Therapeutic Effect And Mechanism Of Recombinant Humanized Collagen On Female Pelvic Floor Dysfunction

BACKGROUND: Pelvic floor dysfunction disease(FPFD)is one of the common refractory diseases in middle-aged and older women,which seriously affects patients’ quality of life.FPFD increased gradually with the increase of birth and age.Therefore,early prevention and treatment are of great significance in preventing the aggravation of the disease.Although pelvic floor rehabilitation training and surgical treatment play a specific role in the prevention and treatment of FPFD,patients’ poor compliance with pelvic floor rehabilitation training and the apparent side effects of pelvic floor surgery seriously affect the treatment effect of FPFD.Therefore,there is an urgent need to find more non-invasive and efficient prevention and treatment methods.The extracellular matrix(ECM)is an essential component of the pelvic floor tissues(vagina,ligaments,fascia),and type Ⅰ and type Ⅲ collagen are the main components of the ECM.Most current studies have shown that the decreased content and increased degradation of type Ⅰ and type Ⅲ collagen in pelvic floor tissues may lead to the development of FPFD.Therefore,promoting collagen synthesis and maintaining the balance of collagen metabolism may be effective for the early prevention and treatment of FPFD.With the development of genetic engineering technology and synthetic biology,recombinant humanized collagen(rhCOL)with specified structure and function has shown potential and received widespread attention as a starting material for surgical implants and regenerative medicine matrices.Recombinant humanized collagen has a triple helix structure with better cell adhesion,water solubility,and biocompatibility than conventional collagen.In treating atrophic vaginitis and skin photoaging,rhCOL promoted collagen synthesis in the vagina and skin mucosa,stimulated local vascular regeneration,and improved the poor extracellular matrix remodelling.It was found that fibroblasts were the primary cells that synthesize collagen in the vagina and skin mucosa.Improving the function of fibroblasts and the state of the extracellular matrix improved the dysfunction of the pelvic floor.Therefore,whether rhCOL can promote collagen synthesis in pelvic floor fibroblasts,maintain the balance of collagen metabolism,improve the poor remodeling of ECM,and play an early role in the prevention and treatment of FPFD is worthy of further study.OBJECTIVES:(1)To study type Ⅰ and Ⅲ collagen expression in sacral ligaments and their isolated fibroblasts from FPFD and non-FPFD patients.(2)To study the effects and mechanism of rhCOL on adhesion,migration,and ECM synthesis of sacral ligament fibroblasts(HULFs).(3)To study the efficacy of rhCOL on the recovery of pelvic floor function in FPFD rats.METHODS:(1)The sacral ligaments of 17 FPFD patients and 15 nonFPFD patients were collected and their HULFs were isolated.Type Ⅰ and Ⅲcollagen expression in the sacral ligaments and HULFs were analyzed using Masson staining,immunohistochemistry,and western blot experiments.(2)To investigate the effects of rhCOL on the biocompatibility,adhesion,extension,migration,and ECM synthesis ability of HULFs from FPFD patients using cell adhesion assay,cytotoxicity assay,scanning electron microscopy,western blot assay,and RT-q PCR assay.(3)Immunofluorescence staining,western blot assay,Co-IP assay,Rho A activity assay and inhibitor assay were used to study the effect of rhCOL on the formation of adherent spots of HULFs,as well as the effect of rhCOL on the activation of FAK/Rho A/ROCK signaling pathway,and the effect of FAK/Rho A/ROCK signaling pathway on the adhesion,extension,migration,and ECM synthesis of HULFs.(4)To investigate the effects of rhCOL on the cellular metabolism of HULFs using metabolomics experiments.(5)The FPFD rat model was constructed.The efficacy of rhCOL on the restoration of pelvic floor function and related pathophysiological changes in FPFD model rats was investigated using urodynamic experiments biomechanical experiments,H&E staining,Masson staining,immunohistochemistry,immunofluorescence,and RT-q PCR experiments.RESULTS:(1)Decreased type Ⅰ and Ⅲ collagen expression was observed in the sacral ligament and its isolated fibroblasts of FPFD patients.(2)rhCOL had good biocompatibility.Compared with bv COLⅠ,rhCOL better promoted the adhesion,extension,and migration of HULFs in FPFD patients and promoted the expression of type Ⅰ and Ⅲ collagen,the main components of ECM.(3)rhCOL promoted HULFs adhesion,migration,and ECM synthesis by activating the FAK/Rho A/ROCK signalling pathway.rhCOL’s ability to promote HULFs adhesion,migration,and ECM synthesis was reversed with FAK and Rho A inhibitors.(4)rhCOLⅠ promoted a significant increase in the secretion of proline,branched-chain amino acids,and glutathione in HULFs from FPFD patients.rhCOLⅢ promoted a substantial increase in the secretion of proline and some branched-chain amino acids in HULFs from FPFD patients.KEGG metabolic pathway analysis suggested that rhCOL could upregulate both the metabolic pathways of arginine and proline in HULFs from FPFD patients and the biosynthetic pathways of valine,leucine and isoleucine in FPFD patients.(5)rhCOL improved urinary function and vaginal resistance in FPFD rats and promoted histological recovery of the vagina and urethra.CONCLUSION: rhCOL promotes adhesion,migration,and ECM synthesis of HULFs by activating FAK/Rho A/ROCK signaling pathway.Perivaginal injection of rhCOL is more effective than bv COL in preventing poor remodeling and pelvic floor function.Therefore,rhCOL is expected to become a non-invasive and efficient new medical biomaterial for the early prevention and treatment of FPFD.