Effects of mechanical stretching on the morphology of extracellular polymers and the mRNA expression of collagens and small leucine-rich repeat proteoglycans in vaginal fibroblasts from women with pelvic organ prolapse.

Pelvic organ prolapse (POP) is the abnormal protrusion of pelvic organs into the vaginal canal or beyond the vaginal opening, which may cause a series of symptoms in the urinary, genital, and bowel tracts that adversely affect the quality of life of affected individuals. Despite being a common disease affecting 41.1% of women aged 50–79 years [ 1 ], the exact etiology and pathogenesis of POP remain poorly understood. Many risk factors related to POP, including vaginal delivery, advanced age, menopause, estrogen deficiency, chronic cough, obesity, constipation, and heavy lifting [ 2 – 4 ], may cause abnormal metabolism and remodeling of the pelvic supportive connective tissues, thereby affecting the mechanical properties of these tissues and resulting in the occurrence and progression of POP. In the supportive system of the pelvic floor, fibrous connective tissues surrounding the pelvic organs form fascia and ligaments to provide mechanical strength to support the vagina and its adjacent organs. Meanwhile, due to their specific anatomical location, these tissues are subjected to constant mechanical tensile loading from abdominal pressure and gravity [ 5 – 7 ]. The fascia and ligaments of the pelvic floor mainly comprise dense connective tissues containing fibroblasts and extracellular matrix (ECM) secreted by fibroblasts. In addition to providing a microenvironment for cells to shape tissue-specific function, the ECM provides mechanical strength and support for tissue. Thus, any hypothesis seeking to explain the etiology and pathogenesis of POP must address the remodeling of fibroblasts and ECM. In a previous study concerning the effects of a 0.1-Hz uniaxial cyclic mechanical stretching (CS) load with 10% elongation on the morphology and cytoskeleton of vaginal fibroblasts in vitro, we confirmed the mechanosensitivity of these fibroblasts and verified that POP fibroblasts exhibit higher sensitivity to surface tension on the culture substrate and lower tolerance to mechanical stretching in terms of cell morphology and F-actin and α-tubulin protein expression. Furthermore, we found that 17-β-estradiol (E 2 ) can improve POP prognosis by inhibiting the mechanical stretching-induced overexpression of F-actin and α-tubulin in healthy fibroblasts and decreased expression of these proteins in POP fibroblasts, thus restraining cell deformation [ 8 ]. The ECM is mainly composed of collagens, proteoglycans and glycoproteins. In the present study, we first observed the effects of a 0.1-Hz uniaxial CS stretching load with 10% elongation on the morphological characteristics of extracellular polymers on the cell surface using scanning electron microscopy (SEM). Collagens are the primary structural components of the ECM, with type I collagen (COL I) and type III collagen (COL III) being responsible for mechanical strength and tissue elasticity, respectively [ 9 , 10 ]. Moreover, collagens are the predominant proteins in vaginal connective tissues [ 11 ]. Thus, we wanted to further investigate the effects of a 0.1-Hz uniaxial CS load with 10% elongation on COL I and COL III mRNA expression using real-time quantitative polymerase chain reaction (RT-PCR). Small leucine-rich proteoglycans (SLRPs) are components of the ECM that are structurally characterized by a specific protein core with leucine-rich repeat (LRR) motifs substituted with one or more covalently linked glycosaminoglycan (GAG) side chains, and SLRPs are classified into five distinct families based on conservation and homology at the protein and genomic levels, the presence of characteristic N-terminal cysteine-rich clusters with defined spacing, and chromosomal organization [ 12 , 13 ]. SLRPs are capable of binding different cell-surface receptors, cytokines, growth factors, and other ECM components to modulate cellular functions, such as collagen fibrillogenesis and matrix assembly, cell proliferation and differentiation, innate immunity and inflammation, and tumor growth and metastasis [ 14 – 17 ]. Decorin (DCN), biglycan (BGN), fibromodulin (FMO), and lumican (LUM) are the best-characterized