Progranulin Promotes the Formation and Development of Capsules Caused by Silicone in Sprague-Dawley Rats

Abstract

Background: Silicone implants are currently the most widely used artificial materials in plastic surgery. Capsule formation following implant application is unavoidable. When the capsule is excessively thick and strongly contracted, it can lead to obvious symptoms, clinically known as capsular contracture. Biological factors have always been the focus of research on the capsule formation. As a growth factor, progranulin (PGRN) plays an important regulatory role in wound healing, tissue fibrosis, tumor proliferation and invasion, and inflammation regulation. At present, the research on the capsule mainly involves the regulation of tissue healing and fibrosis under the influence of inflammation. Because PGRN has a regulatory role in these processes, we believe that the study of both can provide a new theoretical basis and intervention sites for monitoring and inhibiting the development of the capsule.

Methods: In this experiment, the effects of different surgical operations on the content of PGRN in the surgical site and plasma of rats were detected. Sprague-Dawley (SD) rat dermal fibroblasts were co-cultured by recombinant PGRN. The effects of r-PGRN on fibroblasts were detected by 5-ethynyl-2'-deoxyuridine (EdU) assay, wound healing assay and Western blot assay. Finally, the effect of PGRN on capsule formation and contracture was studied by changing the content of PGRN in the prosthesis in rats after operation.

Results: Surgical trauma and silicone implant increased plasma and local PGRN levels in SD rats. PGRN can activate the TGF-β/SMAD signaling pathway in a dose-dependent manner, thereby promoting fibroblast proliferation, differentiation and migration and inhibiting apoptosis and enhancing cell function, thereby promoting capsule formation and contracture.

Conclusion: PGRN promotes the formation and contracture of the silicone implant capsule in SD rats by activating the TGF-β/SMAD signaling pathway. This discovery may provide new therapeutic targets and detection indicators.

Keywords: TGF-β; fibroblast; fibrosis; implantation; silicone implants.

Figure 1

Surgical trauma and implant can increase plasma and local PGRN levels in SD rats, and the two have a synergistic effect. (A) The content of PGRN in the plasma of rats in different groups on days 3, 7, 14 and 112 were detected by ELISA analysis. (B) The local PGRN content in SD rats in different groups on day 14 were detected by Western blot analysis. Data are shown as the mean ± SEM (n = 4) (*p < 0.05 vs control).

Figure 2

Vimentin immunofluorescence staining. Cells were isolated from dermal tissue. Vimentin (+) staining in the cytoplasm showed green fluorescence, and nuclei were blue. (200 x scale bar = 50 µm, 400 x scale bar = 25 µm).

Figure 3

R-PGRN promotes fibroblast migration. Compared with the migration rate in the control group (0.2928 ± 0.03589), r-PGRN promoted the migration of fibroblasts in the experimental groups (0.4167 ± 0.032, 0.4705 ± 0.02451, 0.5193 ± 0.02790, and 0.4282 ± 0.01435). This promoting effect had a certain dose dependence in a concentration range of 0–100 ng/mL. Data are shown as the mean ± SEM (n = 3) (scale bar = 100 µm; *p < 0.05 vs control).

Figure 4

R-PGRN promotes fibroblast proliferation and inhibits apoptosis. (A) EdU fluorescence assay of cell proliferation. EdU(+) staining is shown as red fluorescence, which indicates cell division behavior, and nuclei are blue (scale bar = 25 µm). (B) The expression of apoptosis-related proteins BCL-2 and BAX was detected by Western blot. r-PGRN had a certain dose-dependent anti-apoptotic effect on fibroblasts. Actin served as a control. Data are shown as the mean ± SEM (n = 3) (*p < 0.05 vs control).

Figure 5

R-PGRN promotes the functional activation and differentiation of fibroblasts by activating the TGF-β/SMAD signaling pathway. (A) The expression of collagen types I (COL-I) and III (COL-III), α-SMA, TGF-β1 and p-SMAD2&3 in different groups were detected by Western blot analysis. Actin served as a control. (B) The expressions of COL-I and COL-III, α-SMA, TGF-β1 and p-SMAD2&3 in different groups were detected by Western blot analysis after pretreatment with HY-10431 or not. GAPDH served as a control. Data are shown as the mean ± SEM (n = 3) (*p < 0.05 vs control).

Figure 6

Increasing the local PGRN content promotes capsular formation and contracture. (A) The implant and the fibrous capsule on it at day 112 after operation. (B) H&E and Masson’s trichrome staining. The collagen in the capsule were stained by Masson’s trichrome (blue) (scale bar = 500 µm). (C) The expression levels of proteins COL-I, α-SMA, MMP-1, TIMP-1, TGF-β1 and p-SMAD2&3 in capsular tissues in different groups were detected by Western blot analysis. GAPDH served as a control. Data are shown as the mean ±SEM (n = 3) (*p < 0.05 vs control).