Author Contributions
H.-G.J.: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data curation, writing—original draft preparation, writing—review and editing, Visualization, Project administration; C.-Y.B.: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data curation, writing—original draft preparation, writing—review, and editing; H.S.S.: Conceptualization, Validation, Formal analysis, writing—review and editing, Supervision, Project administration, and funding acquisition; D.L.: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing—review and editing, Supervision, Project administration, and funding acquisition. All authors have read and agreed to the published version of the manuscript.
Figure 1.
A Venn diagram showing potential targets for osteoarthritis treatment with Scutellaria baicalensis. SB: Scutellaria baicalensis, OA: osteoarthritis.
Figure 1.
A Venn diagram showing potential targets for osteoarthritis treatment with Scutellaria baicalensis. SB: Scutellaria baicalensis, OA: osteoarthritis.
Figure 2.
The therapeutic mechanisms of OA in SB are represented by the drug compound–target–disease network. OA: osteoarthritis, SB: Scutellaria baicalensis.
Figure 2.
The therapeutic mechanisms of OA in SB are represented by the drug compound–target–disease network. OA: osteoarthritis, SB: Scutellaria baicalensis.
Figure 3.
The PPI network of hub targets from SB against OA. PPI: protein–protein interaction; SB: Scutellaria baicalensis, OA: osteoarthritis.
Figure 3.
The PPI network of hub targets from SB against OA. PPI: protein–protein interaction; SB: Scutellaria baicalensis, OA: osteoarthritis.
Figure 4.
GO and KEGG enrichment analysis of OA treatment. ((A–C): GO analysis of OA targets. (D): KEGG enrichment analysis of OA targets). GO: Gene ontology, KEGG: Kyoto Encyclopedia of Genes and Genomes, OA: osteoarthritis.
Figure 4.
GO and KEGG enrichment analysis of OA treatment. ((A–C): GO analysis of OA targets. (D): KEGG enrichment analysis of OA targets). GO: Gene ontology, KEGG: Kyoto Encyclopedia of Genes and Genomes, OA: osteoarthritis.
Figure 5.
The high-performance liquid chromatogram of the Scutellaria baicalensis extracts at 275 nm: baicalein, wogonin, and acacetin. Retention times are 28.350 min, 32.985 min, and 33.309 min, respectively. The x-axis indicates the retention time; the y-axis indicates the absorbance unit. A Luna C18 column (250 mm × 4.6 mm, 5 μm; Phenomenex, USA) is used for chromatic separation at 30 °C.
Figure 5.
The high-performance liquid chromatogram of the Scutellaria baicalensis extracts at 275 nm: baicalein, wogonin, and acacetin. Retention times are 28.350 min, 32.985 min, and 33.309 min, respectively. The x-axis indicates the retention time; the y-axis indicates the absorbance unit. A Luna C18 column (250 mm × 4.6 mm, 5 μm; Phenomenex, USA) is used for chromatic separation at 30 °C.
Figure 6.
The effects of SB on hind limb weight bearing in an osteoarthritis rat model induced by monosodium iodoacetate. (A) The weight-bearing ratio of monosodium iodoacetate rats treated with 3 mg/kg indomethacin and 80 and 240 mg/kg SB during a period of 0–24 days was examined, and (B) the incapacitance meter tester examined the AUC. *** p < 0.001 compared. control, ### p < 0.001 vs. sham. INDO 3: indomethacin 3 mg/kg, SB: Scutellaria baicalensis, AUC: area under the curve.
Figure 6.
The effects of SB on hind limb weight bearing in an osteoarthritis rat model induced by monosodium iodoacetate. (A) The weight-bearing ratio of monosodium iodoacetate rats treated with 3 mg/kg indomethacin and 80 and 240 mg/kg SB during a period of 0–24 days was examined, and (B) the incapacitance meter tester examined the AUC. *** p < 0.001 compared. control, ### p < 0.001 vs. sham. INDO 3: indomethacin 3 mg/kg, SB: Scutellaria baicalensis, AUC: area under the curve.