members of the SLRP family and have been extensively studied. DCN was found to inhibit downstream oncogenic signaling in several solid tumors by binding and antagonizing various receptor tyrosine kinases [ 18 ]; LUM was reported to have anticancer activity by effectively regulating the estrogen receptor-associated functional properties of breast cancer cells, the expression of matrix effectors, and the epithelial-to-mesenchymal transition [ 19 ]. As biologically active components of the ECM, SLRPs work in concert to assemble collagen fibrils into a functioning ECM, contribute to the development of connective tissue mechanical properties [ 20 ], and protect collagen fibrils from proteolytic cleavage by various collagenases and thus play roles in guiding and stabilizing the formation and maturation of collagen fibrils [ 21 , 22 ]. Altered expression and structural deficiency of SLRPs impact matrix assembly and tissue function. Knockout of both DCN and BGN genes leads to larger and heterogeneous fibril diameters in tendons, and acute ablation results in tendon failure at lower loads, as well as decreased stiffness [ 23 ]. Alterations were observed early in LUM-deficient tendons, and a severe phenotype was acquired at maturation in FMD-deficient tendons during fibrillogenesis [ 24 ]. Therefore, in the present study, we also examined the effects of a 0.1-Hz uniaxial CS load with 10% elongation on DCN, BGN, FMO, and LUM mRNA expression using RT-PCR. We hypothesized that the mechanical stretching caused by the increase in intra-abdominal pressure and gravity applied to the pelvic floor plays an important role in the occurrence and progression of POP. In turn, the excessive mechanical stretching load may impair the integrity and mechanical properties of the pelvic supportive connective tissues by changing the secretion and organization of the reconstructed ECM, including COL I and COL III and the SLRP members DCN, BGN, FMO, and LUM, eventually resulting in POP. Thus, the purpose of the present study was to investigate the mechanism of POP by observing the effects of a 0.1-Hz uniaxial CS load with 10% elongation on the morphological characteristics of extracellular biopolymers on the cell surface and on the mRNA expression of COL I and COL III as well as the SLRP members DCN, BGN, FMO, and LUM in POP fibroblasts in vitro. Furthermore, we aimed to evaluate the effect of estrogen therapy (ET) in association with the improvement of the prognosis of POP.

Fibroblasts isolated from the vaginal wall connective tissues of women with POP and unaffected women all exhibited a stellate, bipolar, or spindle-shaped morphology when observed on an inverted microscope ( Fig 1A ). When observed using SEM, the fibroblasts exhibited an obvious long fusiform shape with a slightly raised nuclear area ( Fig 1B ) and coarse protrusions or slender filopodia that formed connections between adjacent cells ( Fig 1C ). (A) Primary cultured normal fibroblasts after 72 h at passage 0 (bar = 100 μm). (B) Slightly raised nuclear area of normal fibroblasts at passage 4 (bar = 20 μm). (C) Filopodial connections between adjacent cells of normal fibroblasts at passage 4 (bar = 5 μm). Images were acquired on an inverted microscope (A) and SEM (B) (C). SP staining of all fibroblasts isolated from the vaginal wall connective tissues of women with and without POP showed strong cytoplasmic expression of vimentin, with an IS of 12 (+++); negative cytoplasmic expression of cytokeratin, with an IS of 0 (−); negative cytoplasmic expression of α-smooth muscle actin, with an IS of 0 to 1 (−); and negative cytoplasmic expression of the negative control, with an IS of 0 (−). These findings revealed that the isolated fibroblasts in this study were of connective tissue origin. Immunofluorescent staining for COL I, COL III, DCN, BGN, FMO, and LUM in vaginal fibroblasts from the unaffected and POP groups was also performed. Fibroblasts from both groups showed green immunofluorescent staining patterns for COL I ( Fig 2A and 2A' ) and COL III ( Fig 2B and 2B' ) and red immunofluorescent staining patterns for DCN ( Fig 2C and 2C' ), BGN ( Fig 2D and 2D' ), FMO ( Fig 2E and 2E' ), and LUM ( Fig 2F and 2F' ), confirming the presence and distribution of these proteins in fibroblasts from the two groups. The