2.1 Investigation of the mechanism of HSDTT against KOA using network pharmacology
2.1.1 Data preparation
Huashi Dingtong decoction(HSDTT) was consist of Squama Manis (Chuan-Shan-Jia,CSJ) ,Cinnanmomi Cortex(Rou-Gui,RG), Rhizoma Dioscoreae (Shan-Yao,SY) , Angelicae Sinensis Radix (Dang-Gui,DG) ,Cortex Acanthopanacis (Wu-Jia-Pi,WJP), Radix Angelicae Biseratae (Du-Huo,DH) ,Notopterygii Rhizoma Et Radix(Qiang-Huo,QH) , Atractylodes lancea(Thumb.)DC (Cang-Zhu,CZ) ,licorice (Gan-Cao,GZ). Each ingredient of Chinese herbs were seached from other papers, Chemical database (http://www.organchem.csdb.cn)[14] and TCMSP database (https://tcmspw.com/index.php)[15]. All the 2D structure of ingredients were download from TCMSP database and pubchem(https://pubchem.ncbi.nlm.nih.gov/)[15,16]. The inclusion criterias of these ingredients are assessed by SwissADME (http://www.swissadme.ch/index.php)[17]. If the uploading ingredient in SwissADME meet the rules that it shows “high” in GI absorption of “Pharmacokinetics ” and more than two “yes” in “Druglikeness”,we will bring it into our study[18]. KOA-related genes were obtained from TTD database (https://db.idrblab.org/ttd)[19],OMIMdatabase(https://www.omim.org/)[20],Genecardsdatabase(https://www.genecards.org)[21].
2.1.2 Drug-ingredients-targets network
The targets, which “probability”>0, of included active ingredients were selected by Swiss TargetPrediction (http://www.swisstargetprediction.ch/)[22]. The drugs-ingredients-targets network was established by Cytoscape 8.0.0.The degree of each node in the network was analyzed by using Network Analyzer Tool in cytoscape[23].
2.1.3 Protein-protein interaction (PPI) network construction and analysis
The selected targets were uploaded in STRING databases(http://string-db.org) to confirm the potential proteins interactions[24]. Then the file named ‘interaction’ was put into Cytoscape 8.0.0 to construct the PPI network. The degree and combind-score analyzed by ‘Network Analyzer Tool’ indicated the importance of each target. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed in R with the clusterProfiler package[25].
2.2 Chemicals and reagents
Huashi Dingtong decoction was purchased from TCM Pharmacy of the third hospital affiliated to Fujian University of Traditional Chinese Medicine. Ethyl carbamate and Hematoxylin-Eosin staining kits was obtained from Beijing Solarbio Science & Technology Co., Ltd (Beijing,China). 500 U of type II collagenase from clostridium histolyticum was purchased from Sigma-Aldrich. 0.9% Sodium Chloride Injection was from Jiangxi Kelun Pharmaceutical Co.,Ltd (Jiangxi,China). ELISA kits of IL-1β,TNF-α,COX-2,PGE-2 was provided by Shanghai Westang Bio-tech co.,Ltd (Shanghai,China).
2.3 Preparation of HSDTT
HSDTT is consist of Squama Manis (Chuan-Shan-Jia,6g), Cinnanmomi Cortex(Rou-Gui,1g), Rhizoma Dioscoreae (Shan-Yao,9g), Angelicae Sinensis Radix (Dang-Gui,6g), Cortex Acanthopanacis (Wu-Jia-Pi,9g), Radix Angelicae Biseratae (Du-Huo,6g), Notopterygii Rhizoma Et Radix(Qiang-Huo,6g), Atractylodes lancea(Thumb.)DC(Cang-Zhu,6g), licorice(Gan-Cao,3g). The botanical name of these medicines has been checked with http://www.organchem.csdb.cn,respectivly.
2.4 Animals experiments, grouping and model establishment
32 male Sprague-Dawley (SD) rats weighing 180-220g were provided by Shanghai SLAC Laboratory Animal Co.,Ltd (Shanghai, China),the certificate number is SCXK 2017-0005. Rats housed in SPF facility were randomly divided into 3 groups: HSDTT group, Model group, Control group.There were 12 rats in Control group and Model Group, 8 rats in HSDTT group. Rats in HSDTT group were received HSDTT decoction at a dose of 4.68g/kg/d by intragastric administration for 28 days after model successfully established. The other two groups were recived saline for the same days. The dose of intragastric administration was calculated by the surface area of the body.
KOA model was established by intra-articular injection with type II collagenase (0.4mg/ml). Rats in model group and HSDTT group were injected (0.4ml) into the medial side of the right knee joint of the hind limbs in the first, fourth day. The control group was injected with 0.4mL saline. After the model estabilshed successfully for 1 week, four rats were randomly selected to identificate and evaluate the model in each group except HSDTT group. The other rats were sacrificed after intragastric administration for 28 days. The procedures of this reseach complied with the Chinese Animal Welfare Law and approved by Fujian University of Chineses Traditional Medicine (Protocol ID:NO. FUTCM-2019028).
2.5 Measurement of the Knee Joint Diameter
The transverse diameters of the right knees were measured at day0, day7,day14,day21,day28,day35 by digimatic caliper (Mitutoyo, Kanagawa, Japan).The knee joints were flexed at 90° and measured the distance between the left and right highest points of the knee.
2.6 Hematoxylin-eosin staining (H&E)
The right hind knee joint of rats were dissected and fixed in 10% formalin for 72h at room temperature.Then tissues were decalcified in ethylenediaminetetraacetic acid for 6 weeks. Subsequently, knee joints were routinely sliced (5 μm) after dehydration and paraffin imbedding. Finally, the slices were stained with hematoxylin-eosin staining kit and observed under a light microscope (magnification, 100×).
2.7 Enzyme-linked immunosorbent assays (ELISA)
The contents of IL-1β and TNF-α in the synovium and COX-2 and PGE-2 in synovial fluid were measured with ELISA kits. In brief, the procedure to test for IL-1β,TNF-α,COX-2 and PGE-2 was followed by the manufacturer's instructions. Each group was selected 3 rats to assay and all the samples were assessed for three times.
2.8 Polymerase Chain Reaction (PCR)
The cartilage tissues were used to isolate total RNA by RNAiso reagents. PrimeScript®RT Master Mix Perfect Real Time kit was performed to reverse transcription of total RNA. PCR amplification was carried out using gene specific.PCR primers were provided by Shanghai biosune co. (Shanghai, China),which were used as follows:Collagen type Ⅱ Forward primer:5’-CGCTCAAGTCCCTCAACAAC-3’. Collagen type Ⅱ Reverse primer: 5’-TATCCAGTAGTCACCGCTCTTC-3’. p53 Forward primer: 5’-CAGCACATGACGGAGGTTGT-3’. p53 Reverse primer:5’-TCATCCAAATACTCCACACGC-3’. p38 Forward primer: 5’-TGTGATTGGTCTGTTGGATGTG-3’. p38 Reverse primer: 5’-TGGATTATGTCAGCCGAGTGTA-3’.
2.9 Statistical analysis
All the experimental data are expressed as the mean ± SD. Two-sample t-test or Wilcoxon rank sum test were used to compare the difference between the two groups by software SPSS20.0. The significant difference was dicided by p<0.05.