Animals
A total of 48 healthy male Wistar rats were provided by Guangzhou Hospital of Traditional Chinese Medicine, Guangzhou (Guangdong, China). According to the national standard, all rats were raised for 7 days before the experiment, and the appropriate environment were as follows: enough food and water ad libitum, temperature of 20-24°C, humidity of 50-60%, lights period from 8:00 AM to 8:00 PM. All the animal protocols have also been approved by the Animal Ethics Committee of Guangzhou Hospital of Traditional Chinese Medicine.
Establishment of TAO model
As described in previous research[21], 48 male Wistar rats were randomized into two groups: sham group (n=8) and TAO model group (n=40). The rats were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg, Sigma-Aldrich, Cat# P-010). After the rats were fixed in the supine position, the inner femur of the left lower limb was shorn and skinned to expose femoral artery. Blood flow was blocked by artery clipping, the femoral artery in the sham group was injected with 0.1 ml normal saline, and the femoral artery in the TAO model group was injected with 0.1 ml sodium laurate solution (10 mg/ml). After 20 mins of injection, the artery clamp was removed and the wound has been stitched up.
Grouping
The rats were divided into sham group (n=8), TAO group (n=8), TAO+NP group (n=8), TAO+MFS group (n=8), TAO+NP+NDDM group (n=8) and TAO+MFS+NDDM group (n=8). Sham group (healthy rats, administered saline alone, 2 ml/day for 7 d); TAO group (TAO model rats, dministered saline alone, 2 ml/day for 7 d); NP group (TAO model rats, notoginseng powder, 2 ml/day for 7 d, lavage administration; the ulcer was washed and bandaged with 0.2% Anerdian); MFS group (TAO model rats, maifusheng, 4 ml/day for 7d, lavage administration; the ulcer was washed and bandaged with 0.2% Anerdian); NP+NDDM group (TAO model rats, notoginseng powder, 2 ml/day for 7 d, lavage administration; the ulcer was treated by nibble debridement and dressing method); MFS+NDDM group (TAO model rats, maifusheng, 4 ml/day for 7 d, lavage administration; the ulcer was treated by nibble debridement and dressing method). Rats were fed and observed for 14 days after gavage, blood was collected from the orbits on day 1, day 7 and day 14, at the same time, partial serum was isolated on the 14th day. The experimental rats were put to death in a CO2 euthanasia chamber, all rats were killed and got the muscles on the inside of hind legs at 14 day. The tissues were fixed with 4% paraformaldehyde (Merck KGaA, Darmstadt, Germany, Cat. #1.04005).
Classification of gangrene
According to the grading standard, level 0: normal; level Ⅰ, the ganrene is confined to the toenail; level Ⅱ: the ganrene is confined to the toes; level Ⅲ: the ganrene is confined to the podoculi; level Ⅳ, the ganrene exceeds the ankle joint[22]. Three time points (1 day, 7 days and 14 days) were recorded after intragastric administration.
Hemorheology detection
Blood samples were collected from the eye socket of rats at day 1, day 7, and day 14 after intragastric administration, and put into EDTA-containing Vacutainer (BD, Franklin Lakes, NJ), respectively. Blood platelet count (BPC), white blood cell count (WBC), fibrinogen, plasma viscosity (PV), Red blood cell count (RBC), hemoglobin (Hb), neutrophil counts (NC) and erythrocyte sedimentation rate (ESR) levels were measured using automatic biochemistry analyzer (Roche, cobas c 311) and Hemorheology system (HLIFE, Shandong, China, LB-2A).
RNA extraction and real-time quantitative PCR (RT-qPCR) assay
Total RNAs were extracted from the muscles on the inside of the right hind limbs using TRIzol reagent (#9109, Takara, Japan). The quality of total RNAs was examined using NanoDro2000c (Thermo Scientific). 2 μg RNAs were used to synthesize cDNA using the Reverse Transcription kit (Takara, Japan). The PCR amplification was performed using SYBR GREEN PCR Master Mix (Applied Biosystems). The results were counted by 2−DDCt method [23]. The sequences of primers were shown in Table 1.