identified fibroblasts were used at the fifth passage in the following experiments and were divided into 8 groups: a normal group (N) and a POP group (P) without CS or E 2 [(N-E 2 -CS) and (P-E 2 -CS)], groups C and P without E 2 but with CS [(N-E 2 +CS) and (P-E 2 +CS)], groups C and P with E 2 but without CS [(N+E 2 -CS) and (P+E 2 -CS)], and groups C and P with both CS and E 2 [(N+E 2 +CS) and (P+E 2 +CS)]. (A) Green staining for COL I in normal fibroblasts. (A') Green staining for COL I in POP fibroblasts. (B) Green staining for COL III in normal fibroblasts. (B') Green staining for COL III in POP fibroblasts. (C) Red staining for DCN in normal fibroblasts. (C') Red staining for DCN in POP fibroblasts. (D) Red staining for BGN in normal fibroblasts. (D') Red staining for BGN in POP fibroblasts. (E) Red staining for FMO in normal fibroblasts. (E') Red staining for FMO in POP fibroblasts. (F) Red staining for LUM in normal fibroblasts. (F') Red staining for LUM in POP fibroblasts. Bar = 50 μm. When viewed on a light microscope, static-cultured fibroblasts presented a randomly swirling distribution ( Fig 3A and 3B ), whereas stretching-cultured fibroblasts grew perpendicular to the force ( Fig 3A' and 3B' ), with no significant differences in cell surface morphology observed between normal and POP fibroblasts. When observed via SEM, some polymers secreted by the fibroblasts were distributed on the cell surface. Under static-culture conditions, the polymers on the normal fibroblast surfaces were randomly distributed in clusters of plum flower-like patterns ( Fig 4A ), whereas on the POP fibroblast surfaces, the polymers resembled stretched strips, with stretching along the long axis of the cells ( Fig 4B ). When the cells were stretched, the polymers on the normal fibroblast surfaces also resembled stretched strips, again with stretching along the long axis of the cells ( Fig 4A' ), whereas those on the POP fibroblast surfaces resembled a broken network ( Fig 4B' ). (A) Static-cultured normal fibroblasts with a random swirling distribution. (B) Static-cultured POP fibroblasts with a random swirling distribution. (A') Stretched normal fibroblasts aligned perpendicular to the force. (B') Stretched POP fibroblasts aligned perpendicular to the force. Bar = 100 μm. The arrow indicates the stretching direction. (A) Plum flower-like distribution of polymers on normal fibroblast surfaces. (B) Stretched strip-like distribution of polymers on POP fibroblast surfaces. (A') Stretched strip-like distribution of polymers on stretched normal fibroblast surfaces. (B') Broken network-like distribution of polymers on stretched POP fibroblast surfaces. Compared with (N-E 2 -CS) group, (P-E 2 -CS) group exhibited significantly higher COL I mRNA expression (2 -ΔΔCt = 1.73 ± 0.31, P = 0.030). Similar differences in mRNA expression were noted for DCN, BGN, FMO, and LUM between these two groups (2 -ΔΔCt = 2.25 ± 0.29, P = 0.001; 2 -ΔΔCt = 2.10 ± 0.40, P = 0.014; 2 -ΔΔCt = 2.57 ± 0.43, P = 0.002; 2 -ΔΔCt = 1.41 ± 0.16, P = 0.022), whereas no significant difference was observed in COL III mRNA expression between the two groups (2 -ΔΔCt = 1.14 ± 0.25, P = 0.594) ( Fig 5A ). The bars and error bars indicate the mean and SE, respectively. Data represent the mean ± SE of triplicate trials. * P < 0.05; ** P < 0.01, *** P < 0.001. Following the application of stretching, normal fibroblasts exhibited a significant increase in COL I mRNA expression, with a significant difference specifically being found between the (N-E 2 +CS) and (N-E 2 -CS) group (2 -ΔΔCt = 2.28 ± 0.28, P < 0.001). A similar difference in BGN mRNA expression was observed between these two groups (2 -ΔΔCt = 1.99 ± 0.45, P = 0.041), whereas no significant difference was observed in COL III, DCN, FMO, or LUM mRNA expression in the (N-E 2 +CS) group compared with the (N-E 2 -CS) group (2 -ΔΔCt = 1.10 ± 0.32, P = 0.746; 2 -ΔΔCt = 1.42 ± 0.24, P = 0.096; 2 -ΔΔCt = 1.28 ± 0.36, P = 0.439; 2 -ΔΔCt = 0.89 ± 0.19, P = 0.552) ( Fig 5B ). However, POP fibroblasts subjected to stretching force exhibited no significant increase in COL I mRNA expression, with the data specifically revealing no significant difference between the (P-E 2 +CS) and (P-E 2 -CS) groups (2 -ΔΔCt = 1.45 ± 0.26, P = 0.105). In contrast, significant decreases