R. isatidis powder was purchased from Qixin Chinese Medicine Granules Co., Ltd. (cat. no. 2016-0041). DMEM high-glucose (cat. no. MA0571) and Trypsin-EDTA (cat. no. MB4376) were purchased from Dalian Meilun Biotechnology Co., Ltd. RPMI-1640 (cat. no. 11875093), Antibiotic-Antimycotic (100X; cat. no. 15240096) and fetal bovine serum (FBS; cat. no. 16000-044), were purchased from Gibco (Thermo Fisher Scientific, Inc.). The Griess reagent I and Griess Reagent II (cat. no. S0021) were purchased from Shanghai Beyotime Biotechnology Research Institute, Shanghai, China. Lipopolysaccharide (LPS; cat. no. L2880) was purchased from Sigma-Aldrich (Merck KGaA). The CellTiter 96^®AQ_ueous One Solution Cell Proliferation Assay was purchased from Promega Corp. (cat. no. G3580). IFN-γ Mouse ‘Femto-HS' High Sensitivity Uncoated ELISA (cat. no. 88-8314), IL-10 Mouse Uncoated ELISA (cat. no. 88-7105), TNF-α Mouse Uncoated ELISA (cat. no. 88-7324), IL-6 Mouse ELISA Kit (cat. no. BMS603-2TEN), Pierce^™ Coomassie (Bradford) Protein Assay kit (cat. no. 23200), TLR-4 monoclonal antibody (mAb; cat. no. MA5-16216) and β-actin mAb (cat. no. MA5-15452) were all purchased from Invitrogen (Thermo Fisher Scientific, Inc.). RAW264.7 macrophages were purchased from Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. A total of 16 specific-pathogen-free female BALB/c mice (age, 8 weeks; weight, 20-22 g; Sino-British SIPPR/BK Lab Animal Ltd.) were kept in a temperature-controlled room (temperature, 18-29˚C; relative humidity, 40-70%) under a 12 h light/dark cycle with free access to food and water. All animal procedures were approved and carried out in accordance with the Institutional Guidelines for Animal Experiments of the Animal Care and Use Committee at the Zhejiang Academy of Medical Sciences [Hangzhou, China; approval no. SYXK (Zhe) 2017-0008].

RIP was extracted by water extraction and alcohol precipitation. The extraction procedure was performed as previously described (27). R. isatidis (2 g) powder in 30 ml distilled water was incubated in a thermostatic water bath at 55˚C overnight. Following filtration through defatted cotton, the suspension was centrifuged 2,000 x g for 10 min at 4˚C by Allegra X-12 centrifuge (Beckman Coulter, Inc.) to remove residues. The supernatant was concentrated via rotary evaporation and precipitated with 95% alcohol for 72 h at room temperature, followed by centrifugation at 2,000 x g for 10 min at 4˚C. The precipitates were redissolved and dialyzed for 48 h at room temperature. The non-dialysate was lyophilized using Heto PowerDry LL3000 (Thermo Fisher Scientific, Inc.) at -40˚C for 48 h to obtain RIPs. The carbohydrate content of RIP was determined using the phenol-sulfuric acid method with glucose as a standard, as previously described (30). The protein content of RIP was determined using a Bradford Assay with BSA (cat. no. C506031; Sangon Biotech Co., Ltd.; 25-2,000 µg/ml) as a standard.

RAW264.7 cells were cultured in high-glucose DMEM containing 10% FBS at 37˚C and 5% CO₂ under saturated humidity.

RIP cytotoxicity was quantified using cell viability. RAW264.7 cells (2x10⁴ cells/well) were cultured at 37˚C in 96-well plates with 200 µl 10% FBS high-glucose DMEM for 24 h and 5% CO₂ under saturated humidity, and then incubated at 37˚C with different concentrations of RIP (0.02, 0.04, 0.08, 0.16, 0.32, 0.63, 1.25, 2.5, 5 and 10 mg/ml) in high-glucose DMEM for 24 h. Cell viability was determined using the CellTiter 96^®AQ_ueous One Solution Cell Proliferation Assay. Briefly, 40 µl/well CellTiter 96^® AQ_ueous One Solution reagent was added to the 96-well plates and incubated for 4 h at 37˚C. The optical density (OD) value at a wavelength of 490 nm was analysed using Epoch Microplate Spectrophotometer (BioTek Instruments, Inc.). High-glucose DMEM alone served as negative control.

