Phosphate-buffered saline (PBS), fetal bovine serum (FBS), and high-glucose DMEM were obtained from Gibco (USA). TRIzol was from Takara Bio (Japan). The commercial ELISA kits were from NeoBioscience Technology Co., Ltd. (China) to analyze IL-10, IL-8, IL-6, Monocyte Chemoattractant Protein-1 (MCP-1), and TNF-α. The ROS Assay Kit was acquired from Applygen (China). The Chinese company Nanjing Jiancheng Bioengineering Institute provided the Nitric Oxide Assay Kit. The HiScript III RT SuperMix for qPCR (+gDNA wiper) and ChamQ Universal SYBR qPCR Master Mix were bought from Vazyme (China). The IL-10, IL-8, IL-6, TNF-α, and iNOS antibodies were acquired from Proteintech (China). The streptavidin-peroxidase (SP) immunohistochemistry (IHC) kit was purchased from Bioss (China).

The Polygonum hydropiper L. was supplied by Tai Hua Pharmaceutical Co. Ltd. Professor Renbin Huang of Guangxi Medical University's School of Pharmacy verified the authenticity of the plant material. Tao et al.'s technique comprised the extraction and purification of the FNB in accordance with the specified protocol (5). According to the HPLC study, FNB had a total flavonoid concentration of 55.3%. Its rutin, quercetin glycoside, and quercetin concentrations were 21.9%, 8.2%, and 20.6%, respectively (5). The solution was prepared in complete DMEM, filtered using a 0.22 μm mesh, and stored at 4°C for later application.

PCV2 was supplied by Nanjing Agricultural University (China). Using the Reed-Muench assay, PCV2 titers were found to be 10^4.5 TCID₅₀/0.1 mL. RAW264.7 cells were procured from the Shanghai Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences. Cells were grown in DMEM with 10% heat-inactivated FBS in an incubator at 37°C with 5% CO₂.

The grouping and treatment of the model establishment experiment are shown in Table 1, with four replicates per group. RAW264.7 cells (1 × 10⁵ cells/well) were inoculated in 12-well plates. A total of 500 μL of PCV2 dilutions at varying concentrations (MOI 1, 0.1, 0.01, and 0.001) were added to the PCV2 infection groups. At the same time, an equal volume of serum-free DMEM culture media was administered to the Control group. Following a 2-h treatment, the cells were cultured in each well with 1 mL of cell maintenance medium (DMEM culture media containing 2% fetal bovine serum) following three PBS washes. Lastly, samples were collected at various intervals.

The grouping and treatment for the experiment on the intervention effect of FNB on inflammatory responses are shown in Table 2, with 4 replicates per group. RAW264.7 cells (1 × 10⁵ cells/well) inoculated into 12-well plates. The PCV2, FNB (high-, medium-, and low-dosage), and Rutin groups were administered 10^4.5 TCID₅₀ of the viral solution. In contrast, the Control group was supplemented with equivalent serum-free DMEM culture media. Following a 2-h treatment, the cells underwent three washes with PBS. In the Control and PCV2 groups, 1 mL of cell maintenance solution (DMEM with 2% FBS) was placed in each of the wells. The FNB and the Rutin groups were each supplemented with 1 mL of DMEM containing 80, 40, and 20 μg/mL of FNB and 40 μg/mL of Rutin, respectively, and then placed in an incubator for continued culture. Subsequently, the supernatant or cells were collected from the culture medium at 4, 8, 12, and 24 h for further experiments.

The animal experiment referred to Chen's research (8). A total of 108 SPF-grade KM mice, each weighing 20 ± 2 g, comprising an equal distribution of males and females, were acquired from the Animal Experiment Center of Guangxi Medical University (China). Before the experiment, the mice were acclimatized in an environment maintained at 25°C and 65% humidity for 7 days to mitigate environmental stress. Table 3 indicates that the animals were randomly allocated to several groups, namely, Control, PCV2, Rutin (100 mg/kg·BW), and FNB (25, 50, 100, 200, and 400 mg/kg·BW) groups, as well as a highest dose FNB control group (not infected with the virus). Each group contained 12 mice. Mice received daily inoculations of 1 mL PCV2 via intraperitoneal injection (0.3 mL/mouse), nasal drops (0.2 mL), and gavage (0.5 mL/mouse) for 3 consecutive days. FNB was gavaged into the mice from day 4 to day 6. On day 7, the animals were euthanized via asphyxiation under the guidelines established by the Guangxi University Animal Ethics Committee. Blood and lung specimens were obtained for subsequent study.

The influence of FNB on RAW264.7 cell viability was assessed using the CCK8 assay. Briefly, a 96-well plate containing a Control group and 7 varying concentrations of FNB groups (12.5, 25, 50, 100, 200, 400, and 800 μg/mL), each with eight repetitions, was inoculated with 5 × 10⁴ cells/mL at 100 μL/well. After adding 100 μL of FNB to each well, the wells were cultured for 24 h in a culture incubator. The growth medium was then taken out 2 h before the culture's end, 100 μL of serum-free DMEM with 10% CCK-8 was placed in each of the wells, and the culture was maintained for 2 h at 37°C with 5% CO₂. Lastly, a microplate reader was used to detect the OD value at 450 nm, and the CCK8 kit's instructions were followed to determine the maximum safe concentration of FNB on RAW264.7 cells.

Following the supplier's instructions, the supernatant or serum was collected for the measurement of MCP-1, IL-10, IL-8, TNF-α, IL-6, ROS, and NO levels while employing enzyme-linked immunosorbent assay (ELISA) and related commercial assay kits.

