Effect of puerarin on collagen metabolism of fibroblasts in pelvic tissue of women with pelvic organ prolapse

Abstract

The aim of the present study was to investigate the protective effect of puerarin on pelvic organ prolapse (POP) and the underlying mechanisms that regulate the metabolism of human parametrial ligament fibroblasts (HPLFs). HPLFs obtained from the pelvic tissue of patients with (n=10) or without (n=8) POP during hysterectomy were isolated by enzymatic digestion and subsequently identified by immunocytochemistry in a previous study of the authors. Following this, cultured HPLFs were treated with 0.01, 0.10 or 1.00 mmol/l puerarin, followed by detection of proliferation rate by Cell Counting kit‑8 assay. Following incubation with puerarin for 48 h, mRNA and protein expression levels of tissue inhibitor of metalloproteinase‑1 (TIMP‑1), matrix metalloproteinase (MMP)‑2 and ‑9, and collagen (COL)I and III in HPLFs were quantified by reverse transcription‑quantitative polymerase chain reaction, and western blot and gelatin zymography analyses, respectively. MMP‑2 and ‑9 expression levels were increased, whereas expression levels of TIMP‑1, and COL I and III were decreased, in patients with POP compared with healthy controls. Following puerarin treatment, the expression levels of TIMP‑1, and COL I and III were enhanced, whereas MMP‑2 and ‑9 were inhibited. In conclusion, the present study demonstrated evidence increased degradation of the extracellular matrix in pelvic tissues of patients with POP compared with controls, and the protective effect of puerarin against POP via its anti‑degradation effect on collagen. These results provide evidence for puerarin as a novel approach for the treatment of POP.

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“…A modified enzyme digestion method was used in the present study for the establishment of the primary cell culture, which was similar to previous research methods (19). The tissues were washed 3–5 times with PBS (HyClone; GE Healthcare Life Sciences) containing 1% double-antibiotic solution (100K U/ml penicillin G and 100 mg/ml streptomycin; Hangzhou Ginom Biomedical Technology Co., Ltd.) in order to clear the underlying blood, axungia and necrotic tissue.…”

“…Vimentin is commonly used as a fibroblast marker to confirm the purity of cultured fibroblasts, as described in a previous study (19). After cells had migrated out of the vaginal wall tissue and become stable, the cells were applied to a glass slide.…”

Mechanical Stretching induces the apoptosis of parametrial ligament Fibroblasts via the Actin Cytoskeleton/Nr4a1 signalling pathway

“…Samples were obtained from uterosacral ligaments (USLs), and fibroblasts were cultured and purified as described previously 14 . Briefly, the USL tissues were cut into pieces (approximately 1 mm 3 ), placed in culture bottles and digested with modified collagenase type I (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and trypsinase (Sigma‑Aldrich, St. Louis, MO, USA).…”

“…The derived cells were characterized by their spindle‑like morphology, and identified by immunohistochemical staining, which indicated positive staining for vimentin and negative staining for cytokeratin and α-SMA, as previously described 14 . Cells from passage 3‑6 were cultured in chamber slides to 50% confluence and subsequently washed, fixed, and treated with 0.4% Triton X-100.…”

Mechanical stretching induces fibroblasts apoptosis through activating Piezo1 and then destroying actin cytoskeleton

“…We cultured and puri ed hAVWFs according to our previous description [11]. Brie y, the anterior vaginal wall tissues were cut into pieces (approximately 1 mm 3 ), placed in culture bottles and digested with Type I Collagenase (ThermoFisher, catalog No.…”