Treatment of Pelvic Organ Prolapse by the Downregulation of the Expression of Mitofusin 2 in Uterosacral Ligament Tissue via Mesenchymal Stem Cells

Abstract

Background: The relationship between pelvic organ prolapse (POP), an aging-related disease, and the senescence-related protein mitofusin 2 (Mfn2) has rarely been studied. The aim of the present study was to explore the therapeutic effects of the downregulation of Mfn2 expression by stem cells on POP through animal experiments. Methods: First, a rat POP model was constructed by ovariectomy and traction. The rats in the non-pelvic organ prolapse (NPOP) and POP groups were divided into four groups for negative controls (N1−N4, N1: NPOP-normal saline; N2: NPOP-untransfected stem cells; N3: NPOP-short hairpin negative control (NPOP-sh-NC); N4: NPOP-short hairpin-Mfn2 (NPOP-sh-Mfn2)), and four groups for prolapse (P1−P4, P1: POP-normal saline; P2: POP-untransfected stem cells; P3: POP-sh-NC; P4: POP-sh-Mfn2), respectively. Stem cells were then cultured and isolated. The expression of Mfn2 was inhibited by lentivirus transfection, and the stem cells were injected into the uterosacral ligament of the rats in each group. The expression levels of Mfn2 and procollagen 1A1/1A2/3A1 in the uterosacral ligaments of the rats were observed at 0, 7, 14, and 21 days after injection. Results: Compared to the rats in the NPOP group, the POP rats had significant prolapse. The Mfn2 expression in the uterosacral ligaments of the POP rats was significantly increased (p < 0.05, all), and the expression of procollagen 1A1/1A2/3A1 was significantly decreased (p < 0.001, all). The POP rat model maintained the same trend after 21 days (without stem cell injection). At day 14, compared to the rats in the N1 group, the Mfn2 expression in the uterosacral ligament of the rats in the N4 group was significantly decreased (p < 0.05, all), and the expression of procollagens was significantly increased (p < 0.05, all). Similarly, compared to the rats in the P1 group, the Mfn2 expression in the uterosacral ligament of the rats in the P4 group was significantly decreased (p < 0.05, all), and the expression of procollagens was significantly increased (p < 0.05, all). Similarly, on day 21, the Mfn2 mRNA and protein expression in the uterosacral ligament of the POP and NPOP rats was significantly decreased (p < 0.05, all), and the expression of procollagens was significantly increased (p < 0.05, all) in the rats in the sh-Mfn2 group (N4, P4) compared to the rats in the saline group (N1, P1). Conclusions: The downregulation of Mfn2 expression by stem cells decreased the expression of Mfn2 and increased the expression of procollagen1A1/1A2/3A1 in the uterosacral ligament of the POP rats; this effect was significant 14−21 days after the injection. Thus, Mfn2 may be a new target for POP control.

Keywords: bone marrow stem cells; mitofusin 2; pelvic organ prolapse; procollagen; rat model; uterosacral ligament.

Figure 1

Establishment of the POP rat model. (A) From left to right, after one, five, and 10 stretching cycles. (B) After the model was established, the uterosacral ligaments in rats in the POP and NPOP groups were collected on days 0 and 21 for qRT-PCR detection. (C) After the model was established, the uterosacral ligaments in the rats in the POP and NPOP groups were collected on days 0 and 21 for Western blot detection. ** p < 0.01, *** p < 0.001.

Figure 2

Culture and identification of rat BMSCs. (A–C) The positive markers were used to differentiate the stem cells from other cells. The positive rates of CD29, CD90 and CD106 were 92.5%, 100% and 91.1%, respectively. Red line represents the antibody to be tested, and blue or orange line represents the homotypic control. (D,E) The negative markers CD11b and CD45 were used to distinguish mesenchymal stem cells from hematopoietic stem cells. The positive rates of CD11b and CD45 were 0.080% and 0.030%, respectively. Red line represents the antibody to be tested, and blue line represents the homotypic control. (F) Adipogenic induction and Oil Red O staining; (G) Osteogenic induction and Alizarin Red S staining.

Figure 3

Inhibition of Mfn2 expression. (A) Viral titer detection after the inhibition of Mfn2. Left: bright field, 200×; Right: green fluorescence field, 200×; detected cells: 293T cells. (B) After the sh-Mfn2 and sh-NC lentiviruses were transfected into stem cells, the mRNA level of Mfn2 was detected by qRT-PCR. (C) After the sh-Mfn2 and sh-NC lentiviruses were transfected into stem cells, the protein expression level of Mfn2 was detected by western blot. * p < 0.05. All of the experiments were repeated three times.

Figure 4

Therapeutic effect of stem cells on POP rats. (A) On day 0 of the injection treatment, the uterosacral ligaments of the rats in the eight groups were collected for mRNA detection; (B) mRNA analysis on day 7 post-stem cell injection; (C) mRNA analysis on day 14 post-stem cell injection; (D) mRNA analysis on day 21 post-stem cell injection; (E–H) Western blot analysis on days 0, 7, 14, and 21 post-stem cell injection. * p < 0.05, ** p < 0.01, *** p < 0.001. All of the experiments were repeated three times.