Matrine inhibits IL-1β secretion in primary porcine alveolar macrophages through the MyD88/NF-κB pathway and NLRP3 inflammasome

Abstract

Our previous studies demonstrated that matrine directly acts on the replication process of porcine reproductive and respiratory syndrome virus (PRRSV). Matrine inhibits viral replication and is also associated with the NF-κB signalling pathway. These results suggest that matrine has antiviral and anti-inflammatory effects. However, the specific anti-inflammatory mechanism of matrine is still unclear. In this study, we investigated the anti-IL-1β mechanism of matrine, as IL-1β is a major inflammatory cytokine, in porcine alveolar macrophages (PAMs) stimulated with 4 μg PRRSV 5'-untranslated region (UTR) RNA and 1 μg/mL LPS. After 5'UTR RNA and LPS co-stimulation of PAMs for 12 h, the expression of IL-1β, IL-6, IL-8 and TNF-α was significantly increased. The results also showed that co-stimulation induced the expression of MyD88, and activated the NF-κB signalling pathway and NLRP3 inflammasome. Furthermore, matrine treatment downregulated MyD88, NLRP3 and caspase-1 expression, inhibited ASC speck formation, suppressed IκBα phosphorylation, and interfered with the translocation of NF-κB from the cytoplasm to the nucleus. These results suggest that matrine plays an important role in PAMs co-stimulated with PRRSV 5'UTR RNA and LPS via its effect on NF-κB and the NLRP3 inflammasome. These findings lay the foundation for the exploration of the clinical application of matrine in PRRSV disease.

Figure 1

Inflammatory response in PAMs induced by PRRSV 5′UTR RNA and LPS. A, B PAMs were transfected with different doses of 5′UTR RNA (1, 2, and 4 μg/well) along with 1 μg/mL LPS. qRT-PCR and Western blot results showed that compared with PAMs in other groups, PAMs in the 4 μg 5′UTR RNA and 1 μg/mL LPS co-stimulation group produced higher levels of IL-1β (p < 0.05). C After co-stimulation, the relative expression level of IL-1β mRNA obtained was similar at the 12 and 24 h time-points. D 5′UTR RNA and LPS co-stimulation induced IL-1β expression in cells and supernatants. E, F 5′UTR RNA and LPS co-stimulation induced increased levels of IL-6, IL-8 and TNF-α mRNA and protein. Expression was normalized to that of GAPDH. Different letters (a, b, c, d, and e) on data indicate significant differences between groups (p < 0.05).

Figure 2

Matrine inhibited the secretion of inflammatory mediators. A Matrine inhibited the mRNA expression of IL-1β, IL-6, IL-8 and TNF-α. After 6 h of co-stimulation, different doses of matrine (0.4, 0.2 and 0.1 mg/mL) or 0.04 mg/mL dexamethasone were added to the cells for an additional 6 h of incubation. The expression levels of IL-1β, IL-6, IL-8 and TNF-α genes in PAMs from all treatment groups were measured using qRT-PCR. B, C Matrine reduced the protein levels of IL-1β, IL-6, IL-8 and TNF-α. Expression was normalized to GAPDH expression. ^#Indicates that the expression was significantly different compared to that of the group with 5′UTR RNA and LPS co-stimulation (p < 0.05).

Figure 3

Matrine inhibited NF-κB activation. The protein levels of IκBα, p-IκBα, cytoplasmic p65 and nuclear p65 were measured by Western blot assay (upper), and GAPDH was used as an internal control for normalization. The expression of nuclear p65 was normalized to that of TBP using ImageJ software (lower). ^#Indicates that the expression was significantly different compared to that of the group with 5′UTR RNA and LPS co-stimulation (p < 0.05).

Figure 4

Effect of matrine on TLR4, DHX36 and MyD88 expression. PAMs from all treatment groups were collected, and total RNA or protein was extracted and assessed by qRT-PCR or Western blot, respectively. A The expression of the TLR4, DHX36 and MyD88 genes. The results showed no changes in TLR4 gene expression levels among the different groups (p > 0.05), but DHX36 and MyD88 gene expression levels were significantly increased in the 5′UTR RNA and LPS co-stimulation group (p < 0.05). Compared with the co-stimulation group, the matrine-treated group showed reduced expression of DHX36 and MyD88 genes (p < 0.05). B The expression of TLR4, DHX36 and MyD88 protein levels. Expression was normalized to that of GAPDH. ^#Indicates that the expression was significantly different compared to that of the group with 5′UTR RNA and LPS co-stimulation (p < 0.05).

Figure 5

Matrine inhibited NLRP3 inflammasome activation in PAMs. A The mRNA expression of NLRP3, ASC and caspase-1 was detected by qRT-PCR. B, C Protein levels in PAMs were assessed with antibodies against NLRP3 and caspase-1 by immunoblotting (left panel). Quantification of the protein expression was performed by densitometric analysis of the bands (right panel). D Effects of matrine on ASC protein expression. Expression was normalized to GAPDH expression. E ASC specks (FITC) and nuclei (DAPI) were visualized using a confocal fluorescence microscope. ASC is evenly distributed in the cytoplasm and nucleus (control), and 5′UTR RNA and LPS co-stimulation induced the formation of ASC specks near the nuclear membrane. Matrine inhibited the formation of ASC specks. ^#Indicates that the expression was significantly different compared to that of the group with 5′UTR RNA and LPS co-stimulation (p < 0.05).

Figure 6

Proposed model illustrating the anti-IL-1β activity of matrine. 5′UTR RNA and LPS co-stimulation induces the recruitment of MyD88 to activate the NF-κB signalling pathway, which in turn initiates NF-κB-dependent pro-IL-1β, IL-6, IL-8 and TNF-α transcription and also induces the activation of the NLRP3 inflammasome. Matrine treatment downregulates MyD88, NLRP3 and caspase-1 expression, inhibits ASC speck formation, suppresses IκBα phosphorylation, and interferes with the translocation of NF-κB from the cytoplasm to the nucleus.