PNS was purchased from Guangxi Wuzhou Pharmaceutical Group Co., Ltd., (China Food and Drug Administration approval no. Z20025652), which was mostly made up of ginsenoside Rb1 (29.7%), ginsenoside Rd (7.3%), ginsenoside Rg1 (28.0%), notoginsenoside R1 (6.9%) and ginsenoside Re (3.8%).

The present study obtained approval from the Ethics Committee of Youjiang Medical College for Nationalities Institutional Review Board. A total of 60 male Sprague-Dawley (SD) rats of a specific pathogen-free grade (age, 7-weeks; weight, 200±20 g) were purchased from Changsha Tianqin Biotechnology Co., Ltd. [License Number: SCXK(Xiang)2014-0011]. SD rats were housed under 50–60% humidity, 12/12 h light/darkness, and 20±2°C room temperature with free access to drinking water and food. SD rats were randomly divided into three groups: Control group (rats without any treatment, n=10), Sham operation group (sham, n=10) and MCAO group (model, n=40). SD rats were fasted overnight, but allowed ad libitum access to water in the Youjiang Medical College [License Number: SYXK(Gui)2017-0004]. Rats were anesthetized for surgery by inhalation of isoflurane (4% isoflurane for induction and 2% for maintenance). After 3 min, SD rats were checked to ensure they were fully anesthetized, this was performed using the following methods: i) induction box was shaken gently, if the rat's body fell over to the side-lying position and it did not try to restore its prone position, it indicated that the animal was fully anesthetized; and ii) the toes of the rat were lightly clamped by tweezers, if the rat did not react after pinching, the rat was judged to be in a deep anesthesia. Then, MCAO rat models were established as previously described by Longa et al (18). Briefly, the common carotid artery (CCA), internal carotid artery (ICA) and external carotid artery (ECA) were carefully exposed after anesthesia. Then, the CCA and ECA were tied with a loose surgical knot. The ICA was tied with a loose knot with a silk suture. A small incision was made in the ECA and a silicon-coated monofilament was introduced through the CCA into the ICA 20 mm beyond the carotid bifurcation to occlude the MCA for 1 h. Following surgery, the rats were housed individually and closely monitored for changes in behavior and vital signs. Neurological scores were assessed when rats awoke according to neurological deficit scores, where a score of 3–5 indicates that the model was successfully established (18). Then, MCAO rats were randomly divided into four groups: i) MCAO group; ii) 50 mg/kg PNS treatment group; iii) 100 mg/kg PNS treatment group; and iv) 150 mg/kg PNS treatment group. PNS treatment groups were given PNS by intragastric treatment once every 2 days. The rats in the control and sham groups were treated with the same volume of normal saline. Rats in the sham groups underwent an operation to expose the CCA, ICA and ECA after anesthesia but the ligation was not performed. Rats in the control group did not undergo surgery.

In order to minimize pain, the following characteristics were considered humane endpoints to the study that required immediate intervention: Infection at the surgical site, wound dehiscence, rapid weight loss (>20% body weight loss), becoming cachectic, self-harming, biting or aggressiveness, and difficulty eating, drinking or moving around freely. No rat was euthanized throughout the experiment. After treatment for 7 days, the rats were euthanized with pentobarbital (intraperitoneal injection, 120 mg/kg). Cervical dislocation was used to confirm death.

After treatment, the neurological deficit was evaluated by two researchers who were unaware of the groupings using a neurological deficit score as previously described (18,19). Then, the extent and localization of the cerebral lesion were determined by TTC staining. Briefly, the brain was dissected in the coronal plane at the bifurcation of MCA and anterior cerebral artery, and the cerebral lesion was visualized by TTC staining. Briefly, 2-mm thick brain tissue sections were stained with 2% TTC solution at 37°C for 30 min, followed by fixation with 4% paraformaldehyde. TTC-stained sections were photographed with a D5600 camera (Nikon). The digital images were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc.).

Total RNA from sham, MCAO and 100 mg/kg PNS treatment groups was extracted using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.). RNA concentration was measured using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, Inc.) at 260/280 nm and a Bioanalyzer 4200 (Agilent Technologies, Inc.). The libraries were prepared using a VAHTS mRNA-seq v2 Library Prep Kit for Illumina^® (cat. no. NR601-01; Vazyme Biotech Co., Ltd.). The reverse transcription was conducted at 25°C for 10 min, 42°C for 15 min, then 70°C for 15 min. Adapters were ligated at the 3′ and 5′ ends, and templates were amplified using PCR. The PCR products were further purified using VAHTS™ RNA Clean Beads (cat. no. N412-01; Vazyme Biotech Co., Ltd.) according to the manufacturer's protocol and used for sequencing. mRNAs were analyzed by deep sequencing using a HiSeq X Ten Reagent kit v2.5 (cat. no. FC-501-2521; Illumina, Inc.) on an Illumina HiSeq X sequencing platform (pair-ended; 150-bp reads; Illumina, Inc.).

