Matrine and icariin can inhibit bovine viral diarrhoea virus replication by promoting type I interferon response in vitro

Madin–Darby bovine kidney (MDBK) cells were cultured in Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium (DMEM/F12, 1:1) (Gibco Life Technologies, Grand Island, NY, USA) with 10% foetal bovine serum (FBS; Gibco-BRL, Carlsbad, CA, USA) and 1% penicillin–streptomycin. The cells were cultured in a humidified atmosphere at 37°C and 5% CO₂. The NADL BVDV reference strain was procured from the China Institute of Veterinary Medicine (Nangjing, China). The tissue culture infective dose (TCID₅₀) for NADL on MDBK cells was determined using the Spearman–Kärber method and was found to be l0^−4.5/0.1 mL. Matrine (catalogue No. S2322; purity 99.72%) and icariin (catalogue No. S2312; purity 99.05%) were purchased from Selleck Biotechnology (Houston, TX, USA). Icariin was diluted in cell maintenance solution to final concentrations of 128 nM, 64 nM, 32 nM, 16 nM, 8 nM, 4 nM, 2 nM, and 1 nM. Matrine was diluted to 1 mg/mL in dimethyl sulphoxide (DMSO), and further diluted in cell maintenance medium to achieve final concentrations of 512 ng/mL, 256 ng/mL, 128 ng/mL, 64 ng/mL, 32 ng/mL, 16 ng/mL, 8 ng/mL and 4 ng/mL. The final solution was passed through a 0.22 μm filter membrane and stored at 4°C.

The virus was diluted with DMEM/F12 medium and incubated with cells at 37°C for 1.5 h, then the cells were washed three times with phosphate-buffered saline (PBS) to remove the residual virus solution, and DMEM/F12 containing 2% FBS and 1% penicillin–streptomycin was added.

Ninety-six-well culture plates were inoculated with approximately 4 × 10⁴ MDBK cells. Once the degree of cell confluence reached 70–90%, the culture medium was discarded, and the cells were washed with PBS three times. The concentration gradients of matrine (512 ng/mL, 256 ng/mL, 128 ng/mL, 64 ng/mL, 32 ng/mL, 16 ng/mL, 8 ng/mL and 4 ng/mL) and icariin (128 nM, 64 nM, 32 nM, 16 nM, 8 nM, 4 nM, 2 nM and 1 nM) were added to the culture medium, and the cells were incubated at 37°C with 5% CO₂. After 24 h, 10 μL of cell-counting kit-8 solution (Beyotime Biotechnology, Shanghai, China) was added to each well, and the plates were incubated at 37°C for 3 h. Dimethyl sulphoxide was the control in the matrine-treated test. The half-maximal inhibitory concentrations (IC₅₀) of matrine and icariin on MDBK cells were calculated based on the optical density values at 450 nm. For the calculation of half-maximal effective antiviral concentration (EC₅₀), the same procedure was followed but with BVDV at 100 × the 50% tissue culture infectious dose (TCID₅₀) added to the cells in the presence of matrine and icariin. Finally, the therapeutic index (TI) of matrine and icariin was calculated based on the IC₅₀ and EC₅₀ using the formula: TI = IC₅₀/EC₅₀.