Figure 7.
Photographs of the knee joint cartilage of rats with osteoarthritis induced by MIA. (A) The representative image indicates cartilage erosion with 3 mg/kg indomethacin, and 80 and 240 mg/kg SB were administered to MIA rats. The arrows indicate the cartilage degradation spot. (B) The macroscopic score. ### p < 0.001 vs. sham, ** p < 0.05 vs. control, *** p < 0.001 vs. control by a one-way analysis of variance, Dunnett’s test. INDO 3: indomethacin 3 mg/kg, MIA: monosodium-iodoacetate, SB: Scutellaria baicalensis.
Figure 7.
Photographs of the knee joint cartilage of rats with osteoarthritis induced by MIA. (A) The representative image indicates cartilage erosion with 3 mg/kg indomethacin, and 80 and 240 mg/kg SB were administered to MIA rats. The arrows indicate the cartilage degradation spot. (B) The macroscopic score. ### p < 0.001 vs. sham, ** p < 0.05 vs. control, *** p < 0.001 vs. control by a one-way analysis of variance, Dunnett’s test. INDO 3: indomethacin 3 mg/kg, MIA: monosodium-iodoacetate, SB: Scutellaria baicalensis.
Figure 8.
(A) The IL-1β, (B) IL-6, and (C)TNF-α serum of MIA rats. Rats were treated with 80 and 240 mg/kg SB for 24 d. ### p < 0.001 vs. sham, *** p < 0.001 vs. control by a one-way analysis of variance, Dunnett’s test. INDO 3: indomethacin 3 mg/kg, MIA: monosodium-iodoacetate, SB: Scutellaria baicalensis.
Figure 8.
(A) The IL-1β, (B) IL-6, and (C)TNF-α serum of MIA rats. Rats were treated with 80 and 240 mg/kg SB for 24 d. ### p < 0.001 vs. sham, *** p < 0.001 vs. control by a one-way analysis of variance, Dunnett’s test. INDO 3: indomethacin 3 mg/kg, MIA: monosodium-iodoacetate, SB: Scutellaria baicalensis.
Figure 9.
The effect on analgesic responses. (A) After 30 min of oral administration with ibuprofen (200 mg/kg body weight) and SB extract (160 and 480 mg/kg body weight), all mice were intraperitoneally injected with 0.7% acetic acid 10 min before counting. The number of writhing responses in acetic acid-induced ICR mice was eight per group; ### p < 0.001 vs. ibuprofen, *** p < 0.001 vs. control by a one-way analysis of variance, Dunnett’s test. (B) Hot plate responses of paw withdrawal latency. SB: Scutellaria baicalensis.
Figure 9.
The effect on analgesic responses. (A) After 30 min of oral administration with ibuprofen (200 mg/kg body weight) and SB extract (160 and 480 mg/kg body weight), all mice were intraperitoneally injected with 0.7% acetic acid 10 min before counting. The number of writhing responses in acetic acid-induced ICR mice was eight per group; ### p < 0.001 vs. ibuprofen, *** p < 0.001 vs. control by a one-way analysis of variance, Dunnett’s test. (B) Hot plate responses of paw withdrawal latency. SB: Scutellaria baicalensis.
Figure 10.
Effects of SB on (A) cell viability and (B) LPS-stimulated NO production, (C–T) mRNA (MMP-1, 3, 8, and 13), JUN, FOS, IL-1β, TNF-α, IL-6, COX-2, Ptger2, NOS2, TGF-β1, and IL-4, and the protein expression of (U) MMP-1, MMP-13, NF-κB p65, and NOS2 in RAW264.7 cells. Cells were treated with dexamethasone (1 µg/mL), SB (10, 30, 100, and 300 µg/mL), and LPS (500 ng/mL) for 24 h. ### p < 0.001 vs. sham, * p < 0.05 vs. control, ** p < 0.01 vs. control, *** p < 0.001 vs. control by a one-way ANOVA, Dunnett’s test. DEX 1: dexamethasone 1 µg/mL, LPS: lipopolysaccharide, NO: nitric oxide, NT: non-treated, ANOVA: analysis of variance.
