Study On The Relationship Of Hypoxia And Vulvar Diseases And The Treatment Of Vulvar Lichen Sclerosus With Kebai Cream

Part I Study on the relationship of hypoxia and vulvar diseasesBackgroundVulvar diseases of skin and mucosa include mainly non-neoplastic epithelial disorders (NNEDs), vulvar intraepithelial neoplasia (VIN) and vulvar squamous cell carcinoma (VSCC). NNEDs, including vulvar lichen sclerosus (VLS), vulvar squamous cell hyperplasia and other dermatoses, are chronic lesions with the characteristics of depigmenting and denaturation in vulvar skin and mucosa. 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. 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 approaches 4%-6% in women with VLS, which therefore is regarded as a pre-malignant lesion. 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 (uVIN), also known as warty-basaloid, and the differentiated type (dVIN). The former is related to high-risk human papillomavirus (HPV), and the latter is not related to HPV. The dVIN perhaps is a transitory process from VLS to VSCC. The two types of VIN are all regarded as pre-malignant lesions. VSCC, an invasive carcinoma, is constructed by squamous cells of different degree of differentiation. Squamous cell carcinomas (SCCs), the most common vulvar carcinomas, account for about 80%-90% of all vulvar malignancies and 5% of all gynaecological carcinomas. However, after the age of 75 the incidence of vulvar carcinoma peaks to approximately 25%, 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. Despite a stable pattern in the incidence of vulvar carcinoma, the incidence of uVIN and vulvar carcinoma is increasing in women aged 50 years and younger. This might be due to a higher incidence of HPV infection of the genital tract and/or to an increased awareness of uVIN. VSCC develops following two different pathways, which have their own premalignant lesions. In the absence of HPV, VSCC can develop in a background of lichen sclerosus (LS), dVIN or both. The other pathway leading to VSCC is associated with HPV and the HPV-associated premalignancy is uVIN. Several factors have been linked to vulvar diseases of skin and mucosa development, but the exact pathogenesis remains to be determined, and the clinical management is very difficult.In the processes associated with both development of some physiologial or pathological phenomenon and progression to invasive disease, hypoxic microenvironment perhaps plays an important role. Hypoxia can lead to the expression of a series of genes and proteins, induce angiogenesis and glycolysis, up-regulate the expression of some growth factors and invasion-associated proteins, and cause the malignant transformation, invasion, even metastasis. The expression of hypoxia-inducible factor-1 and its target genes is the prognosis factors of many kinds of malignant tumors, and prognosticates the therapy resistance and the effect of targeted therapy. Hypoxia is associated with tumorigenesis but has not yet been identified in VSCC or in vulvar premalignant lesions.ObjectiveWe aimed to observe the expression of hypoxia induced factors such as hypoxia-inducible factor-1α(HIF-1α), glucose transporter-1 (GLUT-1), carbonic anhydrase IX (CA IX) and vascular endothelial growth factor (VEGF) and cell proliferation marker Ki-67 in control, VLS, VIN, and VSCC, and research the relationship of the expression of the proteins and clinical pathology data. We investigated the role of hypoxia in the development of vulvar diseases and canceration, and explored the correlation of hypoxia and proliferation. The clinical-pathological prognostic factors of VSCC were investigated.MethodsFor immunohistochemical evaluation, archival formalin-fixed, paraffin-embedded vulvar specimens from 2002 to 2007 were retrieved from the Department of Pathology and the Department of Dermatology at the Qilu Hospital of Shandong University. Samples from 41 LSs, 10 VINs, 25 SCCs and 12 control subjects were collected to detect the expression of hypoxia induced factors including HIF-1α, GLUT-1, CA DC and VEGF, and proliferation marker Ki-67. We analyzed the clinical pathology data of the patients with VSCC.Results1. By immunohistochemistry, GLUT-1 expression showed similar significant differences in VLS and VIN than control while immunoreactive cells in SCC were significantly more than LS and VIN (P<0.05).2. CA IX expression was negative in all control and LS sections, while similar significant differences in VIN and SCC (P>0.05).3. VSCC had significant staining of VEGF followed by VIN, LS and normal vulva. The similar immunoreaction of VEGF was showed in LS and normal vulva (P> 0.05).4. Ki-67 expression showed similar significant differences in VIN and VSCC (P> 0.05) while immunoreactive cells in control was significantly less than vulvar diseases (P<0.05).5. Nuclear staining of HIF-1αwas not found in any tissue.6. In all groups, the correlation of the expression of hypoxia induced factors and proliferation marker Ki-67 was not seen. The direct correlation of GLUT-1 and VEGF can be obviously seen, but the correlation of CA IX and VEGF was not detected.7. The survival rate of the patients with VSCC in 1, 3, 5 years is 75%, 70%, 64% respectively. No prognosis factor was detected.Conclusion1. Hypoxia is involved in the development of the vulvar diseases and the malignant progression of VSCC. GLUT-1 may represent an early stage of malignant transformation in vulvar diseases, while CA IX and VEGF perhaps a late stage.2. In the retrospective studies, GLUT-1 may be a more sensitive endogenous hypoxia marker than HIF-1α. HIF-1αis instable and a false-negative expression can be seen. CA IX is not sensitive hypoxic index in vulvar tissues.3. A correlation was found between hypoxia and proliferation.4. Vulvar squamous cell carcinoma is a invasion malignant tumor, whose prognosis is better. Hypoxia related factors arenot ideal prognostic factors of VSCC. Part II Study of Kebai cream 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 prepubertal girls and menopausal women, but in the internal reports, it occurs mainly in the fourth decades, followed by prepubertal girls. 