The sequencing data was filtered using SOAPnuke (version 1.5.2), and clean reads were obtained after removing: i) reads containing sequencing adapters; ii) reads with low-quality base ratio >20% (base quality ≤5); and iii) reads with unknown base (N base) ratio >5%. Differentially expressed genes were selected as having >two-fold differences between their geometrical mean expression in the compared groups, and a statistically significant P-value (<0.05) by analysis of DEseq2. Gene Ontology (GO) analysis of differentially expressed genes was performed with an R package with cluster profiler using P<0.05 to define statistically enriched GO categories (20–22). Pathway analysis was used to determine the significant pathway of the differential genes according to Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/) (23–25). Protein-protein interactions (PPI) were analyzed with the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING; http://string-db.org) (26). Hub genes were analyzed by CytoHubba app in Cytoscape (27).

cDNA synthesis was carried out using PrimeScript™ RT reagent kit (Takara Biotechnology Co., Ltd.). The conditions of reverse transcription were as follows: 30°C for 10 min and 42°C for 60 min, followed by 85°C for 10 min. RT-qPCR was performed using the SYBR^® Premix ExTaq™ II kit (Takara Biotechnology Co., Ltd.) on a 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The RT-qPCR thermocycling conditions consisted of an initial denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 65°C for 32 sec. The relative mRNA expression levels were calculated using the 2^−ΔΔCq method (28). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal reference gene. The sequences of primers are shown in Table I.

Experimental data are presented as the mean ± standard deviation. All the statistical analyses were performed using SPSS 19.0 statistical software (IBM Corp.) with one-way ANOVA followed by a Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Initially, no neurological deficit and infarction were observed in the Control and Sham groups. Compared with the Model group, it was found that PNS treatment attenuated the neurological deficit and infarction area (Fig. S1). Additionally, aberrant mRNA expression profiles were analyzed between the Sham and Model groups, and between the Model and PNS-treated groups. According to the cut-off criteria 1,104 genes of interest were selected, where 690 genes were upregulated and 414 were downregulated in the Model group compared with the Sham group. Additionally, 817 genes of interest were selected, where 390 genes were upregulated and 427 were downregulated in the PNS-treated group compared with the Model group. Aberrant mRNA expression profiles between the Sham and Model groups, and between the Model and PNS-treated groups were identified using hierarchical cluster analysis of the data (Fig. 1). The 10 most upregulated and downregulated genes between the Sham and Model groups, and between the Model and PNS-treated groups are shown in Table II.

GO functional enrichment analysis revealed that when comparing the Sham and Model groups, the upregulated genes involved 3,232 ‘biological processes’, 477 ‘cellular components’ and 830 ‘molecular functions’. The downregulated genes involved 3,240 ‘biological processes’, 350 ‘cellular components’ and 561 ‘molecular functions’. When comparing the Model and PNS-treated groups, GO functional enrichment analysis revealed that the upregulated genes involved 2,610 ‘biological processes’, 352 ‘cellular components’ and 445 ‘molecular functions’. The downregulated genes involved 3,963 ‘biological processes’, 384 ‘cellular components’ and 723 ‘molecular functions’. The top 10 significant ‘biological process’, ‘cellular component’ and ‘molecular functions’ identified by GO enrichment analysis for differentially expressed genes between the Sham and Model groups, and between the Model and PNS-treated groups are shown in Fig. 2. GO analysis suggested that genes upregulated following PNS treatment were primarily related to ‘condensed chromosome’, ‘RAGE receptor binding’ and ‘meiotic chromosome segregation’. Downregulated genes after PNS treatment were mainly involved in ‘presynapse’, ‘glutamate receptor binding’, ‘neuromuscular process’. KEGG analysis was performed to enrich the signaling pathways of differentially expressed genes between the Sham group and Model group or between the Model group and PNS-treated groups. KEGG analysis revealed that the upregulated genes between the Sham and Model groups were involved in 258 signaling pathways, whereas the downregulated genes between the Sham and Model groups were involved in 164 signaling pathways. Additionally, KEGG analysis revealed that the upregulated genes between the Model and PNS-treated groups were involved in 258 signaling pathways, whereas the downregulated genes between the Model and PNS-treated groups were involved in 226 signaling pathways. The top enrichment results are shown in Fig. 3. KEGG analysis indicated that upregulated genes after PNS treatment were primarily involved in the ‘Herpes simplex virus 1 infection’, ‘Oocyte meiosis’ and ‘Cell cycle’, whereas downregulated genes after PNS treatment were related to ‘Glutamatergic synapses’, ‘Rap1 signaling pathway’ and ‘Calcium signaling pathway’.