in DCN and FMO mRNA expression were found in the (P-E 2 +CS) group compared with the (P-E 2 -CS) group (2 -ΔΔCt = 0.75 ± 0.10, P = 0.019; 2 -ΔΔCt = 0.74 ± 0.12, P = 0.037), whereas no significant difference was observed in COL III, BGN, or LUM mRNA expression between the (P-E 2 +CS) and (P-E 2 -CS) groups (2 -ΔΔCt = 1.04 ± 0.20, P = 0.859; 2 -ΔΔCt = 1.34 ± 0.23, P = 0.152; 2 -ΔΔCt = 0.96 ± 0.18, P = 0.816) ( Fig 5C ). When cultured in the presence of concomitant E 2 treatment and stretching, the normal fibroblasts still exhibited significantly increased COL I mRNA expression, with a significant difference specifically being found between the (N+E 2 +CS) and (N+E 2 -CS) groups (2 -ΔΔCt = 2.60 ± 0.32, P < 0.001), whereas COL III mRNA expression was significantly lower in the (N+E 2 +CS) group than in the (N+E 2 -CS) group (2 -ΔΔCt = 0.62 ± 0.14, P = 0.017). In addition, DCN mRNA expression was significantly increased in the (N+E 2 +CS) group compared with the (N+E 2 -CS) group (2 -ΔΔCt = 1.58 ± 0.19, P = 0.009), whereas no significant difference was observed in BGN, FMO or LUM mRNA expression between the (N+E 2 +CS) and (N+E 2 -CS) groups (2 -ΔΔCt = 1.45 ± 0.34, P = 0.201; 2 -ΔΔCt = 1.16 ± 0.33, P = 0.637; 2 -ΔΔCt = 1.05 ± 0.20, P = 0.789) ( Fig 5B' ). With respect to the effects of concomitant E 2 exposure and stretching on POP fibroblasts, both COL I and BGN showed significantly higher mRNA expression in POP fibroblasts in the presence of E 2 and stretching, with significant differences observed between the (P+E 2 +CS) and (P+E 2 -CS) groups (2 -ΔΔCt = 2.03 ± 0.35, P = 0.009; 2 -ΔΔCt = 1.51 ± 0.24, P = 0.047). Moreover, the application of E 2 abrogated the decrease in the DCN and FMO mRNA expression induced by stretching, with the data indicating a non-significant difference between the (P+E 2 +CS) and (P+E 2 -CS) groups (2 -ΔΔCt = 1.39 ± 0.19, P = 0.054; 2 -ΔΔCt = 1.49 ± 0.28, P = 0.097). In contrast, COL III, FMO and LUM mRNA expression was not significantly different between the (P+E 2 +CS) group and the (P+E 2 -CS) group (2 -ΔΔCt = 0.97 ± 0.24, P = 0.915; 2 -ΔΔCt = 1.49 ± 0.28, P = 0.097; 2 -ΔΔCt = 1.26 ± 0.20, P = 0.225) and was similar to that observed in normal fibroblasts under stretching without E 2 application ( Fig 5C' ).

In conclusion, the morphological distribution of the extracellular polymers on the surface of POP fibroblasts revealed higher sensitivity to tension stimuli and lower tolerance to mechanical stretching. Furthermore, the higher sensitivity to tension stimuli was found to be reflected by the transcription of COL I, and the transcription of DCN, BGN, FMO, and LUM was found to be up-regulated as COL I transcription increased. Meanwhile, POP fibroblasts were found to possess lower tolerance to mechanical stretching in terms of COL I transcription and defects in the compensatory function of BGN for DCN and LUM for FMO, thereby affecting the structure and function of COL I in response to mechanical stretching and ultimately resulting in POP. In healthy women, ET can preserve the integrity of the pelvic supportive connective tissues by up-regulating COL I and DCN transcription and maintaining the normal compensatory function of BGN to preserve the structure and function of COL I following its increased expression to prevent the occurrence of POP. Once POP has occurred, ET can also strengthen the pelvic supportive connective tissues by up-regulating COL I transcription and can support the structure and function of COL I by inhibiting the down-regulation of DCN and FMO transcription induced by mechanical stretching and by recovering the compensatory function of BGN to inhibit the progression of POP. Generally, the higher sensitivity and lower tolerance to stretching possessed by fibroblasts and the metabolism of COL I and certain SLRPs, as well as other ECM components, likely cause the occurrence and progression of POP, and the use of ET will improve prognosis. In future studies, the effects of different CS amplitudes/frequencies on collagen and SLRP expression in fibroblasts from pelvic supportive connective tissues need to be examined, and effective ET concentrations need to be further explored. Furthermore, whether supplementation with certain SLRPs can inhibit the occurrence and development of POP should be investigated.