The total RNA was extracted from MDBK cells using Trizol reagent (TransGen Biotech, Beijing, China) and following the manufacturer’s instructions. A PrimeScript II 1^st Strand cDNA Synthesis Kit was used to conduct the reverse transcription reaction (TaKaRa, Kusatsu, Japan). The primers were BVDV 5′UTR (forward primer 5′-ATG CCCWGTAGGACTAGCA-3′ and reverse primer 5′-TCA ACTCCATGTGCCATGTAC-3′), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (forward primer 5′-GATTGT CAGCAATGCCTCCT-3′ and reverse primer 5′-GGTCAT AAGTCCCTCCACGA-3′), IFN-α (forward primer 5′-GTG AGGAAATACTTCCACAGACTCACT-3′ and reverse primer 5′-TGARGAAGAGAAGGCTCTCATGA-3′), IFN-β (forward primer 5′-ACAGAGTCACCCACCTCACC-3′ and reverse primer 5′-GACCAGGTGTGTGTCAGTCC-3′), IRF3 (forward primer 5′-GGCTTGTGATGGTCAAGGTT-3′ and reverse primer 5′-TGCAGGTCGACAGTGTTCTC-3′), RIG-I (forward primer 5′-CCTCAGTTGGCGTTGGAGAT-3′ and reverse primer 5′-GAGCCTCTGTCTCTGCCATC-3′) and TLR3 (forward primer 5′-TCTGTCCCTGAGCAG CAACC-3′ and reverse primer 5′-AAGGTCCAGCGT GGTGAGAT-3′). The threshold cycle (Ct) values of the target genes were normalised to GAPDH and compared to the BVDV positive control (without drugs) using the 2^−ΔΔCt method. The protocol of the quantitative reverse-transcription (RT-q) PCR was described in a previous study (25). The experiment was conducted in triplicate for each reaction.

To perform the viral replication cycle test, the monomer concentrations based on the results of cytotoxicity test and on the morphological observations of cells were selected. Specifically, matrine was used at 128 ng/mL and icariin at 8 nM.

In this group the virus and medicine were co-incubated before being added to the cells. When the MDBK cells grew into monolayers on six-well plates, the culture medium was discarded and the cells were washed with PBS three times. The matrine and icariin were incubated separately and simultaneously mixed with equal volumes of BVDV (100 TCID₅₀) at 4°C for 1 h. The mixture was added to the cells and incubated at 37°C for 1.5 h with full shaking every 15 min. Then the liquid was discarded, the cells were washed with PBS three times and 2 mL of maintenance solution was added, after which the cells and solution were placed in the incubator for 24 h at 37°C and 5% CO₂. After washing with PBS three times, whole cell lysates were collected for RT-qPCR detection.

In this group the medicine and virus were added simultaneously to the cells. Madin–Darby bovine kidney cell monolayers grown on six-well plates were precooled at 4°C for 30 min and then washed three times with PBS at 4°C. Matrine and icariin solutions were added separately to each well, along with an equal volume of BVDV (100 TCID₅₀) and incubated at 4°C for 1.5 h, with full shaking every 15 min. Then the liquid was discarded and 2 mL of maintenance solution was added to the cells, which were cultured in the incubator for 24 h at 37°C and 5% CO₂. After washing with PBS three times, the cell lysates were collected for the RT-qPCR assay.

In this group the cells were incubated with the extracts first and infected with the virus next. When the MDBK cell grew into monolayers on six-well plates, the culture medium was discarded and the cells were washed with PBS three times. A 500 μL volume of icariin or matrine solution at the maximum safe concentration (maximum concentration with no effect on cell viability) was added to each well and incubated at 37°C for 3 h. The solution was then discarded, and 500 μL of 100 TCID₅₀ BVDV solution was added and incubated at 37°C for 1.5 h, with full shaking every 15 min. The residual viruses were discarded, 2 mL of maintenance solution was added, and the cells were placed in the incubator at 37°C for 24 h. The cell lysates were collected for the RT-qPCR.

In this group the cells were infected by virus first and treated with the extracts next. When the MDBK cells grew into monolayers on six-well plates, the culture medium was discarded and the cells were washed with PBS three times. Then, 500 μL of 100 TCID₅₀ BVDV solution was added to each well. The cells were placed in the incubator at 37°C for 1.5 h, with full shaking every 15 min. The liquid was then discarded, 2 mL of matrine or icariin of different concentrations were added, and the cells were placed in the incubator at 37°C for 24 h. The cell precipitations were collected and analysed with the RT-qPCR.

Similar to the pre-treatment group method described above, cell supernatants were collected from MDBK cells at 24 h after BVDV infection. The cell supernatants were centrifuged to remove particulates and aggregates. The concentrations of IFN-β in MDBK cells were measured using bovine IFN-β ELISA kits (Enzyme-linked Biotechnology, Shanghai, China) according to the manufacturer’s instructions, and the cell precipitation was collected at 24 h, 48 h and 72 h for RT-qPCR detection. All samples were measured in triplicate.

