Reagent
The powdered form of Indigo naturalis was prepared from the leaves of B. cusia (Nees) Bremek and purchased from Shanghai Ley’s Pharmaceutical Co., Ltd., with the place of production of Xianyou county, Fujian province, China. The material was identified and authenticated by Dr. Daofeng Chen, chief of Department of Natural Medicine, School of Pharmacy, Fudan University. The voucher specimen of indigo naturalis (DFC-LF-201509) was deposited in Department of Natural Medicine, School of Pharmacy, Fudan University.
Ribavirin was purchased from Shanghai Meryer chemical Co., Ltd.. ELISA kits for mouse tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), regulated upon activation normal T cell expressed and secreted factor (RANTES), interleukin-10 (IL-10), and interferon induced protein-10 (IP-10) were purchased from Shanghai Boatman Biotechnology Co. Ltd.. ELISA kits for interferon-α (IFN-α), interferon β (IFN-β), interferon γ (IFN-γ), myeloperoxidase (MPO), methylene dioxyamphetamine (MDA) were purchased from Shanghai Beyotime Biotechnology Co., Ltd.. The anti-HMGB-1 and anti-TLR4 antibodies were purchased from Abcam (San Francisco, CA, USA).
Herbal extract preparation
The indigo naturalis powder (2 kg) were packaged with 4 layers of etamine, and then extracted with boiling water for 1 h with three times. The water extract was concentrated under reduced pressure and lyophilized to produce 82 g of aqueous extract of indigo naturalis (INAE).
In vitro antiviral evaluation
The influenza A virus (H1N1 A/FM/1/47) was denoted by vice professor Haiyan Zhu, department of microbiological and biochemical pharmacy, school of pharmacy, Fudan University. According to the reference and procedures in our lab, the virus was suspended in Dulbecco’s modified Eagle’s medium (DMEM), propagated in lung of mice and stored at -80◦C. The 50% lethal dose (LD50) of virus was determined in mice infected with serial dilutions of virus [18].
In vitro antiviral evaluation was conducted as described with minor modification [19]. Madin-Darby canine kidney (MDCK) cells were cultured in DMEM supplemented with 10% fetal bovine serum (Hyclone, CA, USA), streptomycin and penicillin. Briefly, MDCK cells were cultured in 96-well plates (1×105 cells per well) till cells fulfilled 90% percent of the bottom. INAE were diluted and prepared in concentrations of 0, 10, 25, 50, 100, 200 and 400 μg/mL. The antiviral process was investigated based on three ways of action: added the drugs 2 h before, incubated the drugs with 100 TCID 50 influenza, or 2 h after virus infection. The cells with only infection viruses were served as virus control. After 3 days incubation, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was added into the wells. Following 4 h incubation, the optical density value (OD value) of supernatant was tested. Inhibition rate (%) = [(OD of INAE – mean OD of virus control) / (mean OD of cells control - mean OD of virus control)] × 100%.
In vitro anti-inflammatory effect
BALB/c mice were administrated with 5% mercaptoethanol acid sodium by intra-peritoneal injection and scarified 4 days later to harvest peritoneal macrophages. Macrophages were suspended with RPMI-1640 culture medium containing 10% fetal bovine serum and antibiotics. The cells were cultured in 96 cell plate (1 × 106 cells per well) for 2 h. The serial dilutions of INAE, lipopolysaccharide (LPS, 1 μg/ml), and dexamethasone (positive control, 10 μM) were added and incubated for 24 h. A sample of cells without LPS was set aside to serve as a control. The supernatant was collected at the end of the incubation. Nitrogen oxide (NO) production in the peritoneal macrophage was determined by measuring the OD value of supernatant following the instruction of method using Griess reagent [20].
animals
Specific pathogen-free male BALB/c mice (14 ~ 16 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. [SCXK (Hu) 2012-0002]. The mice were housed in collective cages under a 12 h light/dark room, with free access to food and water. The air temperature was maintained at 22 ± 2◦C with relative humidity of 50 ± 10%. Experiments were carried out according to the guideline for the care of laboratory animals of national institutes of health. All study protocols were approved by the animal ethical committee of school of pharmacy, Fudan University (approval No. 2015-10-SY-CDF-01).