Figure 10.
Effects of SB on (A) cell viability and (B) LPS-stimulated NO production, (C–T) mRNA (MMP-1, 3, 8, and 13), JUN, FOS, IL-1β, TNF-α, IL-6, COX-2, Ptger2, NOS2, TGF-β1, and IL-4, and the protein expression of (U) MMP-1, MMP-13, NF-κB p65, and NOS2 in RAW264.7 cells. Cells were treated with dexamethasone (1 µg/mL), SB (10, 30, 100, and 300 µg/mL), and LPS (500 ng/mL) for 24 h. ### p < 0.001 vs. sham, * p < 0.05 vs. control, ** p < 0.01 vs. control, *** p < 0.001 vs. control by a one-way ANOVA, Dunnett’s test. DEX 1: dexamethasone 1 µg/mL, LPS: lipopolysaccharide, NO: nitric oxide, NT: non-treated, ANOVA: analysis of variance.
Figure 11.
Changes of cytokines at knee joint cartilage tissue with 3 mg/kg indomethacin and 80 and 240 mg/kg SB treatment. (A–W) The mRNA expression of matric metalloproteinases (MMPs) (MMP-1, 3, 8, and 13), JUN, FOS, IL-1β, TNF-α, IL-6, COX-2, Ptger2, NOS2, TGF-β1, type II collagen, IL-4, and TIMP-1 determined by quantitative real-time PCR. (X) The protein expression of MMPs (MMP-1, 3, 8, and 13), NF-κB p65, NOS2, and IL-1β measured with Western blot analysis. ### p < 0.001 vs. sham, * p < 0.05 vs. control, ** p < 0.01 vs. control, *** p < 0.001 vs. control by a one-way ANOVA and Dunnett’s test. INDO 3: indomethacin 3 mg/kg, MIA: monosodium-iodoacetate, ANOVA: analysis of variance, SB: Scutellaria baicalensis, PCR: polymerase chain reaction.
Figure 11.
Changes of cytokines at knee joint cartilage tissue with 3 mg/kg indomethacin and 80 and 240 mg/kg SB treatment. (A–W) The mRNA expression of matric metalloproteinases (MMPs) (MMP-1, 3, 8, and 13), JUN, FOS, IL-1β, TNF-α, IL-6, COX-2, Ptger2, NOS2, TGF-β1, type II collagen, IL-4, and TIMP-1 determined by quantitative real-time PCR. (X) The protein expression of MMPs (MMP-1, 3, 8, and 13), NF-κB p65, NOS2, and IL-1β measured with Western blot analysis. ### p < 0.001 vs. sham, * p < 0.05 vs. control, ** p < 0.01 vs. control, *** p < 0.001 vs. control by a one-way ANOVA and Dunnett’s test. INDO 3: indomethacin 3 mg/kg, MIA: monosodium-iodoacetate, ANOVA: analysis of variance, SB: Scutellaria baicalensis, PCR: polymerase chain reaction.
Table 1.
Chemical compounds of Scutellaria baicalensis extracts.
Table 1.
Chemical compounds of Scutellaria baicalensis extracts.
