2.1 A total of 1493 genes for cerebral ischemia-reperfusion were found in Genecards, and some genes were shown in Table 1:
Table1 partial cerebral ischemia-reperfusion gene targets
Cerebral ischemia reperfusion partial genes
|
APP
|
LPL
|
TGFBR2
|
PLAU
|
PRKCA
|
CNTF
|
IRF5
|
MMP10
|
KRIT1
|
EDNRA
|
TSPO
|
NES
|
TGFA
|
MAOB
|
SLC11A2
|
CCL20
|
CST3
|
EDNRB
|
HTRA2
|
SLC12A2
|
MT3
|
GCKR
|
IRAK4
|
ITGA6
|
F2
|
SMAD4
|
SLC9A1
|
SMAD2
|
ITGAL
|
NRG1
|
MIR544A
|
KCNK2
|
IL6
|
RELA
|
GPT
|
EGFR
|
TNFRSF12A
|
CSF1
|
NPR1
|
RYK
|
TNF
|
TGFBR1
|
SLC6A4
|
LEP
|
OPRK1
|
LIF
|
PPARD
|
CNRIP1
|
NOS3
|
ADA
|
CSF1R
|
NOTCH1
|
PRKAA1
|
CLDN5
|
ABCG1
|
CHRDL1
|
COL4A1
|
MIR155
|
PF4
|
BAD
|
AOC1
|
UCN
|
NMT1
|
WNT9A
|
ACE
|
ACTB
|
PTGS1
|
STAT1
|
GCLM
|
ADAMTSL1
|
MIR191
|
CARTPT
|
F5
|
DRD2
|
PRKCE
|
IL2
|
MIR211
|
IRF1
|
CAMK2N1
|
SERPING1
|
NOS2
|
TIMP3
|
PRKAA2
|
BCL2L1
|
DCN
|
NEFL
|
S100A6
|
KCNA5
|
ENG
|
MIR145
|
TOMM40
|
ESR1
|
RTN4
|
HSP90AA1
|
MAP2K7
|
MIR675
|
SOD1
|
PIK3CG
|
MIR34A
|
IKBKG
|
BCL2L11
|
CCK
|
MIR193A
|
MIR134
|
MTHFR
|
CDKN2A
|
PDP1
|
SMPD1
|
G6PC1
|
PRKCD
|
CYP2C9
|
TSPAN18
|
ICAM1
|
PPARA
|
REN
|
GATA4
|
S100A9
|
SLC1A3
|
TGM1
|
SLIT2
|
PIK3CA
|
NLRP3
|
PTK2B
|
IGF1R
|
NTS
|
IL5
|
APOO
|
PTP4A2
|
MPO
|
CDON
|
HP
|
HYOU1
|
CAST
|
C1S
|
AASS
|
CFP
|
GAD1
|
AGER
|
SELL
|
VIP
|
KIT
|
SLC22A3
|
PPP1CA
|
STIP1
|
TP53
|
FN1
|
ADORA3
|
LRP2
|
GUSB
|
MIR221
|
ERBB4
|
TIPARP
|
VEGFA
|
MYH7
|
TNFRSF1A
|
TRAF6
|
S100A8
|
CCND1
|
PPIG
|
MIR342
|
PTEN
|
CD40
|
IL17A
|
GCG
|
GPX3
|
CHGA
|
JUP
|
PRDX2
|
IL10
|
CNR1
|
SYNGAP1
|
MIR142
|
BRCA1
|
MMP14
|
ROCK2
|
CREB3L1
|
MEF2C
|
TTN
|
MME
|
FASLG
|
GSTM1
|
GRM5
|
E2F1
|
ACSS2
|
CASP3
|
STAT3
|
NGB
|
CTSG
|
SLC24A3
|
IDO1
|
FNDC3B
|
ESM1
|
PLAT
|
TNNI3
|
HTR2A
|
SULT1A3
|
LEPR
|
CHKA
|
CCL26
|
ROBO1
|
SMARCA4
|
MIF
|
THBS1
|
NT5E
|
C1R
|
GDF15
|
LBP
|
PIM1
|
MAPT
|
TNNT2
|
TGIF1
|
CAV1
|
PER2
|
TRPM7
|
AK1
|
HSPA1L
|
GFAP
|
PCNA
|
GDNF
|
GABBR2
|
PDE4A
|
ADK
|
S1PR2
|
TRPA1
|
EDN1
|
MBP
|
LAMB1
|
PLSCR1
|
CUL3
|
GJB1
|
COL6A1
|
CHEK1
|
PSEN1
|
NFKB1
|
HSPG2
|
OGG1
|
SIRT3
|
UCP2
|
ARG2
|
SLC3A2
|
SERPINC1
|
XIAP
|
MT-CO1
|
A2M
|
CYSLTR1
|
RETN
|
ZFYVE9
|
MYH10
|
TLR4
|
IL13
|
RPS27A
|
RHOB
|
ABCB7
|
YAP1
|
MIR130B
|
NCK1
|
HIF1A
|
KCNMA1
|
ADIPOQ
|
SLC29A1
|
ANO1
|
TNC
|
ADAMTS20