Western blot assay
The muscles on the inside of the right hind limbs were added a little liquid nitrogen and quickly crushed. Proteins were extracted using RIPA buffer (Beyotime, Cat. No. P0013B) including PMSF (Genebase, #329-98-6/ #115-39-9). Bicinchoninic acid (BCA) kit (Thermo Scientific) was utilized to determine the concentrations of proteins. Total proteins (30 μg) were separated using 10% SDS-PAGE, transferred onto nitrocellulose membranes (Millipore, USA). After blocking with 5% skimmed milk (BD Biosciences), the membranes were incubated with primary antibodies overnight at 4˚C. next day, the membranes were incubated with HRP Goat anti-Rabbit IgG (1: 20000, BOSTER, cat#BA1054) or HRP Goat anti-Mouse IgG (1: 20000, BOSTER, cat# BA1051) for 40 mins. Finally, the membranes were treated with the enhanced chemiluminescent reagents (MILLIPORE, WBKLS0500), he results were observed by X-ray films (XBT-1, Eastman Kodak Company, NY, USA), and the data were analyzed by Image-Pro Plus 6.0. The primary antibodies included IL-17 (1:1000, Abcam, ab77171), IFN-γ (1:1000, Abcam, ab77246), IL-1β (1:1000, Abcam, ab2105), IL-6 (1:1000, Abcam, ab208113), TNF-α (1:1000, Abcam, ab6671) and GAPDH (1:2000, Abcam, ab9482).
Enzyme-linked immunosorbent assay (ELISA) detection
After 14 days, serum was isolated from all the rats blood. According to the protocols of manufacturer, the concentrations of IL-17, IFN-γ, IL-β, IL-6 and TNF-α were measured using commercial ELISA kits as per the instructions of manufacturers. The ELISA kits included IL-1β (Cusabio, CSB-E08055r), IL-6 (Cusabio, CSB-E04640r), IL-17 (Cusabio, CSB-E07451r), IFN-γ (Cusabio, CSB-E04579r) and TNF-α (Cusabio, CSB-E11987r).
Immunohistochemistry (IHC) assay
The samples were embedded with paraffin, and cut into 4 μm section. After dewaxing and hydration, the antigen retrieval was performed with sodium citrate buffer at 95˚C. The sections were treated with 3% hydrogen peroxide for 10 mins. And then the sections were blocked with 3% BSA at 37˚C for 30 mins and incubated with anti-CD3 (1:150, Abcam, ab16669) and anti-CD20 (1:400, Bioss, bs-20639R) overnight at 4˚C. Next day, the sections were incubated with goat Anti-Rabbit IgG H&L (HRP, 1:1000, Abcam, ab6721) for 50 mins at room temperature. And then the sections were treated with diaminobenzidine (DAB) for 5 mins, hematoxylin for 3 mins and 1% acid alcohol for a few seconds. Finally, the sections were observed under a light microscope.
Haematoxylin and eosin (HE) staining
The medial muscles of the right hind limbs were fixed with 4% paraformaldehyde and dehydrated with alcohol gradient. Then 4-μm sections were stained with H&E solution (H8070, Solarbio, China). The pathology results were obtained under a microscope (Nikon Eclipse Ci, Japan).
Masson staining
The sections were treated with dimethylbenzene and dehydrated with graded ethanol. And then the sections were stained using Masson staining kit (G1006). After staining, the sections were treated with 1% glacial acetic acid for 1 min. finally, the sections were treated with graded ethanol and dimethylbenzene. The results were obtained using a microscope (Nikon Eclipse CI).
Statistical analysis
The results were calculated by GraphPad (Ver. Prism 7, GraphPad Prism Software, La Jolla, CA, USA). All data are expressed as mean ± standard deviation (SD). Differences between groups were analyzed by one-way analysis of variance or Student’s t-test. P < 0.05 was considered as significant.