RAW264.7 cells (1x10⁶ cells/well) were cultured in 6-well plates for 24 h at 37˚C and 5% CO₂ under saturated humidity. Cells were then treated with different concentrations of RIP (0, 0.08, 0.32, 1.25 and 5 mg/ml) or LPS (2 µg/ml) for 24 h. Morphological changes were observed using a phase-contrast microscope (magnification, 20x; Olympus Corporation).

RAW264.7 cells (1x10⁶ cells/well) were cultured in six-well plates with 3 ml 10% FBS high-glucose DMEM for 24 h at 37˚C and 5% CO₂ under saturated humidity. RAW264.7 cells were then incubated at 37˚C with different concentrations of RIP (0, 0.08, 0.32, 1.25 and 5 mg/ml) or LPS (2 µg/ml) for 24 h. NO production in cells was measured by the Griess method according to the instructions provided by the manufacturer (31). Briefly, the cell culture media of the six-well plates were centrifuged (200 x g for 5 min) at 4˚C, the supernatant (50 µl) was distributed in 96-well plate, and 50 µl /well Griess reagent I and 50 µl/well Griess reagent II were added. The reaction was allowed to proceed for 10 min at room temperature. The absorbance was read at 540 nm using Epoch Microplate Spectrophotometer, and the concentrations of NO₂^- were determined using a least squares linear regression analysis of the sodium nitrite standard curve.

All 16 female mice were randomly divided into two groups (8 mice/group) and immunized intramuscularly in their hind legs on day 0. Animals were treated with 50 µl per hind leg of PBS (PBS group) or with RIP (1 mg/ml; RIP group). On day 14, all mice were injected a second time. The body weight of experimental animals was measured every day until the end of the experiment. The animals were euthanized when the defined humane endpoint requirements were met: >15% loss of body weight within 1-2 days or >20% loss of body weight overall (32). On day 28, mice were humanely euthanized via intraperitoneal injection of pentobarbital sodium (50 mg/kg) followed by cervical dislocation (33). Animal death was confirmed by the cessation of heartbeat and respiration. The spleens of mice were collected under sterile conditions. A single-cell suspension of splenocytes was prepared in RPMI-1640 supplemented with 10% FBS and 1X Antibiotic-Antimycotic at 37˚C and 5% CO₂ under saturated humidity.

RAW264.7 cells (2x10⁵ cells/well) were seeded into a 96-well plate and cultured for 24 h at 37˚C and 5% CO₂ under saturated humidity. Cells were subsequently incubated at 37˚C with different concentrations of RIP (0.08, 0.32, 1.25 and 5 mg/ml) or LPS (2 µg/ml) in high-glucose DMEM containing 10% FBS for 24 h. TNF-α and IL-6 levels were determined by ELISA according to the manufacturer's instructions. Splenocytes (2x10⁶ cells/well) were seeded into a 96-well plate and stimulated with inactivated HSV-2 (50 µg/ml) in RPMI-1640 containing 10% FBS and 1X Antibiotic-Antimycotic or RPMI-1640 containing 10% FBS and 1X Antibiotic-Antimycotic only (negative control) and incubated at 37˚C for 48 h. IFN-γ and IL-10 levels of culture supernatant were then determined by ELISA according to the manufacturer's instructions. The absorbance was measured at 450 nm using Epoch Microplate Spectrophotometer.

RAW264.7 cells (3x10⁶ cells/well) were seeded into a six-well plate and cultured in high-glucose DMEM containing 10% FBS for 24 h at 37˚C and 5% CO₂ under saturated humidity, then incubated at 37˚C with different concentrations of RIP (0.08, 0.32, 1.25 and 5 mg/ml) or LPS (2 µg/ml) in high-glucose DMEM containing 10% FBS for 24 h. RAW264.7 cells were subsequently washed twice with PBS and lysed with Mammalian Cell Total Protein Lysis Buffer (cat. no. CW0889M; CoWin Biosciences). Total protein was quantified using Pierce^™ Coomassie (Bradford) Protein Assay kit using BSA as a standard. Proteins (4.5 µg/sample) were separated by 10-12% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% skimmed milk in TBS containing 0.05% Tween-20 (TBST) at 37˚C for 1 h. The membranes were incubated with the following primary monoclonal antibodies in TBST containing 1% skimmed milk at 4˚C overnight: TLR-4 (1:300) and β-actin (1:10,000). After washing the membrane with TBST for a total of three times, the membranes were incubated with HRP-conjugated goat-anti-mouse IgG (1:5,000) secondary antibodies (cat no. D110087; Sangon Biotech Co., Ltd.) at 37˚C for 2 h. After washing the membrane with TBST for a total of five times, the membranes were treated with the W (western blot)-TMB Chromogenic Reagent kit (cat. no. C510025; Sangon Biotech Co., Ltd.) at 37˚C for 15 min. Protein bands were visualized and imaged by western blotting imaging system (Peiqing Science & Technology Co., Ltd.). ImageJ software v1.8.0 (National Institutes of Health) was used to analyze the relative protein expression levels of TLR-4 (97 kDa), with β-actin (42 kDa) as the loading control.