Survival experiment in IAV infected mice
The survival experiment was conducted as described with minor modification [21]. Mice were randomly divided into six groups (n = 10): normal, model, INAE (40, 80 and 160 mg/kg) and positive control (ribavirin,100 mg/kg). Mice were anaesthetized by tail intravenous injection of propofol (0.026 mL/10g) and were intranasal challenged with 6 × LD50 IAV in 30 μL DMEM 2 h before treatment. Normal mice were challenged with 30 μL DMEM. The mice were treated orally with INAE or ribavirin once daily for 7 days. For comparison, normal group and model group were given 0.5% carboxymethyl cellulose sodium (CMC-Na). All groups were monitored for 14 days after virus infection. Body weight, body temperature, and numbers of mice were recorded daily. Lifespan and mortality rate of mice were calculated.
Acute lung injury in IAV infected mice
The experiment was scheduled by six groups (n = 6): normal, model, INAE (40, 80 and 160 mg/kg) and positive control (ribavirin,100 mg/kg). Mice were challenged with 3 × LD50 IAV in 30 μL DMEM 2 h before treatment. Normal mice were challenged with 30 μL DMEM. The mice were orally treated with agents as we mentioned about. The treatment began 2 h after the virus challenge and once daily for 4 days. The mice were sacrificed on the 4th day post infection. The lung tissues were harvested, weighted and then washed with pre-chilled saline. The superior right lobe was cut and placed in 10% neutral formalin buffer for histopathologic evaluation, and the rest parts were snap frozen at -80◦C for cytokines detection [22]. To estimate the severity of lung, liver, thymus and spleen. Organ indexes were calculated as follows: Organs index = Organ weight (mg) / body weight (g) × 100%.
Lung tissues were fixed with 10% neutral formalin buffer. After fixation, the samples were dehydrated and embedded in paraffin. The embedded samples were cut into 5-μm slices and stained with hematoxylin and eosin (H&E). Lesions in lung were observed by optical microscopy. Lung injury were determined through the severity of pneumonia in a blinded fashion [21].
After rehydrated through a graded series of alcohol, the slices were incubated with rabbit antibodies against HMGB-1 (1:200 diluted) and TLR4 (1:250 diluted) at 4◦C overnight. The sections were then incubated with special HRP-conjugated goat anti-rabbit IgG antibody at 37◦C for 30 minutes. Slides were stained with chromogenic substrate solution diaminobezidin (DAB) and counterstained with hematoxylin. The expression of HMGB-1 and TLR4 were visualized under a microscope [23].
Determination of lung virus titer
The frozen lung tissues were thawed and homogenized in PBS at a concentration of 100 mg tissue/1 mL PBS. The supernatant was split into small parts and stored at -80◦C for subsequent use.
MDCK cells were plated at 2 × 106 cells in 96 cell plate till the cells grown to 90% confluent, cells were then infected with serial dilutions of supernatant from lung homogenate and incubated for 2 h at 37◦C with 5% CO2. The supernatant was removed and the wells were washed with PBS and incubated with 200 μL DMEM at 37◦C, 5% CO2 for 3 days. The cell activity was assessed by MTT assays. The lung virus titer was expressed by the inhibition rate of virus replication [24].
Anti-oxidant capacity in supernatant of lung homogenate
The anti-oxidation of INAE was tested through methods of ferric ion reducing antioxidant power (FRAP) kit. Levels of MPO and MDA, oxidant stress products in lung homogenate of IAV infected mice were evaluated by ELISA kits according to the manufacturer’s instructions.
Assessment of cytokines in supernatant of lung homogenate
Levels of IFN-α, IFN-β, IFN-γ, MCP-1, RANTES, IP-10, TNF-α, IL-6, and IL-10 in the supernatant of lung homogenates were determined with ELISA kits according to the manufacturer’s instructions.
Statistical analysis
All parameters were recorded for individuals within all groups and statistical computations were performed with GraphPad prism 6 (GraphPad software Inc., San Diego, CA, USA). Data comparison were carried out with one-way ANOVA and expressed as mean ± S.D. (Standard deviation). Post hoc comparisons were performed using Fishers’s PLSD if any significant changes were found. The P values less than 0.05 were considered as statistically significant.