PPI network analysis is important for understanding the interactions that occur between the proteins. The results of the PPI network analysis are shown in Fig. 4. Based on PPI network analysis, transmembrane protease, serine 2 (Tmprss2), dedicator of cytokinesis 8 (Dock8), 2′-5′ oligoadenylate synthase (Oasl2), mannose receptor, C type 2 (Mrc2), glycoprotein (transmembrane) nmb (Gpnmb), lysosomal protein transmembrane 5 (Laptm5), colony stimulating factor 1 receptor (Csf1r), transmembrane protein 176A (Tmem176a), AABR07035470.1 and WDFY family member 4 (Wdfy4) were the top 10 differentially expressed genes between the Sham and Model groups. Furthermore, the top 10 differentially expressed genes between the Model group and PNS-treated groups were ubiquitin conjugating enzyme E2 variant 2 (UBE2V2), small RNA binding exonuclease protection factor La (SSB), peptidylprolyl isomerase D (Ppid), CCHC-type zinc finger, nucleic acid binding protein (Cnbp), Zinc finger protein 788 (Zfp788), zinc finger protein 182 (Zfp182), AABR07035107.1, polyribonucleotide nucleotidyltransferase 1 (Pnpt1), required for meiotic nuclear division 5 homolog A (Rmnd5a) and C1GALT1-specific chaperone 1 (C1galt1c1).

To analyze the aberrant expression of mRNAs that have important roles in ischemic stroke and the underlying mechanisms of PNS treatment, the overlapping genes of interest identified between Sham and Model groups, and between the Model and PNS-treated groups were analyzed using Venn diagrams, which demonstrated 303 overlapping genes (Fig. 5A). These 303 mRNAs were analyzed using STRING, and the hub genes were analyzed using CytoHubba to identify maximum neighborhood component (MNC) centrality in the Cytoscape software. Based on MNC centrality, the top 10 differentially expressed genes were identified as UBE2V2, small ubiquitin-related modifier 1 (SUMO1), SSB, Finkel-Biskis-Reilly murine sarcoma virus ubiquitously expressed (FAU), centrosomal protein 290 kDa (CEP290), DNA-directed RNA polymerase II subunit K (POLR2K), cullin-4B (CUL4B), matrin-3 (MATR3), vascular endothelial growth factor receptor 2 (KDR) and pre-mRNA-processing factor 40 homolog A (PRPF40A) (Fig. 5B). Additionally, the present study also found that these top 10 genes were involved ‘nucleotide excision repair’, ‘positive regulation of catabolic process’, ‘cell cycle’ and ‘regulation of cell cycle process’ (Fig. 5C). The changes in expression in the top 10 genes are shown in Table III. The results showed that the expression levels of UBE2V2, SUMO1, SSB, CEP290, POLR2K, CUL4B, PRPF40A and MATR3 were significantly reduced in the Model group compared with the Sham group, but was upregulated in the PNS-treated group. Expression of FAU and KDR was significantly increased in the Model group compared with the Sham group, but decreased in the PNS-treated group. Finally, the expression levels of UBE2V2, SUMO1, SSB, FAU, CEP290, POLR2K, CUL4B, MATR3, PRPF40A and KDR were analyzed using RT-qPCR in the Control, Sham, Model, 50 mg/kg PNS, 100 mg/kg PNS and 150 mg/kg PNS treatment groups (Fig. 6). The expression levels of UBE2V2, SUMO1, SSB, CEP290, POLR2K, CUL4B, PRPF40A and MATR3 were significantly reduced, whereas FAU and KDR expression was significantly increased in the Model group compared with the Sham group. PNS treatment appeared to reverse the trends in gene expression regulation; the effects of 100 and 150 mg/kg PNS treatment groups were more significant than that of the 50 mg/kg PNS treatment group.