Evaluation of the antifibrotic potency by knocking down SPARC, CCR2 and SMAD3

Abstract

Background: The genes of SPARC, CCR2, and SMAD3 are implicated in orchestrating inflammatory response that leads to fibrosis in scleroderma and other fibrotic disorders. The aim of the studies is to evaluate synergistic anti-fibrotic potency of the siRNAs of these genes.

Methods: The efficacy of the siRNA-combination was evaluated in bleomycin-induced mouse fibrosis. The pathological changes of skin and lungs of the mice were assessed by hematoxylin and eosin and Masson's trichrome stains. The expression of inflammation and fibrosis associated genes and proteins in the tissues were assessed by real-time RT-PCR, RNA sequencing, Western blots and ELISA. Non-crosslinked fibrillar collagen was measured by the Sircol colorimetric assay.

Findings: The applications of the combined siRNAs in bleomycin-induced mice achieved favorable anti-inflammatory and anti-fibrotic effects. Activation of fibroblasts was suppressed in parallel with inhibition of inflammation evidenced by reduced inflammatory cells and proinflammatory cytokines in the BALF and/or the tissues by the treatment. Aberrant expression of the genes normally expressed in fibroblasts, monocytes/ macrophage, endothelial and epithelial cells were significantly restrained after the treatment. In addition, transcriptome profiles indicated that some bleomycin-induced alterations of multiple biological pathways were recovered to varying degrees by the treatment.

Interpretation: The application of the combined siRNAs of SPARC, CCR2, and SMAD3 genes ameliorated inflammation and fibrosis in bleomycin-induced mice. It systemically reinstated multiple biopathways, probably through controlling on different cell types including fibroblasts, monocytes/macrophages, endothelial cells and others. The multi-target-combined therapeutic approach examined herein may represent a novel and effective therapy for fibrosis.

Keywords: CCR2; Fibrosis; Inflammation; Multiple targets-siRNAs; Peptide nanoparticle; SMAD3; SPARC; Scleroderma.

Graphical abstract

Fig. 1

Examination of the anti-fibrotic effect in lungs of mouse models induced by bleomycin (n = 4) (A) The mRNA levels of collagen Col1a1, Col1a2, and Col3a1 were examined by Q-PCR. (B) Sircol assays to detect the soluble collagen content in the pulmonary tissues. (C) The type I collagen protein was detected by western blotting. COL I: type I collagen protein of the mouse. (D) The protein density in lung tissues was quantified by the Image-QuantTL software (General Electric Company, CT, USA). (E) Representative histological figures of HE and Trichrome staining of mouse lung tissues in the different groups. Sa: saline group, injected with saline (negative control); BLM: BLM group, injection with BLM only on day 0; Nano: Nano group, injection with BLM and PNP; T: therapeutic group, injection with PNP/PRSsi; *: P < .05 and **: P < .01.

Fig. 2

Examination of the anti-fibrotic effect in skin of mouse models induced by bleomycin (n = 5) (A) The mRNA levels of collagen Col1a1, Col1a2, Col3a1, and α-SMA were examined by Q-PCR. (B) Collagen I protein was detected by western blotting. (C) The protein density in skin tissues was quantified by the Image-QuantTL software. (D) Sircol assays to detect the soluble collagen content in the skin tissues. (E) The mRNA expression level of the anti-fibrotic gene, Mmp3 was examined by Q-PCR. (F) Representative histological figures of HE and Trichrome staining of mouse skin samples in the different groups. Sa: saline group, injected with saline; BLM: BLM group, injection with BLM per day from day 0 to day 21; Nano: Nano group, injection with BLM and PNP per day; T: treatment group, injection with PNP/PRSsi treatments. *: P < .05 and **: P < .01. Di-arrowhead indicated the thickness of the dermis.

Fig. 3

Examination of the inflammatory cells and inflammatory cytokines in the BALF of pulmonary fibrosis mouse models (n = 4) (A) White blood cells count and differential count by Wright-Giemsa's staining in the BALF under the optical microscope. The concentration of IL 6 (B) and MMP3 (C) proteins in the BALF were detected by ELISA. Sa: injected with saline (negative control); BLM: injection with BLM only on day 0; Nano: injection with BLM and PNP; T: therapeutic treatments; WBC, white cells count; Neu, neutrophil; Lym, lymphocyte; Mɸ, macrophage. *: P < .05 and **: P < .01. #, P > .05.

Fig. 4

Examination of the cytokines involved in the inflammation and immune response in dermal and pulmonary mouse models by Q-PCR (A) Il 6 and (E) Mmp3 mRNA levels were examined in pulmonary fibrosis mouse models. (B, C) Ifn γ mRNA levels were examined in lung and skin fibrosis models, respectively. (D) Il 10 mRNA levels were examined in dermal fibrosis mouse models. Sa: injected with saline (negative control); BLM: injection with BLM; Nano: injection with BLM and PNP; T: therapeutic treatments. *: P < .05, **: P < .01, ***: P < .001, #: P > .05.

Fig. 5

The gene expression profiles in the control, model and PNP/PRSsi treatment mice in pulmonary fibrosis mouse models. (A) The heatmap of all the differentially expressed genes (DEGs) among the control, model and treatment groups. Horizontal axis was depicted as a z score of each gene expression level with FPKM (fragments per kilobase of transcript per million fragments mapped). Vertical axis represents each of the DEGs. C: Saline group that the mouse models were injected with saline and served as the negative control, M: the mouse model injected with BLM, which served as a model group, T: the mouse model of treatment group, therapeutic injection with PNP/PRSsi. (B) The expression profiles of DEGs in the extracellular matrix (ECM) pathway among three groups were shown. (C) The Venn diagram showed the unique and shared numbers of the DEGs in different comparisons. 1294 DEGs were unique in the comparison between model group and control group, while 53 DEGs were unique in the comparison between PNP/PRSsi treatment group and model group. Meanwhile, 103 DEGs were shared between the two comparisons. (D) Top 15 significant pathways in KEGG enrichment analysis based on DEGs of different comparisons. Right part represents the down-regulated signaling pathways in the treatment group compared with those of in the model group, while the left part represented the up-regulated signaling pathways in model group compared with those in control group. (E) Top 15 down-regulated pathways in BLM group while partly up-regulated in PNP/PRSsi treatment group.