pawar et al. found that curcumin extracted from C. longa L. has an apparent inhibitory effect on aspirin-delayed wound healing.[10] A previous study showed that curcuma rhizome lectin is a mannose-binding protein of the nonseed part of CR that has antifungal, antibacterial, and glucosidase inhibitory properties.[11] Furthermore, it was demonstrated that CR diterpenoid C could play a role in inhibiting proliferation and inducing apoptosis of cancer cells.[12] However, the potential effects of CR on CIRI remain unclear.
Network pharmacology is a strategy developed in recent years to clarify the complex pharmacological problems in the discovery of new drugs for traditional Chinese medicines (TCMs), which could reduce the development costs of drugs.[13],[14],[15] Numerous studies have indicated that network pharmacology is a suitable method for systematically predicting the active components, molecular mechanisms, potential targets, and drug interactions of TCMs.[16],[17] For instance, Jin et al. reported that Astragalus membranaceus might play an important role in the treatment of diabetic retinopathy through multiple targets and metabolic pathways based on network pharmacology.[18] Similarly, a recent study showed that network pharmacology analyses might provide more convincing evidence for the study of the therapeutic effects of Xiao Luo Wan for on uterine fibroids.[19]
Through network and functional enrichment analyses, this pharmacological study was established based on the currently identified active components of CR and potential effects related to CIRI. The information of effects of the active ingredients of CR and targets of CIRI from several databases was first investigated. Furthermore, in vivo experiments were performed to explore the potential effects of water extract of CR (WECR) on PC12 cells induced by hypoxia/reoxygenation (H/R), which is widely applied to model building of ischemia–reperfusion (I/R) injury. For example, researchers had reported a model of H/R using zebrafish larvae, which could be used for rapid in vivo screening and efficacy evaluation for I/R injury.[20] The study provides potential evidence for exploring the active roles of CR in the treatment of CIRI.
Materials and MethodsActive component screening of curcumae radix from chemical components database
All active components of CR were manually collected from the TCM Systems Pharmacology Database and Analysis Platform (https://tcmsp-e.com/?qn = 449) and Bioinformatics Analysis Tool for Molecular mechANism of TCM (BATMAN-TCM, http://bionet.ncpsb.org.cn/batman-tcm/).[21],[22] The active components for CR were further filtered by integrating oral bioavailability (OB) ≥30% and drug-likeness (DL) ≥0.18[23],[24] or by searching literatures related to CIRI.
Prediction for component targets of curcumae radix and targets of cerebral ischemia–reperfusion injury
All potential active components, except for curdione and zingiberene, were loaded into the PubChem Database (http://pubchem.ncbi.nlm.nih.gov/) to acquire canonical SMILES. Canonical SMILES was imported into the SwissTargetPrediction database (http://www.swisstargetprediction.ch/) to estimate the most probable macromolecular targets of a small molecule.[25] However, the targets of potential active components of curdione and zingiberene were acquired using the BATMAN-TCM database. In addition, the potential targets for CIRI were obtained by integrating the results from GeneCards (https://www.genecards.org/) and Online Mendelian Inheritance in Man (OMIM, https://omim.org/).[26],[27]
Construction of compound-target network and pathway enrichment analyses
The above-mentioned potential active compounds of CR and their potential targets were inserted into Cytoscape 3.7.2 to plot the compound-target network.[28] The Search Tool for the Retrieval of Interacting Genes (STRING) database (https://string-db.org/) was used to obtain protein–protein interaction (PPI) data and acquire information on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment.[29] Furthermore, Molecular Complex Detection (MCODE), used to cluster highly related genes from Cytoscape 3.7.2, was applied to search hub genes. GO and KEGG enrichment analysis were presented using ggplot2 to visualize the data for functional analysis.
