Study On The Relationship Of Osteopontin And Matrix Metalloproteinases-9in Vulvar Diseases And The Treatment Of Vulvar Lichen Sclerosus With Yinyang Lotion

Part I Study on the relationship of osteopontin and matrix metalloproteinases-9in vulvar diseasesBackground and significanceVulvar diseases mainly include vulvar carcinoma, vulvar intraepithelial neoplasia (VIN) and vulvar non-neoplastic epithelial disorders (VNED). VNED includes vulvar lichen sclerosus (VLS), vulvar squamous cell hyperplasia and other dermatoses. Vulvar squamous cell carcinoma (VSCC), an invasive carcinoma, is constructed by squamous cells of different degrees of differentiation. Squamous cell carcinomas (SCCs), the most common vulvar carcinomas, account for about90%of all vulvar malignancies and3%-5%of all gynaecological carcinomas. However, over the age of75the incidence of vulvar carcinoma peaks to approximately25%, making it as common as cervical carcinoma. VSCC represents an important medical challenge worldwide whose incidence is increasing at an alarming rate in young females. The diagnosis of VSCC is often delayed and the clinical management is very difficult. Surgery is considered to be the cornerstone of treatment of VSCC, and radio-and/or chemotheraphy only play a limited role. However, postoperative complications of vulvar impact seriously on the patient’s quality of life. Therefore, the research work on the progression and prognosis of VSCC is very important.Vulvar intraepithelial neoplasia (VIN) and vulvar lichen sclerosus (VLS) are all regarded as precancerous lesions. VIN, less seen in the vulva, is a histological diagnosis based on loss of squamous epithelial maturation associated with enlarged, hyperchromatic, pleomorphic nuclei and increased, usually atypical mitoses. There are two types of VIN according to the newest separate criteria:the usual type and the differentiated type. These two types are all regarded as pre-malignant lesions. The former is related to high-risk human papillomavirus and primarily affects younger patients. The latter is not related to HPV. It occurs in older women and leads to mostly differentiated keratinizing squamous cell carcinoma. This type relates to chronic inflammatory vulvar conditions such as VLS and accounts for the majority of all VSCC. VLS, often seen in the department of gynecology, is a chronic inflammatory skin disease, runing a relapsing and remitting course, and the management is difficult. The common symptom is pruritus, micturition difficult, algopareunia and burning sensation. At least one third of patients may be asymptomatic. Typically, the lesions are white plaques and papules, often with areas of erythema, ecchymosis, hyperkeratosis, pallor, fissuring, telangiectasia, hyperpigmentation, bullae, excoriation, oedema and/or ulceration. The lingering effects, most often encountered in long-term cases of VLS, include scarring, narrowing of the introitus which can make intercourse impossible, as well as widespread psychosexual disorder. The risk of developing squamous cell carcinoma of the vulva approaches3%-5%in women with VLS, which therefore is regarded as a pre-malignant lesion.Early detection and treatment of VIN and VLS may prevent development of VSCC and improve its prognosis. The oncogenesis of VLS to VSCC and its connection with VIN also remains to be elucidated. In order to prevent the progression of VSCC, it is important to find a way to predict the development of vulvar precancerous lesions.Progress of malignant tumors rely on the regulation of the extracellular matrix protein, which helps regulate the transfer of genetic modification and physiological changes, and OPN is such an extracellular matrix protein. Osteopontin is a highly phosphorylated glycoprotein and has been shown to be synthesized in a variety of tissues and cells and secreted into body fluids. OPN stimulates various signal transduction pathways through binding to its receptors, such as integrins and CD44variants, and therefore plays an active role in many physiological and pathological processes. It has also been shown that increased OPN expression correlates with tumor progression and metastasis in breast cancer, lung cancer, colon cancer, and in others cancers. It has been shown that OPN enhances the abilities of mobility and chemical invasiveness of malignant tumor cells possibly through regulating the activities of MMP-9, which degradate extracellular matrix. MMP-9is the largest and most important proteases of the matrix metalloproteinase family and plays an important role in the process of extracellular matrix degradation. MMP-9has been shown to be secreted in a variety of cells, such as hematopoietic stem cells, neutrophils, endothelial cells and a variety of tumor cells. The main role of MMP-9is to degrade the basement membrane and extracellular