Data are expressed as the mean and standard deviation of the mean (SD), and the significance of differences between groups was evaluated using Student’s t-test or the one-way analysis of variance followed by Tukey’s post-hoc test. Results with P-values of < 0.05 < 0.01 < 0.001 and < 0.0001 were considered to be statistically significant. All experiments were repeated at least three times individually. Graphs were plotted and analysed using GraphPad Prism software, version 8.0 (GraphPad Software, La Jolla, CA, USA).

Matrine had no significant effect on MDBK cell viability when the concentration was 4, 8, 16, 32, 64 or 128 ng/mL compared with the DMSO control (Fig. 1A), but it significantly decreased the activity of MDBK cells at 256 and 512 ng/mL (P-value < 0.0001). Therefore, 128 ng/mL was considered the maximum concentration of matrine which was non-toxic to MDBK cells and was adopted as the safe concentration. The dose–effect relationship between the logarithm of matrine concentration (X) and the inhibition rate of MDBK (Y) is shown in Fig. 1B. The IC₅₀ of matrine was calculated as 294.9 ng/mL. Icariin had no significant effect on MDBK cell activity when the concentration was 1, 2, 4 or 8 nM compared with the control group (Fig. 1C), but it significantly decreased the activity of MDBK cells at 16, 32, 64 and 128 nM (P-value < 0.0001). Therefore, 8 nM was regarded as the maximum concentration of icariin which was non-toxic to MDBK cells and was adopted as the safe concentration. The dose–effect relationship between the logarithm of the concentration (X) and the inhibition rate (Y) of MDBK is shown in Fig. 1D. The IC₅₀ of icariin was calculated as 124.8 nM.

Fig. 1.

At the safe concentrations, matrine and icariin had inhibitory effects on BVDV, and the viral inhibition rate gradually increased with the rise in concentration, indicating that the anti-BVDV effects of matrine and icariin were dose-dependent (Figs 2A and 2C). Furthermore, matrine at 128 ng/mL had a very highly significant anti-BVDV effect compared to this extract at 4, 8, 16 and 32 ng/mL (P-value < 0.0001), and had a significant anti-BVDV effect compared to 64 ng/mL (P-value < 0.05). Icariin of 8 nM had a very highly significant anti-BVDV effect compared to this extract at 0.25 and 0.5 nM (P-value < 0.0001), a highly significant anti-BVDV effect compared to 1 and 2 ng/mL (P-value < 0.001), and a significant anti-BVDV effect compared to 4 ng/mL (P-value < 0.05). The EC₅₀s of matrine and icariin against BVDV were 43.55ng/mL and 4.48 nM (Figs 2B and 2D), and their TIs were 6.772 and 27.857, respectively.

Fig. 2.

As shown in Figs 3A and 3B, the BVDV 5′UTR gene level in the virucide group was very highly significantly decreased from that in the BVDV-positive group (P-value < 0.0001), illustrating that both matrine and icariin had a direct virucidal effect on BVDV. The co-treatment group was established to identify whether matrine and icariin could inhibit the process of virus adsorption. The results showed that the BVDV 5′UTR gene level in the co-treatment group decreased very highly significantly from that in the BVDV model group (P-value < 0.0001) (Figs 3A and 3B), suggesting that both matrine and icariin could block the replication process of BVDV by inhibiting adsorption. Furthermore, the BVDV 5′UTR gene level in the pre-treatment and post-treatment groups was very highly significantly downregulated from that in the BVDV model group (P-value < 0.0001), suggesting that the two herbal extracts had preventive and therapeutic effects. Therefore, these results indicated that matrine and icariin could inhibit BVDV replication through multiple mechanisms.

Fig. 3.