Pubchem ID | Compound Name | Structure | OB (%) | DL |
---|
6782 | Diisobutyl phthalate | | 43.59 | 0.35 |
31161 | Pedalitin | | 34.02 | 0.31 |
33934 | Diisooctyl phthalate | | 43.59 | 0.39 |
64982 | Baicalin | | 40.12 | 0.75 |
72322 | Coptisine | | 30.67 | 0.86 |
72323 | Jatrorrhizine | | 30.44 | 0.75 |
124211 | Skullcapflavone II | | 69.51 | 0.44 |
156992 | 5,8,2′-Trihydroxy-7-methoxyflavone | | 37.01 | 0.27 |
159029 | Tenaxin I | | 31.71 | 0.35 |
160876 | Epiberberine | | 43.09 | 0.78 |
161271 | Salvigenin | | 49.07 | 0.33 |
182232 | (+)-Epicatechin | | 48.96 | 0.24 |
188308 | Carthamidin | | 33.23 | 0.24 |
188316 | 5-Hydroxy-7,8-dimethoxyflavone | | 44.09 | 0.25 |
222284 | beta-Sitosterol | | 36.91 | 0.75 |
373261 | Eriodyctiol (flavanone) | | 41.35 | 0.24 |
440735 | Eriodictyol | | 71.79 | 0.24 |
457801 | Clionasterol | | 36.91 | 0.75 |
471719 | Negletein | | 41.16 | 0.23 |
5280442 | Acacetin | | 34.97 | 0.24 |
5280666 | Chrysoeriol | | 35.85 | 0.27 |
5280794 | Stigmasterol | | 43.83 | 0.76 |
5281330 | Poriferasterol | | 43.83 | 0.76 |
5281605 | Baicalein | | 33.52 | 0.21 |
5281674 | Norwogonin | | 39.4 | 0.21 |
5281703 | Wogonin | | 30.68 | 0.23 |
5283637 | 22,23-Dihydrobrassicasterol | | 37.58 | 0.71 |
5320315 | Oroxylin A | | 41.37 | 0.23 |
5320399 | Skullcapflavone I | | 76.26 | 0.29 |
5321865 | 5,7,2′,6′-Tetrahydroxyflavone | | 37.01 | 0.24 |
5322059 | Viscidulin II | | 45.05 | 0.33 |
42608119 | 5,7,4′-trihydroxy-8-methoxyflavanone | | 74.24 | 0.26 |
5322078 | 4′-Hydroxywogonin | | 36.56 | 0.27 |
5365674 | 11,13-Eicosadienoic acid, methyl ester | | 39.28 | 0.23 |
5367328 | 1-Monolinolenoyl-rac-glycerol | | 38.14 | 0.31 |
9601691 | Glucobrassicin | | 66.02 | 0.48 |
12303645 | 3-epi-beta-Sitosterol | | 36.91 | 0.75 |
13889022 | Rivularin (flavone) | | 37.94 | 0.37 |
14135323 | (2S)-dihydrobaicalein | | 40.04 | 0.21 |
25721350 | Dihydrooroxylin | | 66.06 | 0.23 |
26213330 | (2R)-5,7-dihydroxy-2-(4-hydroxyphenyl)-6-methoxy-2,3-dihydrochromen-4-one | | 36.63 | 0.27 |
44258628 | 5,7,3′,6′-Tetrahydroxy-6,8,2′-trimethoxyflavone | | 33.82 | 0.45 |
141457867 | 5-Hydroxy-2-(2-hydroxy-5-methoxyphenyl)-6,7,8-trimethoxychromen-4-one | | 104.34 | 0.44 |
162988960 | Carthamidin | | 41.15 | 0.24 |
Table 2.
Top 15 compounds of SB from the Network Analyzer.
Table 2.
Top 15 compounds of SB from the Network Analyzer.