Method of obtaining water extract of curcumae radix
CR was obtained from the First Affiliated Hospital of the Hunan University of Chinese Medicine (Changsha, Hunan, China). WECR was extracted as follows: 50 g of CR was added to a 1000 mL round-bottom flask, and then ultrapure water of 10 times the amount of CR was added to the flask, then heated, boiled, refluxed for 1.5 h in the condenser, cooled down, and filtered with three layers of gauze. Subsequently, 8 times the amount of ultrapure water of CR was added to the flask, and the above-mentioned operations were repeated. Subsequently, the two water extracts were merged and concentrated using a rotary evaporator. Finally, the concentrated sample obtained underwent WECR.
Multiple reagents, including cells of rat pheochromocytoma PC12 cell line, Roswell Park Memorial Institute 1640 medium, phosphate buffered saline (PBS), Cell Counting Kit-8 (CCK-8), 75% alcohol, fetal bovine serum (FBS), trypsin, penicillin-streptomycin solution, dimethyl sulfoxide, paraformaldehyde, Triton-100, malondialdehyde (MDA) ELISA Kit, superoxide dimutase (SOD) ELISA Kit, Hoechst Kit, and glutathione peroxidase (GSH-Px) ELISA Kit were all purchased from Minghua Biotechnology Consulting Service (Changsha, Hunan, China).
The instruments used were New Brunswick an Eppendorf company Galaxy 170R carbon dioxide (CO2) (Eppendorf, Germany), Enspire full wavelength multifunctional enzyme labeling instrument (PerkinElmer, USA), LSM 800 (Zeiss, Germany), and New Brunswick an Eppendorf company Galaxy 48R CO2 (Eppendorf, Germany).
Primary culture of PC12 cells
PC12 cells were cultured adaptively under 5% CO2 at 37°C in a 170R CO2 incubator with 1640 medium containing 5% FBS and 1% penicillin-streptomycin solution. After the cells grew to the bottom of the culture bottle, the culture dish was washed twice with PBS, 0.25% trypsin was added and digested for 2 min, and 1640 culture medium was added to terminate digestion and centrifuged at 1200 rpm for 5 min. Then, the cells were inoculated in the culture bottle, and the cells were used for the experiment when they formed a monolayer.
Model building for water extract of curcumae radix treatment in response to PC12 cell injury induced by hypoxia/reoxygenation
Cell model building was carried out by improving the method reported by Zou et al.[20] The PC12 cells obtained were diluted to 104 cells/mL with 1640 medium and inoculated into 96-well plates, with 200 μL added into each well for 24 h. In the control group, the PC12 cells obtained were replaced with 1640 culture medium containing 5% FBS and cultured for 3 h. Culture medium was changed again and cultured for 3 h. However, the model and experimental group cells were both replaced with 1640 medium without sugar and exposed to hypoxia for 3 h with 1% oxygen (O2) and 5% CO2 in a 48R CO2 incubator. Concentrations of WECR were added to the groups: 0.125, 0.25, 0.5, 1, 2, and 4 mg/mL, respectively, with reoxygenation performed for 24 h. In the above-mentioned steps, H/R was applied to simulate CIRI.
Observation of cell morphology and detection of cell survival, apoptosis level, superoxide dimutase, malondialdehyde, or glutathione peroxidase levels
According to the above-mentioned treatments, the treated PC12 cells were divided into four groups for different experiments. Some cells were observed under a microscope. After cells were again replaced with 1640 medium, 10 μL CCK-8 was added into each well and incubated at 37°C for 3 h, and some cells were used to detect survival. The absorbance of cells in each group at 450 nm was detected using an Enspire full wavelength multifunctional enzyme labeling instrument. In addition, some of the remaining cells were applied to detect apoptosis; specifically, treated cells were fixed with 0.3 mL paraformaldehyde for 45 min in each well, discarding the solution, and washed twice with PBS, and 0.2 mL of 0.25% triton-100 was added into each well for 10 min. Again, the solution was discarded and washed twice with PBS, and then 2 mL Hoechst staining solution (1:400) was added to each well for 15 min. Finally, the cells were washed with PBS 3 times, and 0.3 mL PBS solution was added to each well. Apoptosis was observed under the LSM800. The supernatant of the remaining treated cells was centrifuged at 3000 rpm for 10 min and then analyzed using the MDA ELISA Kit, SOD ELISA Kit, and GSH-Px ELISA Kit to investigate SOD, MDA, and GSH-Px levels, respectively.