It is well known that preventing disease is preferable to treating it. In order to explore whether matrine and icariin could be used as feed additives for the prevention of BVD, the pre-treatment group of 24 h was set as the benchmark, and the 5′UTR gene level of BVDV increased with the continuous replication of BVDV at 48 h and 72 h (Fig. 3C). The levels of BVDV 5′UTR in MDBK cells pretreated with matrine were very highly significantly lower at 24 h, 48 h and 72 h than that in MDBK cells infected with BVDV only (the BVDV-positive group) (P-value < 0.0001) (Fig. 3C). Similarly, the levels of BVDV 5′UTR in MDBK cells pretreated with icariin were significantly lower at 24 h, 48 h and 72 h than that in MDBK cells infected with BVDV only (the BVDV-positive group) (P-value < 0.0001) (Fig. 3C).

At 24 h, BVDV infection downregulated the secretion of IFN-β as compared to the control group without BVDV infection (P-value < 0.01) (Fig. 4A). Compared with the BVDV-only group, there was an increase in IFN-β secretion in the group treated with matrine alone (P-value < 0.01), where it was higher than in the control group (P-value < 0.05). Furthermore, again compared to the BVDV-only group, there was a significant increase in IFN-β secretion in the matrine pretreatment group (P-value < 0.01), where it was higher than the control group (P-value < 0.05). There was no statistical difference between the icariin-treated group and the BVDV group (P-value > 0.05), nor was there a significant difference between the icariin-treated group and the control group (P-value > 0.05). However, the level of IFN-β in the icariin pretreatment group was significantly higher than those in the BVDV group (P-value < 0.01) and the control group (P-value < 0.05). It demonstrated that matrine and icariin could block BVDV’s inhibition of the secretion of IFN-β and significantly upregulate the expression of IFN-β in BVDV infection (Fig. 4A).

Fig. 4.

Regarding the messenger RNA (mRNA) levels of IFN-α and IFN-β their expression levels in the matrine-treated group increased very highly significantly over those in the control group and the BVDV group at 24 h after BVDV infection (P-value < 0.0001) (Figs 4B and 4C). The expression level of IFN-α in the matrine pretreatment group was significantly higher than that in the BVDV group at 24 h (P-value < 0.01), while the level of IFN-β in the matrine pretreatment group was very highly significantly upregulated when measured against those of the other three groups at 24 h (P-value < 0.0001). Interestingly, the levels of IFN-α and IFN-β in the matrine pretreatment group were very highly significantly above those in the other three groups at 48 h after BVDV infection (P-value < 0.0001). Compared with the control group, the mRNA of IFN-α in the BVDV group was very highly significantly downregulated at 24 h (P-value < 0.0001), and highly significantly upregulated at 72 h (P-value < 0.001) (Fig. 4B). The level of IFN-β in the matrine-treated group was very highly significantly higher than those in the control group and the BVDV group at 48 h (P-value < 0.0001), while there were no differences in the mRNA of IFN-α among the matrine-treated group, the control group and the BVDV group (P-value > 0.05). Similarly, the levels of IFN-α and IFN-β in the matrine pretreatment group very highly significantly exceeded those of the other three groups at 72 h after BVDV infection (P-value < 0.0001). The level of IFN-α in the matrine-treated group was raised very highly significantly compared to this level in the control group (P-value < 0.0001), and was also significantly higher than the level in BVDV group at 72 h (P-value < 0.01). The mRNA of IFN-β in the matrine-treated group was upregulated very highly significantly over that of the control group (P-value < 0.0001), while there was no difference between the levels in the matrine-treated group and the BVDV group (P-value > 0.05).

Compared with the BVDV group, the expression levels of IFN-α and IFN-β in the icariin treated group were very highly significantly upregulated at 24 h (P-value < 0.0001) (Figs 4D and 4E). Very highly significant upregulation was noted of the levels of IFN-α and IFN-β in the icariin pretreatment group compared to those of the other three groups at 24 h and 72 h after BVDV infection (P-value < 0.0001). IFN-α was secreted significantly more in the icariin pretreatment group as compared to in the control group, and also was as compared to its secretion in the icariin-treated group and the BVDV group at 48 h (P-value < 0.01). Compared with the control group, the mRNA of IFN-α in the BVDV group was very highly significantly downregulated at 24 h (P-value < 0.0001), and significantly and very highly significantly upregulated at 48 h and 72 h (respective P-values < 0.01 and < 0.0001) (Fig. 4D). The mRNA levels of IFN-β in the icariin pretreatment group were very highly significantly upregulated over those in the other three groups at 48 h (P-value < 0.0001). IFN-β in the icariin treated group displayed very highly significantly upregulated secretion compared to secretion in the control group and the BVDV group at 48 h (P-value < 0.0001) (Fig. 4E). These results suggested that both matrine and icariin could enhance the expression of IFN-α/β to produce an inhibitory effect on BVDV infections.