Compound Name | Degree Centrality | Betweenness Centrality | Closeness Centrality |
---|
Wogonin | 26 | 0.094 | 0.509 |
Baicalein | 19 | 0.062 | 0.471 |
Acacetin | 16 | 0.035 | 0.456 |
beta-Sitosterol | 13 | 0.030 | 0.442 |
5-Hydroxy-7,8-dimethoxyflavone | 12 | 0.011 | 0.438 |
Oroxylin A | 12 | 0.017 | 0.438 |
4′-Hydroxywogonin | 11 | 0.009 | 0.433 |
Chrysoeriol | 11 | 0.009 | 0.433 |
5-Hydroxy-2-(2-hydroxy-5-methoxyphenyl)-6,7,8-trimethoxychromen-4-one | 10 | 0.010 | 0.429 |
Rivularin (flavone) | 9 | 0.007 | 0.425 |
Jatrorrhizine | 8 | 0.004 | 0.421 |
Salvigenin | 8 | 0.004 | 0.421 |
Skullcapflavone I | 8 | 0.003 | 0.421 |
Tenaxin I | 8 | 0.003 | 0.421 |
Viscidulin II | 8 | 0.003 | 0.421 |
Table 3.
The topological characteristics of hub targets from SB against OA.
Table 3.
The topological characteristics of hub targets from SB against OA.
Gene Name | Degree Centrality | Betweenness Centrality | Closeness Centrality | Average Shortest Path Length |
---|
JUN | 22 | 0.209 | 0.560 | 1.786 |
RELA | 16 | 0.086 | 0.519 | 1.929 |
FOS | 15 | 0.060 | 0.494 | 2.024 |
TP53 | 15 | 0.222 | 0.525 | 1.905 |
MAPK14 | 13 | 0.058 | 0.500 | 2.000 |
ESR1 | 13 | 0.111 | 0.472 | 2.119 |
TNF | 12 | 0.078 | 0.483 | 2.071 |
IL6 | 11 | 0.112 | 0.477 | 2.095 |
AKT1 | 10 | 0.067 | 0.457 | 2.190 |
NR3C1 | 9 | 0.008 | 0.457 | 2.190 |
AR | 8 | 0.015 | 0.412 | 2.429 |
CCND1 | 7 | 0.004 | 0.438 | 2.286 |
CXCL8 | 7 | 0.027 | 0.457 | 2.190 |
TGFB1 | 7 | 0.033 | 0.429 | 2.333 |
Table 4.
Monosodium iodoacetate-induced OA model design.
Table 4.
Monosodium iodoacetate-induced OA model design.
Group | OA Inducer (50 μL, Intra-Articular) | Sample (10 mL/kg, P.O.) |
---|
Sham | Saline | DW |
Control | MIA 40 mg/mL | DW |
Indomethacin | MIA 40 mg/mL | indomethacin 200 mg/kg |
SB (low dose) | MIA 40 mg/mL | SB 80 mg/kg |
SB (high dose) | MIA 40 mg/mL | SB 240 mg/kg |
Table 5.
Macroscopic score of cartilage erosion.
Table 5.
Macroscopic score of cartilage erosion.
Grade | Cartilage Appearance |
---|
0 | Normal appearance in cartilage surface |
1 | Slight yellowish discoloration of the surface or slight fibrillation |
2 | Erosion reaching the superficial or middle layers of the cartilage |
3 | Extensive erosions reaching down to the subchondral bone |
4 | Massive erosions with extensive exposure of subchondral bone |
Table 6.
mRNA primer sequence for OA-induced cartilage tissues.
Table 6.
mRNA primer sequence for OA-induced cartilage tissues.