|
AOX1
|
PRNP
|
ABCA1
|
IL1A
|
PTGIR
|
MIR199A1
|
PNPO
|
NPR3
|
GBA3
|
CTNNB1
|
ITGB2
|
NCF1
|
GCLC
|
UGCG
|
IL1R1
|
MAPKAPK2
|
BIRC2
|
IL1B
|
PLA2G7
|
CDKN3
|
LOC111365141
|
SLC22A2
|
BMP7
|
ZFAS1
|
MEF2D
|
COL3A1
|
MAPK8IP1
|
SH2B3
|
APLNR
|
CXCL11
|
YY1
|
CYP2C8
|
SLC6A19
|
CRP
|
ITGB1
|
GAPDH
|
CIITA
|
RPS6KB1
|
CCN2
|
CPT1A
|
MCM6
|
COL4A2
|
CAMK2A
|
VCP
|
FGF7
|
AGRN
|
HABP2
|
HTR2B
|
PTHLH
|
BCL2
|
TERT
|
AOC3
|
SLC22A8
|
STK11
|
CASP7
|
DLD
|
THY1
|
BDNF
|
BMP6
|
SST
|
P2RY1
|
MMP13
|
MIR107
|
SMPD2
|
SLC25A3
|
XDH
|
DNM1L
|
GSS
|
CGB5
|
CAPN2
|
LGALS3
|
CA9
|
EIF4EBP1
|
NOS1
|
FABP3
|
FGFR2
|
CYGB
|
AIM2
|
APEX1
|
PRKAB1
|
SLC22A4
|
CCL2
|
F8
|
SDHB
|
SERPINA4
|
MAP2K4
|
ACE2
|
TBXAS1
|
STMN1
|
CBS
|
IGFBP3
|
COX5A
|
MIR143
|
TAT
|
MIAT
|
PPIA
|
ATP2A1
|
SLC1A2
|
IL12A
|
ARID1B
|
CD55
|
UNC5B
|
IL15
|
MCAM
|
ATG9A
|
HMOX1
|
CCL3
|
IL2RB
|
MIR186
|
FOXO1
|
MIR140
|
HSF1
|
SLC27A4
|
MMP9
|
CCL5
|
VIPR1
|
TRPV1
|
NDUFA13
|
BDKRB2
|
TNFRSF10B
|
EGR3
|
SELE
|
HSPB6
|
FKBP1A
|
KLK1
|
MIR24-1
|
NAXE
|
TXN2
|
ENTPD2
|
EPO
|
AIF1
|
WT1
|
SLC16A1
|
IL22
|
RIPK1
|
STAT5A
|
UCP1
|
2.2 A total of 194 genes related to the components of qufengtongqiao formula were found in Genecars.Some genes are shown in Table 2:
Table2 Gene targets of effective components of Qufeng Tongqiao Prescription
Gene targets of effective components of Qufeng Tongqiao Prescription
|
PGR
|
MMP2
|
MET
|
COL3A1
|
F7
|
HSD3B1
|
AHR
|
NOS2
|
MMP9
|
PKIA
|
CXCL11
|
ACHE
|
CHRNA2
|
PSMD3
|
PTGS1
|
MAPK1
|
JUN
|
CXCL2
|
MAOB
|
PRKCB
|
SLC2A4
|
AR
|
IL10
|
IL4
|
DCAF5
|
RELA
|
BIRC5
|
NR1I3
|
SCN5A
|
EGF
|
ATP5F1B
|
CHEK2
|
NCF1
|
DUOX2
|
INSR
|
PTGS2
|
RB1
|
ND6
|
CLDN4
|
OLR1
|
NOS3
|
DIO1
|
ESR2
|
IL6
|
HSD3B2
|
PPARA
|
ADRB1
|
HSPB1
|
PPP3CA
|
CHEK1
|
TP63
|
IKBKB
|
PPARD
|
HTR3A
|
SULT1E1
|
GSTM1
|
PRSS1
|
ELK1
|
AKT1
|
HSF1
|
ADRA2C
|
MGAM
|
GSTM2
|
NCOA2
|
NFKBIA
|
BCL2
|
CRP
|
ADRB2
|
IL2
|
AKR1C3
|
CHRM3
|
POR
|
BAX
|
CXCL10
|
ADRA1D
|
CCNB1
|
SLPI
|
CHRM1
|
ODC1
|
TNFSF15
|
CHUK
|
OPRM1
|
PLAT
|
MMP3
|
CHRM2
|
CASP8
|
AHSA1
|
SPP1
|
KCNH2
|
THBD
|
EGFR
|
ADRA1B
|
TOP1
|
CASP3
|
RUNX2
|
CHRM5
|
SERPINE1
|
VEGFA
|
GABRA1
|
RAF1
|
MAPK8
|
RASSF1
|
CHRM4
|
COL1A1
|
CCND1
|
GRIA2
|
SOD1
|
MMP1
|
E2F1
|
OPRD1
|
IFNG
|
BCL2L1
|
ADH1B
|
PRKCA
|
STAT1
|
E2F2
|