Statistical analysis
Statistical analyses were performed using SPSS21.0 software (Statistical Product Service Solutions). Data are presented as mean ± standard deviation and were calculated from three replicates. One-way ANOVA was used to compare the differences among groups, and the least significant difference test was used when the variance was coincident, whereas Tamhane's T2 was used when the variance was uneven. P < 0.05 was considered statistically significant differences, and P < 0.01 was considered highly significant. The steps of this study are shown in [Figure 1].
ResultsPotential active components and targets of curcumae radix against cerebral ischemia–reperfusion injury
In our study, 21 potential active components were identified in CR, and these are shown in [Table 1]. Some of these components were filtered by OB ≥30% and DL ≥0.18, including naringenin, zedoalactone A, oxycurcumenol, (E)-5-hydroxy-7-(4-hydroxyphenyl)-1-phenyl-1-heptene, (5R,6E)-5-hydroxy-1,7-diphenyl-6-heptene-3-one, curcumenolactone C, 3-Epi-beta-Sitosterol, and beta-sitosterol. Other components were obtained by searching the literature related to CIRI involving curcumin, demethoxycurcumin, vanillic acid, (L)-alpha-terpineol, (−)-alpha-pinene, (Z)-caryophyllene, eugenol, zerumbone, p-coumaric acid, cinnamaldehyde, myrcene, zingiberene, and curdione.[30],[31],[32],[33],[34],[35],[36],[37],[38],[39],[40],[41] The 21 components of CR resulted in 2169 targets. After removing duplicate targets, 918 potential nonrepetitive targets were identified. The above-mentioned targets were further intersected with 272 target genes for CIRI obtained from GeneCards (relevance scores >10) and OMIM. Finally, 73 genes were identified as targets of the 21 components of CR against CIRI. The relevance between the active components of CR and potential targets is shown in [Figure 2].
Figure 2: The compound-target network for CR active components and candidate targets related to CIRI. Green circles represent components and yellow triangles represent gene targets, CR: Curcumae radix, CIRI: Cerebral ischemia–reperfusion injuryGene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of candidate targets for curcumae radix treating cerebral ischemia–reperfusion injury
GO enrichment analysis results were obtained from the STRING database of the 73 target genes. The top 10 terms were selected to draw the histograms for biological process (BP), cellular component (CC), and molecular function (MF) [Figure 3]. The top 10 enrichment results of BP and the target of CR treatment of CIRI were response to oxygen-containing compounds, positive regulation of signaling, positive regulation of MF, regulation of biological quality, positive regulation of cell communication, response to external stimuli, response to stress, positive regulation of signal transduction, response to organic substances, and positive regulation of response to stimuli [Figure 3]. According to the CC analysis, the endomembrane system, cytoplasmic vesicle, vesicle lumen, membrane raft, secretory granule lumen, plasma membrane, vesicle, secretory granule, secretory vesicle, and cell surface were the top 10 results [Figure 3]. The top 10 MF enrichment results were protein binding, signaling receptor binding, identical protein binding, enzyme binding, enzyme binding, signaling receptor activator activity, binding, phosphatase binding, protein phosphatase binding, and receptor ligand activity [Figure 3]. In addition, the KEGG analysis results were analyzed, and the top 15 count values were selected to draw a bubble chart [Figure 4]. The top 15 relevant pathways involved in CR protection against CIRI were cancer, AGE-RAGE signaling pathway in diabetic complications, proteoglycans in cancer, hypoxia-inducible factor (HIF-1) signaling pathway, fluid shear stress and atherosclerosis, Chagas disease, Kaposi sarcoma-associated herpesvirus infection, MAPK signaling pathway, Alzheimer's disease, hepatitis B, microRNAs in cancer, inflammatory mediator regulation of TRP channels, relaxin signaling pathway, human cytomegalovirus infection, and toxoplasmosis.