MMP-1 | F | AACTTGGGTGAAGACGTCCA |
R | TCCTGTCACTTTCAGCCCAA |
MMP-3 | F | GTACGGCTGTGTGCTCATCC |
R | TCAGCCCAAGGAACTTCTGC |
MMP-8 | F | TCTGTTCTTCTTCCACACACAG |
R | GCAATCATAGTGGCATTCCT |
MMP-13 | F | ACCTTCTTCTTGTTGAGTTGGA |
R | CTGCATTTCTCGGAGTCTA |
JUN | F | CCAACCAACGTGAGTGCAAG |
R | GAG GGCATCGTCGTAGAAGG |
FOS | F | TACTACCATTCCCCAGCCGA |
R | GCGTATCTGTCAGCTCCCTC |
IL-1β | F | AACTCAACTGTGAAATAGCAGC |
R | TCCACAGCCACAATGAGTG |
TNF-α | F | GCATGATCCGAGATGTGGAA |
R | GATGAGAGGGAGCCCATTTG |
IL-6 | F | TCCGCAAGAGACTTCCAGC |
R | CCTCCGACTTGTGAAGTGG |
COX-2 | F | GTTCCAACCCATGTCAAAAC |
R | TGTCAGGAATCTCGGCGTAG |
Ptger2 | F | TGTGTGTACTGTCCGTCTGC |
R | CAGGGATCCAGTCTCGGTGT |
TGF-β1 | F | AGGAGACGGAATACAGGGCT |
R | CCACGTAGTAGACGATGGGC |
Type II collagen | F | TGGCCTTGGTGGAGGAAA |
R | AGGACCAGGGAGGCCTCTTT |
IL-4 | F | CGTGATGTACCTCCGTGCTT |
R | GTGAGTTCAGACCGCTGACA |
TIMP-1 | F | TTTCCCTGTTCAGCCATCCC |
R | TAGCCCTTCTCAGAGCCCAT |
GAPDH | F | CTTGTGACAAAGTGGACATTGTT |
R | TGACCAGCTTCCCATTCTC |
Table 7.
mRNA primer sequence for LPS-stimulated RAW264.7 cells.
Table 7.
mRNA primer sequence for LPS-stimulated RAW264.7 cells.
MMP-1 | F | ATGCCTAGCCTTCCTTTGCT |
R | TTCCAGGTATTTCCAGACTG |
MMP-3 | F | AAGTTCCTCGGGTTGGAGAT |
R | ACCAACATCAGGAACACCAC |
MMP-8 | F | CAATCAATTCCGGTCTTCGA |
R | GGTTAGCAAGAAATCACCAGA |
MMP-13 | F | AACCAAGATGTGGAGTGCCT |
R | GACCAGACCTTGAAGGCTTT |
JUN | F | ACAGAGCATGACCTTGAACCT |
R | GTGATGTGCCCATTGCTGGA |
FOS | F | GGACTTTTGCGCAGATCTGT |
R | GGTGGGGAGTCCGTAAGGAT |
IL-1β | F | CCAGCTTCAAATCTCGCAGC |
R | GTGCTCATGTCCTCATCCTGG |
TNF-α | F | GAGAAGTTCCCAAATGGCCT |
R | AGCCACTCCAGCTGCTCCT |
IL-6 | F | CACTTCACAAGTCGGAGGCT |
R | CAAGTGCATCATCGTTGTTC |
COX-2 | F | ATCCATGTCAAAACCGTGGG |
R | TTGGGGTGGGCTTCAGCAG |
Ptger2 | F | CTGGTAACGGAATTGGTGC |
R | TGGCCAGACTAAAGAAGGTC |
NOS2 | F | ACCAAGATGGCCTGGAGGAA |
R | CCGACCTGATGTTGCCATTG |
TGF-β1 | F | GGACTCTCCACCTGCAAGAC |
R | TGTTGTACAAAGCGAGCACC |
IL-4 | F | ACGGAGATGGATGTGCCAA |
R | TGCGAAGCACCTTGGAAGC |
GAPDH | F | ATGGTGAAGGTCGGTGTG |
R | GCCGTGAGTGGAGTCATAC |
Table 8.
Antibodies.
Antibody | Dilution Rate | Company |
---|
MMP-1 | 1:700 | Proteintech |
MMP-3 | 1:1000 | Abcam |
MMP-8 | 1:1000 | Abcam |
MMP-13 | 1:2000 | Proteintech |
IL-1β | 1:1000 | Abcam |
NF-κB p65 | 1:1000 | Cell Signaling |
NOS2 | 1:1000 | Abcam |
β-actin | 1:1000 | Cell Signaling |