As directed by the manufacturer, TRIzol was utilized for RNA extraction from cells and lungs. Reverse transcription was used for transcribing RNA into cDNA. Using the cDNA as the template, RT-qPCR amplification was performed through a SYRB super-mix solution and a CFX96^TMReal-Time PCR Detection solution (Bio-Rad, USA). Table 4 contains primer sequences for β-actin, MCP-1, TNF-α, IL-10, IL-8, IL-6, and iNOS. The relative mRNA levels, adjusted to β-actin, were determined through the comparative 2^−ΔΔct technique.

For the immunohistochemistry study, lung tissues preserved in 4% paraformaldehyde were embedded in paraffin wax and sliced into 3.5 μm thick pieces. The expression level of the relevant protein was then determined according to the IHC kits.

Data were analyzed with SPSS v.22.0, using the LSD test and one-way analysis of variance (ANOVA). Data are shown as mean ± standard deviation. p < 0.05 was considered statistically significant.

Using commercial assay kits, the concentrations of inflammatory cytokines were evaluated in vitro in RAW264.7 cells to develop an inflammatory model in RAW264 induced by PCV2 (Figure 1). At 12 and 24 h post-infection (hpi), all measured inflammatory cytokines showed a substantial increase (p < 0.05, p < 0.01) following infection with 10⁰ PCV2 dilutions in comparison to the Control. This suggests that an in vitro inflammatory model can be established using 10⁰ PCV2 dilutions to treat RAW264.7 cells when the PCV2 titer is 10^4.5 TCID₅₀/0.1 mL. Therefore, we selected this condition to carry out subsequent experiments.

CCK-8 assays were utilized for measuring cell viability. At 24 h post-treatment (hpt), FNB at 12.5, 25, and 50 μg/mL elevated cell viability (p < 0.01; Figure 2) while FNB at 100 and 200 μg/mL had no substantial impact (p > 0.05). However, FNB at 400 and 800 μg/mL notably reduced viability (p < 0.01). Therefore, to examine their intervention effects on inflammatory responses induced by PCV2 infection, FNB concentrations of 20, 40, and 80 μg/mL were finally selected as high, medium, and low dosages, respectively.

Commercial assay kits were used to explore the intervention impacts of FNB on inflammatory cytokine levels in cells, including MCP-1, IL-8, IL-10, TNF-α, IL-6, ROS, and NO (Figure 3). The efficient development of an in vitro inflammatory model was demonstrated by the considerable up-regulation of all evaluated inflammatory factors induced by PCV2 infection relative to the Control (p < 0.05, p < 0.01).

At 4 or 8 hpt, the PCV2-induced increase in IL-6 and MCP-1 decreased considerably (p < 0.01) after exposure to 20, 40, and 80 μg/mL FNB. At 4 and 8 hpt, 40 μg/mL of FNB significantly reduced IL-10 and IL-8 levels (p < 0.01). TNF-α and NO levels were substantially lower than those of the PCV2 group after 8, 12, and 24 h following 20 μg/mL therapy (p < 0.01 or p < 0.05). At 4, 8, 12, and 24 h, ROS levels were significantly reduced (p < 0.01) by 20, 40, or 80 μg/mL FNB. An inflammatory response was triggered upon PCV2 infection, seen in raised inflammatory factor levels. Treatment with FNB significantly reduced cell TNF-α, MCP-1, IL-8, IL-6, IL-10, ROS, and NO levels, indicating that FNB could prevent inflammatory responses induced by PCV2.

Using RT-qPCR, the mRNA levels of the cytokines were further investigated. Following infection with PCV2, significant (p < 0.01) up-regulation of all six inflammatory factors was observed. Transcription was decreased by 40 and 80 μg/mL FNB administration, as evidenced by lower mRNA levels of MCP-1, IL-8, IL-10, IL-6, TNF-α, and iNOS at 12 hpt (p < 0.05, p < 0.01) than the PCV2 groups (Figure 4). These findings revealed that FNB could prevent PCV2 from activating genes and that PCV2 produced an inflammatory response by encouraging the expression of associated genes.

To evaluate the effects of FNB on inflammatory responses in vivo, we infected mice with PCV2 and treated them with FNB. The levels of IL-10, IL-6, TNF-α, MCP-1, and IL-8 in mouse sera were assessed by ELISAs. As shown in Figure 5, PCV2 infection significantly increased those inflammatory cytokines (p < 0.05 or p < 0.01). FNB administration at 100, 200, and 400 mg/kg·BW substantially decreased the increase in those inflammatory cytokines (p < 0.05 or p < 0.01).

We also investigated the intervention effects of FNB on PCV2 infection-induced inflammatory responses at the gene expression level. All inflammatory cytokines listed had significantly higher mRNA expression when infected with PCV2 (p < 0.01). Moreover, FNB was able to substantially down-regulate mRNA expression at doses of 25, 50, 100, 200, and 400 mg/kg·BW (Figure 6).

Consistent with the earlier studies, we investigated the intervention effects of FNB on inflammatory responses produced by PCV2 infection using immunohistochemistry methods. The proteins IL-8, IL-10, TNF-α, IL-6, and iNOS were expressed in the lung tissues of both the PCV2 group and the Control group of mice, demonstrating a diffuse distribution in the lungs and localized expression in the cytoplasm of cells, alveolar spaces, blood vessels, and tiny bronchial epithelial cells (Figure 7A). Relative to the controls, the protein iNOS, IL-10, IL-8, TNF-α, and IL-6 expression levels in the lung tissue of PCV2-infected mice were substantially higher (p < 0.01). Administration of 25, 50, 100, 200, and 400 mg/kg·BW FNB markedly reduced the protein iNOS, IL-10, IL-8, TNF-α, and IL-6 expressions levels in the alveolar space, fine bronchi, and cell cytoplasm of mice (p < 0.01; Figure 7B).