This study was approved by the medical ethics committee of Beijing Chaoyang Hospital, Capital Medical University, on January 23, 2013 (project identification code: 13-S-11). From January 24, 2013 to December 26, 2013, a total of 12 participants were recruited. Six women (aged 52–69 years) undergoing pelvic floor construction surgery with advanced POP (stage III-IV by POP quantification) constituted the case group, and six women (aged 49–67 years) undergoing benign gynecologic hysterectomy due to fibroids, dysfunctional bleeding, or ovarian cysts were the controls. In addition to the influence of innate immunity, inflammation, and malignant tumors on SLRP expression as described above [ 13 – 19 ], endometriosis is a disease involving chronic inflammation and fibrosis of the parametrium and uterine ligaments [ 25 ]. To avoid the interference of these diseases on the experimental results, patients with endometriosis, gynecologic malignancies, pelvic inflammatory conditions, connective tissue disorders, or emphysema were excluded. All participants provided verbal and written informed consent. After informed consent was obtained, a 1-cm 2 , full-thickness area of the vaginal wall was procured from the anterior wall near the vaginal apex of the POP and control patients during surgery. The connective tissue beneath the anterior vaginal wall approximates the vaginal fornix, which was previously considered to be a representative portion of the endopelvic fascia [ 26 ]. The procedures used to isolate and culture vaginal connective tissue fibroblasts were the same as those described in our previous report [ 8 ]. Briefly, the excised vaginal wall samples were immediately placed in 4°C sterile Dulbecco’s phosphate-buffered saline (DPBS, HyClone, South Logan, UT, USA) with 1% penicillin/streptomycin (HyClone, South Logan, UT, USA) and sent to the laboratory within 2 h. Connective tissue blocks were first separated from the vaginal wall samples and minced into 1-mm 3 pieces and then digested for 24 h with 0.5% collagenase type I (Sigma-Aldrich, St. Louis, MO, USA) in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA) in a 5% CO 2 humidified incubator at 37°C. Finally, the fine sand-like tissue pieces resulting from collagenase digestion were suspended and centrifuged, and the sediment was reconstituted and cultured in Dulbecco’s modified Eagle’s medium (supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin). At the fourth passage, the derived cells were identified using the immunohistochemical streptavidin-peroxidase (SP) method, and the staining results were assessed using an index of staining (IS), as in our previous report [ 8 ]. Cells at 50% confluence cultured in chamber slides were fixed with 4% paraformaldehyde, treated with 0.4% Triton X-100, blocked with 3% hydrogen peroxide, and then incubated overnight with mouse anti-human vimentin monoclonal antibody (1:200), mouse anti-human cytokeratin monoclonal antibody (1:200), mouse anti-human α-smooth muscle actin monoclonal antibody (1:50), or DPBS (as the negative control) at 37°C (all antibodies were from Zhongshan Goldbridge Biotechnology, Beijing, China). Subsequently, the cells were incubated with the PV-6000 polymer detection system for immunohistological staining (Zhongshan Goldbridge Biotechnology, Beijing, China), and immunoreactivities were revealed using a 3,3′-diaminobenzidine tetrahydrochloride substrate kit (Zhongshan Goldbridge Biotechnology, Beijing, China). In addition, the cells were counterstained with Meyer’s hematoxylin and finally identified by examining the IS values. COL I, COL III, BGN, DCN, FMO, and LUM protein expression in the isolated fibroblasts was confirmed by immunofluorescent staining according to the suggested protocol of Wen Y [ 27 ]. Specifically, cells at 50% confluence cultured in chamber slides were fixed with 4% paraformaldehyde, treated with 0.4% Triton X-100, and then incubated overnight with different conjugated primary antibodies, including FITC-conjugated rabbit anti-collagen (I or III) polyclonal IgG antibody (1:200), rhodamine-conjugated rabbit anti-DCN (or anti-BGN, anti-FMO, or anti-LUM) polyclonal IgG antibody (1:200), or DPBS (as the negative control) at 37°C (all antibodies were from Bioss, Beijing, China). Finally, nuclei were stained with Hoechst 33342 (Enzo Life Sciences, Farmingdale, NY, USA), and the cells were observed on an Olympus BX51 fluorescence microscope equipped with an Olympus DP72 camera (Olympus Optical Co Ltd, Tokyo, Japan). Mechanical stretching experiments were performed according to our previous