matrix, maintaining physiological homeostasis of extracellular matrix process. It has been shown that MMP-9degradation of extracellular matrix, promoting tumor angiogenesis, thereby affecting the outcome and prognosis of cancer patients in the process of tumor invasion and metastasis. However, to the best of our knowledge, the expression of OPN and MMP-9in VSCC, VIN and VLS has not yet been elucidated so far.In this study, we detect the expression and clinical significance of OPN and MMP-9in VSCC, VIN and VLS by the immunohistochemical method and chromogenic in situ hybridization.ObjectiveWe aimed to observe the expression of OPN and MMP-9in VSCC, VIN, VLS, and control. We investigated the role of OPN and MMP-9in the development of vulvar diseases and canceration, and investigated the relationship of OPN and MMP-9to the clinicopathologic factors and prognosis. The clinical-pathological prognostic factors of VSCC were investigated.MethodsFor immunohistochemical evaluation and chromogenic in situ hybridization, archival formalin-fixed, paraffin-embedded vulvar specimens from2002to2007were retrieved from the Department of Pathology and the Department of Dermatology at the Qilu Hospital of Shandong University. Samples from25VSCC,21VIN,21VLS and13control were collected to detect the expression of OPN and MMP-9. Immunohistochemical staining for OPN and MMP-9were performed using the streptavidin-peroxidase method.We analyzed the clinical pathology data of the patients with VSCC. All statistical analyses were performed with SPSS13.0statistical software. The x2test was performed to examine the association between OPN, MMP-9and variable clinicopathologic factors. Kaplan-Meier method was used to calculate the survival curves, and log-rank test was used to compare the statistical significance of difference between the survivals of patient subgroups.Results1. OPN and MMP-9expression increase with the degree of malignancy of vulvar lesions by the immunohistochemical method and chromogenic in situ hybridization (P <0.05).2. OPN staining was found in the extracellular matrix in VSCC and VIN by the immunohistochemical method. The expression of MMP-9was notable increase in normal epidermis adjacent to VSCC when compared with normal skin by the immunohistochemical method.3. OPN overexpression and high MMP-9were no significantly associated with tumor relapse by the immunohistochemical method and chromogenic in situ hybridization (P<0.05).4. Univariate survival analysis showed that patients with OPN overexpression had a significantly poor overall survival at5years after operation by the immunohistochemical method (P=0.033).5. Univariate survival analysis showed that patients with MMP-9overexpression had a significantly poor overall survival at5years after operation by chromogenic in situ hybridization (P=0.037).6. Univariate survival analysis showed that patients with OPN overexpression had a significantly poor disease-specific survival at5years after operation by the immunohistochemical method (P=0.048)7. The x2test showed that OPN overexpression was significantly associated with high MMP-9by the immunohistochemical method (P=0.000).8. The x2test showed that OPN overexpression was significantly associated with high MMP-9by chromogenic in situ hybridization (P=0.001).Conclusion1. OPN and MMP-9are involved in the development of the vulvar diseases and the malignant progression of VSCC.2. OPN protein overexpression is significantly associated with poor survival and may have clinical potential as a promising predictor to identify individuals with poor prognostic potential.3. MMP-9may represent an early stage of malignant transformation in VSCC, while OPN perhaps a late stage. Part Ⅱ Study of Yinyang lotion in the treatment of vulvar lichen sclerosusBackgroundVulvar lichen sclerosus (VLS), often seen in the department of gynecology, is a chronic inflammatory skin disease, runs a relapsing and remitting course, and the management is difficult. VLS occurs at all ages, and has a bimodal peak incidence in menopausal women. The common symptom is pruritus, micturition difficult, burning sensation and algopareunia. At least one third of patients may be asymptomatic. Typically, the lesions are white plaques and papules, often with areas of erythema, ecchymosis, hyperkeratosis, pallor, fissuring, telangiectasia, hyperpigmentation, excoriation, oedema and/or ulceration. The lingering effects, most often encountered in long-term cases of VLS, include scarring, narrowing of the introitus which can make intercourse impossible, as well as widespread psychosexual disorder. The risk of developing squamous cell carcinoma of the vulva approaches3%-5%in women with VLS. The histological diagnosis is the only method to make a definite diagnosis. Up to now, the pathogenesis of VLS is incompletely understood. The autoimmunity, endocrine