ADRA1A
|
IL1A
|
FOS
|
ADH1C
|
HIF1A
|
HMOX1
|
ACPP
|
SLC6A3
|
MPO
|
CDKN1A
|
LYZ
|
RUNX1T1
|
CYP3A4
|
CTSD
|
SLC6A4
|
TOP2A
|
EIF6
|
RXRA
|
ERBB2
|
CYP1A2
|
IGFBP3
|
RXRB
|
ABCG2
|
CASP9
|
SLC6A2
|
ACACA
|
CYP1A1
|
IGF2
|
KDR
|
NFE2L2
|
PLAU
|
ESR1
|
CAV1
|
ICAM1
|
CD40LG
|
MAP2
|
NQO1
|
ADCYAP1
|
PPARG
|
MYC
|
SELE
|
IRF1
|
NR3C2
|
PARP1
|
RXRG
|
MAPK14
|
F3
|
VCAM1
|
ERBB3
|
ADRA2A
|
PSMG1
|
CASP1
|
GSK3B
|
GJA1
|
NR1I2
|
PON1
|
LTA4H
|
MAP2K4
|
HK2
|
CCNA2
|
IL1B
|
CYP1B1
|
PCOLCE
|
MAOA
|
NR3C1
|
RASA1
|
PYGM
|
CCL2
|
ALOX5
|
NPEPPS
|
CTRB1
|
CACNA1S
|
GSTP1
|
AKR1B1
|
PTGER3
|
HAS2
|
NCOA1
|
CXCL8
|
|
|
Figure 2 Gene targets of Qufengtongqiao Prescription
2.3Venny diagram The crossover genes between cerebral ischemia reperfusion and qufeng Tongqiao formula were analyzed, and a total of 53 key targets of the intersection genes of Venny diagram were obtained, as shown in Figure 1:
The left circle is the target of qufengtongqiao prescription, the right one is the target of cerebral ischemia reperfusion, and the intersection between them in the middle is the key target of treatment.
Table 3 Intersection genes of cerebral ischemia-reperfusion qufengtongqiao prescription
Intersection gene of cerebral ischemia-reperfusion - Qufengtongqiao prescription
|
NOS2
|
ALOX5
|
HSPB1
|
CASP3
|
F3
|
CRP
|
IL6
|
PTGS2
|
VEGFA
|
PLAT
|
MAPK8
|
GJA1
|
CTSD
|
ODC1
|
PPARG
|
FOS
|
THBD
|
HMOX1
|
IL1B
|
CD40LG
|
SOD1
|
MAPK14
|
CASP9
|
SERPINE1
|
ICAM1
|
CCL2
|
PON1
|
HIF1A
|
ACHE
|
MMP2
|
IFNG
|
SELE
|
CXCL8
|
MAP2
|
JUN
|
ADRB2
|
MMP9
|
MPO
|
VCAM1
|
NOS3
|
CASP1
|
IL4
|
SLC6A3
|
MAPK1
|
NFE2L2
|
IL10
|
PARP1
|
COL3A1
|
AKT1
|
KDR
|
BAX
|
BCL2
|
EGF
|
2.4. GO analysis
In order to clarify the mechanism of qufeng Tongqiao prescription in the treatment of cerebral ischemia reperfusion, go enrichment analysis was performed.The top 10 biological processes (BPS) involved in go enrichment analysis are as follows: Response to lipopolysaccharide, and molecule of bacterial origin, cellular response to chemical stress, cellular Response to oxidative stress, oxidative stress, and reactive oxygen species, reactive oxygen species Metabolic process, response to metal ion, cellular response to lipopolysaccharide, regulation of apoptotic signaling Pathway.