study using a cell-stretching device designed and manufactured to apply stress in vitro [ 8 ]. The identified fourth-passage fibroblasts were seeded on a gelatin-coated polydimethylsiloxane membrane with a utilized area of 40 × 20 mm 2 (length × width) and a thickness of 3 mm at a density of 2 × 10 3 cells/cm 2 to assess cell morphology and a density of 2 × 10 4 cells/cm 2 to assay mRNA expression. After being cultured for 24 h, a 0.1-Hz uniaxial CS with 10% elongation and a 12-h stretching duration was applied to the vaginal fibroblasts every day with or without a concomitant dose of 10 −8 M E 2 (Sigma-Aldrich Co, St. Louis, MO, USA) for 72 h. Non-stretched vaginal fibroblasts cultured on gelatin-coated polydimethylsiloxane membranes were used as controls. The viable cells in each group were collected to perform SEM analysis and mRNA detection. SEM analysis was performed to observe the CS-induced changes in cell shape and extracellular polymer structure. For this purpose, cells were washed with DPBS (pH 7.4) and fixed with 2% glutaraldehyde in DPBS (pH 7.4) for 4 h at 4°C. The specimens were dehydrated using an ascending ethanol gradient (50%, 70%, 80%, 90% and 100%), after which the ethanol was replaced with tertbutyl alcohol. After dehydration, the specimens were critical-point dried with CO 2 . Finally, the specimens were sputter-coated with gold in an ion coater for 2 min at an applied current of 50 mA (Eiko IB-3, Eiko Engineering Ltd, Tokyo, Japan) and examined via SEM (S-570, Hitachi, Japan). RNA was extracted from the experimental cells using an RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s standard protocol. The RNA concentration was controlled to an OD260/OD280 ratio of >1.8 using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Reverse transcription was performed using a QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) following the supplier’s recommendations. Briefly, a 20-μL reaction volume, including 1 μg of total RNA, 1 μL of oligo (dT) 15 (10 μM, TIANGEN, Beijing, China), and 10 μL of 2× RT-PCR Buffer (Qiagen, Hilden, Germany), was heated to 65°C for 5 min, quickly chilled, and incubated at 42°C for 1 h to allow the reaction, after which the enzyme was inactivated by heating at 85°C for 1 min. The oligonucleotide primer sequences for COL I and COL III were described previously [ 28 ], as were those for FMO, DCN, and BGN [ 27 ]. We designed the primer sequences for LUM. The housekeeping gene 18S was used as a reference. All the primers were validated using NCBI Primer-BLAST and synthesized by TaKaRa (TaKaRa Biotechnology, Dalian, China) and are listed in Table 1 . RT-PCR was performed using a QuantiTect SYBR Green RT-PCR Kit (Qiagen, Hilden, Germany) and an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Each RT-PCR reaction was performed in a 20-μL total volume containing 10 μL of 2× QuantiTect SYBR Green RT-PCR master mix, 8 μL of 1:4 diluted cDNA template, and 0.5 μL of each of the forward and reverse target-specific primers (10 μmol/L), which were designed to amplify a part of each gene. Amplification was performed as follows: 50°C for 2 min; 95°C for 15 min; and 45 cycles of 94°C for 15 sec and 55°C for 30 sec, followed by 72°C for 40 sec. After PCR, a melting-curve analysis was performed to demonstrate the specificity of the PCR product through the presence of a single peak. A control reaction containing all of the reaction components except for the template was included in all experiments. RT-PCR assays were validated as described previously [ 29 ]. Data analysis was carried out using ABI 7500 SDS System software (version 1.4) (Applied Biosystems, Foster City, CA, USA). COL I, COL III, DCN, BGN, FMO, and LUM mRNA expression levels were normalized to that of the gene 18S based on ΔCt = Ct for the gene of interest—Ct for the housekeeping gene. The data were analyzed using the 2 -ΔΔCt method and are presented as the mean ± standard error (SE); the baseline values were considered to be 1. Statistical analyses were conducted using SPSS for Windows (SPSS, Chicago, IL, USA). Comparisons of multiple groups were performed using one-way analysis of variance, and differences between two groups were determined using Student’s t-test. The results were considered significant at P < 0.05. To guarantee the accuracy of the results, each experiment was performed in triplicate, each independent experiment involved three rounds of RT-PCR detection, and the researcher who collected and processed the original data could not identify individual participants during or after data collection.