Figure 3: GO enrichment analysis significantly enriched in the top 30 of candidate targets. BP: Biological process; CC: Cellular component; MF: Molecular function, GO: Gene ontology
Figure 4: Bubble chart of KEGG enrichment analysis significantly enriched in the top 15 of candidate targets, KEGG: Kyoto Encyclopedia of Genes and GenomesFunction-related protein interaction network
The 36 target genes with high connectivity degrees (MCODE_Score ≥20) were selected as hub genes for CIRI treatment. The hub genes were STAT3, VEGFA, TNF, NOS3, ACE, HMOX1, EDN1, INS, MMP2, ALB, TGFB1, SERPINE1, MAPK8, MPO, CAT, NOS2, APOE, CRP, JAK2, IL1B, TP53, EGFR, APP, PPARG, MTOR, HIF1A, TLR4, MAPK1, CASP3, IL10, CTNNB1, IGF1, MAPK14, IL4, PTGS2, and MMP9. The hub genes are thought to play an important role in the treatment of CIRI. The PPI network among these genes is shown in [Figure 5]. Furthermore, STAT3, IL4, HIFIA, and CTNNB1 were predicted to be important genes with a higher degree of connectivity compared to other genes [Figure 5].
Figure 5: PPI network of hub genes of CR, CR: Curcumae radix, PPI: Protein–protein interactionWater extract of curcumae radix treatment on survival rate effects of PC12 cell injury induced by hypoxia/reoxygenation
To further explore the effect of CR on CIRI, the H/R model was applied to simulate CIRI and investigate the role of WECR treatment on PC12 cell injury induced by H/R. The results show that different concentrations of WECR treatment significantly improved PC12 cell injury, and PC12 cell numbers were markedly increased compared to those in the model group [Figure 6]. Further results of the experiment on PC12 cell survival rate show that compared to the control group, PC12 cells in the model group have significantly lower OD values and survival rates (P < 0.01); compared to the model group, WECR treatment for PC12 cell injury induced by H/R significantly increases their survival rate (P < 0.05) [Table 2].
Figure 6: Cell morphological observation of PC12 cells in each group after treatments via different concentrations of WECR, WECR: Water extract of curcumae radix
Table 2: The cellular OD value and survival rate of PC12 cells in each group after treatments via different concentrations of water extract of curcumae radixWater extract of curcumae radix treatment on apoptosis effects of PC12 cell injury induced by hypoxia/reoxygenation
Based on the CCK-8 experimental results, we hypothesized that WECR would protect against H/R-induced PC12 cell damage. PC12 cells under H/R conditions were observed using Hoechst staining [Figure 7]a. Compared to untreated cells, PC12 cells under H/R conditions were severely damaged, chromosomes were concentrated and broken, and cell membrane structures were blistered to form apoptotic bodies that appeared dark and shiny during Hoechst staining. However, WECR treatment showed a strong protective effect on PC12 cell injury induced by H/R. Furthermore, compared to the model group (except for the WECR_0.125 group), apoptosis levels of cells in the other groups significantly decreased, especially in the WECR_1 group [Figure 7]b.