The top 10 factors involved in cell component (CC) in go enrichment analysis were: Raft, membrane microdomain, membrane region, Caveola, Plasma membrane raft, secretory granule lumen, cytoplasmic Vesicle Lumen, Vesicle Lumen, Neuron projection cytoplasm, Collagen-containing extracellular matrix.
The top 10 factors involved in molecular function (MF) in go enrichment analysis were as follows: Cytokine receptor binding, cytokine activity, DNA-binding transcription factor binding, RNA polymerase II-specific DNA-binding transcription factor binding, Receptor ligand activity, Signaling receptor Activator activity, Phosphatase Binding, Protease Binding, Cysteine-type Endopeptidase activity involved in apoptotic signaling pathway, growth factor receptor binding.
The main BP was response to lipopolysaccharide, the main CC process was membrane raft, and cytokine receptor binding was the main MF process.
2.5 KEGG pathway analysis
In order to clarify the mechanism of qufeng Tongqiao prescription in the treatment of cerebral ischemia reperfusion, we performed KEGG pathway analysis. The results suggested that the first ten pathways in KEGG pathway analysis were age-rage signaling pathway in diabetic complications, Fluid shear stress and atherosclerosis Pathway and Atherosclerosis, TNF signaling Pathway, IL-17 signaling Pathway, Chagas disease, Pertussis, Malaria, Kaposi Sarcoma-associated Herpesvirus infection and HIF-1 signaling Pathway.
Table4 the top ten KEGG pathways
ID
|
Description
|
pvalue
|
p.adjust
|
qvalue
|
Count
|
hsa04933
|
AGE-RAGE signaling pathway in diabetic complications
|
5.75 e-30
|
1.18 e-27
|
3.39 e-28
|
22
|
hsa05418
|
Fluid shear stress and atherosclerosis
|
7.59 e-25
|
7.81 e-23
|
2.24 e-23
|
21
|
hsa05417
|
Lipid and atherosclerosis
|
9.54 e-24
|
6.55 e-22
|
1.88 e-22
|
23
|
hsa04668
|
TNF signaling pathway
|
7.23 e-17
|
3.72 e-15
|
1.07 e-15
|
15
|
hsa04657
|
IL-17 signaling pathway
|
1.92 e-16
|
7.92 e-15
|
2.27 e-15
|
14
|
hsa05142
|
Chagas disease
|
6.34 e-16
|
2.18 e-14
|
6.23 e-15
|
14
|
hsa05133
|
Pertussis
|
1.69 e-14
|
4.97 e-13
|
1.42 e-13
|
12
|
hsa05144
|
Malaria
|
2.60 e-13
|
6.69 e-12
|
1.91 e-12
|
10
|
hsa05167
|
Kaposi sarcoma-associated herpesvirus infection
|
2.99 e-13
|
6.85 e-12
|
1.96 e-12
|
15
|
hsa04066
|
HIF-1 signaling pathway
|
1.48 e-12
|
3.04 e-11
|
8.70 e-12
|
12
|
2.6 PPI protein interaction and analysis
2.6.1 Open string(https://string-db.org/)After UniProt Transformation) and paste the intersection genes in List of Names. Choose HOMO sapiens to map the network of interactions between a molecule and a protein.AKT1 and MAPK14 can be known from the table.AKT1, HSPB1;AKT1, SOD1;AKT1, NOS3;AKT1, CASP3;AKT1, NOS2;AKT1, CASP9;AKT1, CD40LG;AKT1, JUN;AKT1 and BCL2 were the ten most important gene targets.
2.6.2 Screening Hub genes
The PPI interaction table was imported into Cytoscape to construct the network diagram. Sequencing was conducted according to the top 5 genes in Degree value, and the size of the circle was proportional to the that of Degree value. As shown in Figure 4B, the histogram of Degree value is made according to size, likewise in Figure 4C. The sequence of genes from large to small is: MAPK14, FOS, IL6, IL4 and JUN.