factor, enetic factor, metabolism, infection and local factor may be involved in it. The early diagnosis and treatment for VLS is a challenge for gynaecologists. In the past few years, scholars investigated the aetiology, pathogenesy and management of VLS, however, extensively approved management. The local corticosteroids is regarded as the first choice, but its side effects, including atrophia and skin stimulus, make the treatment to run into a predicament. The conbined treatment of traditional Chinese medicine and western medicine was used by internal scholars and got satisfactory results. We used Yinyang lotion to treat VLS on the basis of clinical and empirical study for more than40years in Qilu Hospital of Shandong University, and investigated its clinical curative effect and mechanism of action.ObjectiveTo observe the clinical curative effect of Yinyang lotion on VLS, and investigate the mechanism of action, so as to explore an effective way for the prevention and treatment of VLS, and provide strong theoretical and practical basis for the wide administration of Kebai cream. MethodsWe referred to the medicine teaching material edited by the government (Chinese Gynaecology, Obstetrics and Gynecology), Practical Obstetrics and Gynecology (second edition) and The Clinical Guidlines for the New Chinese Medicine,(Enacted by the Department of Health of China in1993and2002), and instituted the clinical diagnosis, the criterion of differentiation of symptoms and signs for classification of syndrome with traditional Chinese medicine and evaluation criterion of curative effect of VLS.116patients with VLS were divided into case group and control group randomly. The case group (58patients) was treated with Yinyang lotion while the control group (58patients) was treated with clobetasol propionate ointment. The serum from38healthy women and vulvar tissue from13normal women were collected as normal control. The changes of the symptoms and signs such as pruritus, depigment and atrophy, were followed up during the period of the management. We detected the serum of the patients and the healthy women for T cell subsets (mainly CD3+, CD4+, CD8+T cells), IgG, IgA, IgM. The expression of osteopontin (OPN) and the histomorphology change in the prior and post-treatment were observed.Results1. The symptoms and signs of the case group, such as itching, depigment and atrophy, were greatly improved after treatment. And the validity among the types of case group was found no significant difference.2. The total effective rate of case group is82.76%, while the control group is41.38%. By comparation, a statistical meaning can be educed (P<0.05), which is that the curative effect of the case group is better than the control group.3. the expression of CD3+, CD4+and CD4+/CD8+T cells decrease significantly, while the significant increases of CD8+T cells were observed before the treatment (P <0.05). However, significant increases of CD3+, CD4+and CD4+/CD8+T cells, and the significant decreases of CD8+T cells were observed after the treatment with Yinyang lotion (P<0.05). There was no difference in results for T cells expression in the prior and post-treatment with clobetasol propionate ointment (P>0.05). After treatment, the significant difference in the expression of T cells between case group and control group was observed (P<0.05).4. Significant decreases in the level of IgG were observed before treatment(P< 0.05). However, significant increases of IgG were detected after the treatment with Yinyang lotion (P<0.05). There was no difference in results for IgG in the prior and post-treatment with clobetasol propionate ointment (P>0.05). After treatment, the significant difference in level of IgG between case group and control group was observed (P<0.05).5. By immunohistochemistry, significant decreases of OPN immunostaining cells were observed after the treatment with Yinyang lotion (P<0.05). There was no difference in results for OPN expression in the prior and post-treatment with clobetasol propionate ointment (P>0.05).6. Observation by light microscope, after the treatment with Yinyang lotion, showed that lessened hyperkeratinization in the epidermis, more prickle cell layer, appearing rete ridges, improved obviously basal keratinocyte hydropic degeneration and vacuolization, melanocytosis, restored dermal papilla, obvious improvement or loss of edema and homogenization of collagen in the upper dermis, lessened obviously lymphocytic infiltration.Conclusion1. Yinyang lotion has a good clinical curative effect, and is an ideal medicine for theraphy of VLS.2. Changes in immune function may be associated with VLS lesions, Yinyang lotion therapeutic effect may be related to immune regulation.3. The mechanism of Yinyang lotion may adjust the immune function, OPN may be involved in this process.