Figure 7: Effects of WECR on the apoptosis of PC12 cells. (a) Representative immunofluorescence images of PC12 cells in each group after treatments via different concentrations of WECR; (b) the apoptosis levels of PC12 cells in each group after treatments via different concentrations of WECR. **p < 0.01 represents statistically significant difference compared with the control group; #P < 0.05 and ##P < 0.01 represent statistically difference and statistically significant difference compared with the model group, WECR: Water extract of curcumae radixWater extract of curcumae radix treatment on effects of superoxide dimutase, malondialdehyde, and glutathione peroxidase in hypoxia/reoxygenation-stimulated PC12 cells
The effects of WECR on the levels of SOD, MDA, and GSH-Px in H/R-stimulated PC12 cells were also investigated [Figure 8]. The levels of SOD and GSH-Px were significantly decreased, whereas the MDA content was obviously increased in both model cells and WECR-treated PC12 cells compared to the control group. Interestingly, the WECR groups showed significantly reduced MDA content compared to the model cells. The SOD and GSH-Px levels (except for WECR_0.125), were obviously increased in WECR-treated cells compared to the model cells.
Figure 8: Effects of WECR on oxidative stress index of PC12 cell supernatant.(a) Detection of SOD activities of PC12 cell supernatant in each group; (b) detection of MDA activities of PC12 cell supernatant in each group; (c) detection of GSH-PX activities of PC12 cell supernatant in each group. **P < 0.01 represents statistically significant difference compared with the control group; #P < 0.05 and ##P < 0.01 represent statistically difference and statistically significant difference compared with the model group, WECR: Water extract of Curcumae radix, SOD: Superoxide dimutase, MDA: Malondialdehyde, GSH-PX: Glutathione peroxidase Discussion CIRI is one of the main causes of death and acquired disability in adults worldwide.[42] Currently, H/R has been reported as a classic model with optimal relevance to I/R injury.[43] Furthermore, combined therapeutic strategies, together with herbal medicines that could exert their pharmacological effects on multiple targets using their multicomponent framework, were gradually developed, with the limitations of previous therapeutic technologies.[44],[45] Numerous studies have reported that through network pharmacology analysis, the therapeutic effect of medicinal plants could be confirmed from the aspects of multicompound, multiprotein/gene, and multitarget synergistic effects.[46],[47],[48] In this study, the H/R model was used on PC12 cells to simulate CIRI to evaluate the effects, with a network pharmacology approach.
To explore the biologically active ingredients and potential pharmacological mechanisms of CR against CIRI, network pharmacology was first applied. In this study, 21 potentially active components of CR were identified [Figure 1] and [Table 1]. Some of these components have been associated with I/R injury.[49],[50],[51] For instance, Raza et al. found that naringenin might be applied as a potential neuroprotectant to deal with high-risk I/R injury by suppressing the nuclear factor kappa B signaling pathway.[49] Furthermore, beta-sitosterol was demonstrated to have protective effects against myocardial I/R injury.[50] Similarly, oxycurcumenol has been reported to prevent PC12 cells from oxidative stress-induced cell damage and might also reduce injuries caused by I/R injury.[51]
Moreover, functional enrichment analysis of candidate targets against CIRI from active components of CR was also carried out through GO and KEGG annotation [Figure 3] and [Figure 4]. GO enrichment analysis revealed that the response to stress and the regulation of apoptotic processes were significantly enriched in BPs [Figure 3]. Previous studies have reported that I/R injury is mainly treated by alleviating stress damage or reducing the level of apoptosis caused by itself.[52],[53] Therefore, CR might be closely related to stress and apoptotic process induced by I/R injury. Furthermore, KEGG pathway enrichment analysis showed that the HIF-1 signaling pathway, AGE-RAGE signaling pathway in diabetic complications, and relaxin signaling pathway were significantly enriched, and multiple studies showed that they were closely related to I/R injury [Figure 4]. For instance, the reactive oxygen species/HIF-1a signaling pathway was closely related to CIRI by applying tetramethylpyrazine with ultrasound.[46] Furthermore, it was also reported that acute treatment together with relaxin protected the kidney against I/R injury.[54]
In addition, 36 hub genes exhibited a high correlation with CR against CIRI [Figure 5]. More importantly, STAT3, IL4, HIFIA, and CTNNB1 showed high connectivity [Figure 5]. Zhang et al. reported that tetrandrine exerted cardioprotective effects in I/R injury via the JAK3/STAT3/Hexokinase II pathway.[55] Similarly, H9C2 cell autophagy under OGD/R injury was decreased by the downregulation of HIF-1α expression.[56] Furthermore, other hub genes were also investigated to obviously associate with CR against CIRI, such as VEGFA, NOS3, MMP9, HMOX1, MTOR, MMP2, MAPK14, PPARG, MPO, and NOS2 [Figure 5], which had been reported that they might participate in alleviating damage caused by I/R.[56],[57],[58],[59],[60],[61],[62],[63] For example, VEGFA can be regulated by sitagliptin applied to protect rats from I/R injury.[64] Above all, CR with these hub genes might play a major role in inhibiting CIRI.