Table5: Relationship between cerebral ischemia reperfusion and effective components of Qufeng Tongqiao Prescription
Relationship between cerebral ischemia reperfusion and effective components of Qufeng Tongqiao Prescription
|
node1
|
node2
|
coexpression
|
experimentally_
determined_
interaction
|
database_
annotated
|
automated_
textmining
|
combined_
score
|
AKT1
|
MAPK14
|
0.083
|
0.83
|
0.6
|
0.769
|
0.956
|
AKT1
|
HSPB1
|
0
|
0.76
|
0
|
0.982
|
0.995
|
AKT1
|
SOD1
|
0
|
0.09
|
0.9
|
0.569
|
0.957
|
AKT1
|
NOS3
|
0.049
|
0.879
|
0.9
|
0.988
|
0.999
|
AKT1
|
CASP3
|
0
|
0.475
|
0
|
0.882
|
0.935
|
AKT1
|
NOS2
|
0.063
|
0.077
|
0.9
|
0.646
|
0.965
|
AKT1
|
CASP9
|
0
|
0.231
|
0.9
|
0.814
|
0.984
|
AKT1
|
CD40LG
|
0
|
0.056
|
0.9
|
0.493
|
0.947
|
AKT1
|
JUN
|
0
|
0.057
|
0.8
|
0.877
|
0.974
|
AKT1
|
BCL2
|
0.062
|
0
|
0.9
|
0.457
|
0.944
|
2.7 Molecular docking results
Five HUB genes screened from Cytoscape were docked with each active ingredient in qufengtongqiao prescription. The structure of HUB gene was found in PDB database and downloaded. A series of operations were performed to docked hub gene protein with the active ingredient, and finally the ligand and the overall picture were derived.As shown in FIG. 5 and 6, FIG. 5A: IL6-huangqi, FIG. 5B:IL4-huangqi, FIG. 5C: FOs-huangqi, FIG. 5D: MapK14-huangqi, FIG. 5E: Jun-huangqi, FIG. 5F: Jun-gegen, FIG. 5G: Jun-shichangpu, Figure 6H: Jun-lingzhi;Figure 6I: Jun-Renshen, Figure 6J: IL4-Gegen, Figure 6K: MapK14-Shichangpu, Figure 6L: MapK14-Gegen, Figure 6M: mapK14-Chuanxiong.Figure 6L: MAPK14-Gegen, Figure 6M: MAPK14-Chuanxiong Target proteins in cannot docking with drug ligands.
2.8 Cytoscape mapping
The active components of qufengtongqiao prescription, the intersection genes of drugs and diseases, the table of genes in the first ten KEGG pathways related to them and key targets were imported into Cytoscape software, and the disease-drug active component-key target-KeGG pathway map was drawn through a series of operations.FIG. 7 shows that: Based on the screening of drug component targets, network interaction between related targets was found.
On the left is cerebral ischemia-reperfusion disease,the middle one is mol number of Hirulong Huoxue Tongyu Capsule and 56 active ingredients, and the outer blue circle is common gene of cerebral ischemia-reperfusion and Hirulong Huoxue Tongyu Capsule.The first 10 KEGG pathways are on the right.The relationship diagram of cerebral ischemia-reperfusion - Hirulong Huoxue Tongyu capsule -KEGG pathway network was constructed, which was connected by the close relationship in the diagram. The regulation effect of Hirulong Huoxue Tongyu capsule on cerebral ischemia-reperfusion was expounded.
2.9 Behavioral verification
MNSS neurobehavioral evaluation was performed on the rats of SHAM, NS and QFH groups that had been successfully modeled at day 1, 5 and 10, and SPSS analysis was performed on the data. The main clause was first analyzed into one-way anVARIANCE, and the results were statistically mapped and tabulated. It was found that: On the first day, the neurological function of the SHAM group was statistically significant compared with that of the NS group, P <0.001;On the fifth day, the comparison between SHAM group and NS group, NS group and QFH group was statistically significant (P <0.001), p=0.029 in the latter group;On day 10, comparisons between SHAM group and NS group, NS group and QFH group were statistically significant, p<0.001 for the former group and P =0.015 for the latter group. Then, repeated measurement an OVA was used to conduct sphericity test for the data again, and it was found that P =0.048, which could not be analyzed by assuming sphericity. Instead, Greenhouse-Geisler was used for analysis, and it was found that time and Time *group were statistically significant, with p<0.001 and P =0.009, respectively.
Table6 behavioral score
Variables
|
DF
|
SS
|
MF
|
F
|
P
|
Intergroup error
|
2
|
342.1186
|
171.0593
|
98.66594
|
< 0.001
|
Time
|
17
|
29.47327
|
1.733722
|
|
|
Time*Intervence
|
2
|
70.44492
|
35.22246
|
92.66389
|
< 0.001
|
Repeated measurement
|
4
|
7.151034
|
1.787758
|
4.703268
|
0.004
|
error
|
34
|
12.92374
|
0.38011
|
|
|
Note: SS is the sum of squares, MF is the mean square, DF is the degree of freedom, P is the significance p-value, SS is the sum of squares.