To investigate the pathway through which matrine and icariin improved IFN-α/β expression, the mRNA expression levels of RIG-I, TLR3 and IRF3 were examined. Noteworthily, the expression of RIG-I, TLR3 and IRF3 was significantly downregulated by BVDV infection at 24 h in the BVDV-only group (P-value < 0.01) (Figs 5A–F), indicating BVDV could suppress the innate immune response of host cells. However, the expression of RIG-I, TLR3, and IRF3 gradually rose from 48 to 72 h after BVDV infection. As Fig. 5A shows, compared to the control group, the expression level of RIG-I was very highly significantly downregulated in the BVDV group at 24 h after BVDV infection (P-value < 0.0001), but very highly significantly upregulated at 48 h (P-value < 0.0001) and significantly upregulated at 72 h (P-value < 0.05). Importantly, the mRNA level of RIG-I in the matrine-only group increased dramatically over those in the other three groups at 24 h, 48 h and 72 h (P-value < 0.0001). The mRNA level of RIG-I in the matrine pretreatment group (matrine + BVDV) was also upregulated significantly when measured against the levels in the BVDV group and the control group at 24 h and 48 h (P-value < 0.0001). Notably, the mRNA level of RIG-I in the matrine pretreatment group increased significantly than the other three groups at 72 h (P-value < 0.0001). The mRNA expression of TLR3 in the BVDV group underwent significant and very highly significant downregulation, as comparison with the control group expression level showed respectively at 24 h (P-value < 0.01) and 48 h (P-value < 0.0001) (Fig. 5B). The levels of TLR3 in the matrine pretreatment group were highly significantly above those in the other three groups at 24 h and 72 h (P-value < 0.001). Similarly, the mRNA of TLR3 in the matrine-treated group was markedly higher than those in the BVDV group and the control group at each time point (P-value < 0.001). The mRNA expression of IRF3 in the matrine-treated group very highly significantly exceeded its expression in the other three groups at 24 h and 48 h (P-value < 0.0001) (Fig. 5C). The mRNA of IRF3 in the matrine pretreatment group was highly significantly more abundant than in the BVDV group and the control group at 24 h and 48 h (P-value < 0.001). At 72 h after BVDV infection, the mRNA level of IRF3 in the matrine pretreatment group was very highly significantly upregulated compared to those of the other three groups (P-value < 0.0001).

Fig. 5.

Compared to the mRNA expression of RIG-I, TLR3 and IRF3 in the control group and the BVDV group, this expression was upregulated very highly significantly in the icariin-treated group at 24 h and 48 h after BVDV infection (P-value < 0.0001) (Figs 5D–F). Compared to the mRNA expression of RIG-I and TLR3 in the BVDV group, mRNA expression of these receptors in the icariin pretreatment group were upregulated very highly significantly at 24 h and 72 h (P-value < 0.0001). Additionally, the mRNA level of TLR3 in the icariin pretreatment group rose very highly significantly above those of the other three groups at 48 h after BVDV infection (P-value < 0.0001) (Fig. 5E). Notably, the mRNA level of IRF3 in the icariin-treated group was enhanced very highly significantly over those of the other three groups at 48 h (P-value < 0.0001) (Fig. 5F). These results suggested that matrine and icariin upregulated IFN-α/β expression levels by potentiating the expression of RIG-I, TLR3 and IRF3, illustrating that the pathways of RIG-I and TLR3 may be involved in the action of matrine and icariin against BVDV.