Furthermore, different concentrations of WECR were added to the PC12 cell injury induced by H/R to investigate the effect of WECR on CIRI [Figure 6], [Figure 7] and [Table 2]. The results of cell morphology and activity experiments showed that WECR treatment significantly increased the number of PC12 cells and further improved their survival rate [Figure 6] and [Table 2]. A previous study reported that curcumin, an active ingredient of WECR, exerts a neuroprotective role in the nervous system.[65] Likewise, Wu et al. found that germacrone isolated from Rhizoma curcuma may attenuate injuries caused by cerebral I/R in rats.[66] Further investigation of the effect of WECR treatment on apoptosis in PC12 cell injury induced by H/R showed that apoptosis levels of cells significantly decreased in WECR-treated groups, especially the WECR_1 group [Figure 7]. This was similar to the findings of Kandezi et al., which showed that curcumin might be closely related to antiapoptotic roles.[67] The results imply that WECR might enhance cell vitality and inhibit cell apoptosis, further suggesting that WECR might exert protective action against CIRI.
Finally, SOD, MDA, and GSH-Px concentrations were measured as biomarkers to detect the effect of WECR on CIRI [Figure 8]. These factors were considered important indicators for evaluating the damage caused by I/R and were reported to be closely related to some of the hub genes and functional enrichment pathways mentioned above.[68],[69],[70],[71] In this study, WECR groups had significantly reduced MDA content, while it clearly increased the levels of SOD and GSH-Px compared to the model cells. This is similar to the findings of Kang et al., which showed that garcinol protected against CIRI with decreased MDA and increased SOD activity.[68]
ConclusionsIntegrated network pharmacological analysis and pharmacological evaluation were applied to explore the role of CR to treat CIRI. The 21 potential active components of CR, corresponding to 73 targets, were identified to be closely related to CIRI. GO and KEGG analyses were performed for these 73 targets. Furthermore, 36 of 73 targets were clustered into hub genes; STAT3, IL4, HIFIA, and CTNNB1 were predicted to have a higher degree of connectivity with other hub genes. In addition, WECR was used for PC12 cell injury induced by H/R, which showed that WECR could significantly increase cell survival rate and decrease apoptosis levels in cells induced by H/R. The stress factors SOD, MDA, and GSH-Px were also analyzed. This study may provide a candidate natural product for the treatment of CIRI.
Data availability
Data are available upon request.
Authors' contributions
Yu-Hong Wang and Hong-Ping Long reviewed and edited the manuscript. Shang-Xia Zhang was responsible for writing the original draft, figure drawing, and table design. Liu, Yi, Zhao, and Ling analyzed the data. All of the authors have read and approved the final manuscript.
Acknowledgments
We are thankful for the funding support from national major science and technology project for “Significant New Drugs Creation” (2017ZX09309026), the National Natural Science Foundation of China (grant no. 81874464), Hunan postgraduate innovation project (CX20190566), and postgraduate innovation project of Hunan University of Chinese Medicine (2018CX70).
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References
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