All in vitro experiments were performed in triplicate. A total of 8 mice per group were used for in vivo experiments. GraphPad Prism 5.0 software (GraphPad Software, Inc.) was used for data analysis. Data are presented as the mean ± SD. An unpaired Student's t-test was used make statistical comparisons between two groups, and one-way ANOVA followed by Dunnett's or Tukey's post hoc tests were used to make statistical comparisons between three or more groups. P<0.05 was considered to indicate a statistically significant difference.

The obtained RIPs were water soluble polysaccharides. We established in a previous study a glucose standard curve using the phenol-sulfuric acid method: The linear regression equation was y=0.0069x + 0.0397 [with x the mass of glucose (µg) and y the OD value at 450 nm]; R²=0.9955(27). The carbohydrate content of RIP was 87.5% (100 mg crude polysaccharides contained 87.5 mg glucose). Using the Bradford protein assay, absorbance at 595 nm was 0.009, which was not within the BSA standard curve (25-2,000 µg/ml) and the protein content of RIP was lower than the detection sensitivity. The protein content was therefore considered to be weak.

RIP cytotoxicity on RAW264.7 cells was examined (Fig. 1). RAW264.7 cell viability was almost 100% when RIP concentration was ≤1.25 mg/ml, demonstrating that RIPs had no cytotoxicity. Increasing the RIP concentration up to 10 mg/ml decreased cell viability; however, cell viability remained >95%, further demonstrating that RIP had a low cytotoxicity. The water-soluble RIPs extracted from R. isatidis, like other plant polysaccharides, had a low cytotoxicity (14). Therefore, 0.08, 0.32, 1.25 and 5 mg/ml of RIP were chosen for subsequent studies.

Following the addition of LPS and different concentrations of RIP to RAW264.7 cells, morphological changes were observed. In the control group, the RAW264.7 cells were round and clumped together (Fig. 2A). The RAW264.7 cells treated with different concentrations of RIP all displayed morphological changes in a dose-dependent manner. In the 0.08 mg/ml RIP-treated group, the cellular morphology was almost the same as the control group and cells were clustered together, only a few cells has a spindle-like shape (Fig. 2B). In the 0.32 mg/ml RIP-treated group, approximately one fourth of the cells were spindle-like but the round cells still clustered together (Fig. 2C). In the 1.25 mg/ml RIP group, almost half of the cells were spindle-like and independent, not aggregating in groups (Fig. 2D). In the 5 mg/ml RIP-treated group, most of the cells were spindle-like or polygonal in shape and were more dispersed (Fig. 2E). The morphological changes seen in the 5 mg/ml RIP treated group were the same as the LPS-treated group (Fig. 2F).

RIP (0.08, 0.32, 1.25 and 5 mg/ml) stimulated RAW264.7 cells to produce NO as demonstrated in Table I. A RIP concentration of 1.25 mg/ml produced the highest significant concentration of NO (17.38±0.50 µM) in RAW264.7 cells compared with the medium control and all other RIP concentrations (P<0.01). LPS stimulated RAW264.7 cells to produce 59.16±2.97 µM NO.

RIP-stimulated (0.08, 0.32, 1.25 and 5 mg/ml) RAW264.7 cells produced TNF-α in a dose-dependent manner, with 5 mg/ml RIP inducing the highest significant level of TNF-α compared with the control and all other RIP concentrations (P<0.01; Fig. 3A). RIP-stimulated (0.08, 0.32, 1.25 and 5 mg/ml) RAW264.7 cells also produced IL-6 in a dose-dependent manner, with 5 mg/ml RIP inducing the highest significant level of IL-6 compared with the other RIP samples and LPS (2 µg/ml; P<0.01; Fig. 3B).

TLR-4 protein expression levels in RAW264.7 cells treated with 0.08, 0.32, 1.25 and 5 mg/ml RIP were examined using western blotting. TLR-4 protein expression levels increased in a dose-dependent manner and were significantly higher compared with the medium control group (P<0.01; Fig. 4). LPS-treated RAW264.7 cells expressed the highest levels of TLR-4.

Splenocytes were obtained from RIP-treated mice on day 28 and stimulated by inactivated HSV-2. The levels of IFN-γ and IL-10 in the RIP group were significantly higher compared with the PBS group (P<0.01; Table II).