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 approaches 4%-6% 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, genetic factor, endocrine factor, metabolism, infection and local factor may be involved in it. The early diagnosis and treatment for VLS is a challenge for gynaecologists, dermatologists, pathologists and pediatricians. For the past few years, scholars investigated the aetiology, pathogenesy and management of VLS, however, extensively approved management and evaluation criterion of curative effect is absent. 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 Kebai cream to treat VLS on the basis of clinical and empirical study for more than 30 years in Qilu Hospital of Shandong University, and investigated its clinical curative effect and mechanism of action.ObjectiveTo observe the clinical curative effect of Kebai cream 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 in 1993 and 2002), 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.102 patients with VLS were divided into case group and control group randomly. The case group (51 patients) was treated with Kebai cream while the control group (51 patients) was treated with clobetasol propionate ointment. The serum from 30 healthy women and vulvar tissue from 12 normal 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 and erythrocuprein (SOD), malonaldehyde (MDA). The expression of CD3, CD4, CD8, CD68 and hypoxia induced factors (glucose transporter-1 (GLUT-1) and vascular endothelial growth factor (VEGF)) and the histomorphology change in the prior and post-treatment were observed. Meanwhile, the best adaptive type and the toxic and side effect after long-term using of the Kebai cream were researched. Moreover, we followed up to investigate its effect to prevent the canceration. The animal experiment aimed to research the pharmacodynamics of Kebai cream.Results1. The total effective rate of case group is 60.78%, while the control group is 33.33%. By comparation, a statistical meaning can be educed (P<0.05), which showed that the curative effect of the case group is better than the control group.2. 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.3. Significant decreases in the expression of CD3+, CD4+ and CD4+/CD8+ T cells, 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 Kebai cream (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 Kebai cream (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. Significant decreases in the level of SOD, while the significant increases of MDA were observed before the treatment (P<0.05). However, significant increases of SOD, and the significant decreases of MDA were observed after the treatment with Kebai cream (P<0.05). There was no difference in results for SOD and MDA in the prior and post-treatment with clobetasol propionate ointment (P>0.05). After treatment, the significant difference in the expression of SOD and MDA between case group and control group was observed (P<0.05).6. Using immunohistochemistry, before treatment, significant numbers of CD3+ and CD4+ lymphocytes were observed in the dermal band of inflammatory cells in approximately equal proportions. Less numerous CD3+ and CD4+ lymphocytes also occurred adjacent to the dermoepidermal junction and occasionally in the lower epidermis. Less numbers of CD8+ lymphocytes and cells staining with the monocyte/macrophage marker CD68 were detected. Significant difference in the expression of CD3 and CD4 in the prior and post-treatment in both groups were observed (P<0.05). There was no difference in the expression of CD3 and CD4 after treatment between both groups (P>0.05).7. By immunohistochemistry, significant increases in the expression of GLUT-1, while the significant decreases of VEGF staining in the endothelial cells and pericytes associated with microvessels were observed before treatment (P<0.05). However, significant decreases of GLUT-1 immunostaining cells, and the significant increases of VEGF immunostaining cells were observed after the treatment with Kebai cream (P<0.05). There was no difference in results for GLUT-1 and VEGF expression in the prior and post-treatment with clobetasol propionate ointment (P>0.05).8. Observation by light microscope, after the treatment with Kebai cream, 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.9. Ultrastructural changes in VLS tissue showed that the thickness of the basement membrane (BM) of the epithelium differed as partly thickening, thinning or breaking. There were sparse hemidesmosomes on the BM; some substances in the epithelium intruded into the dermis from BM breakage. Mitochondria of basal cells were swollen, with loss of cristae and vacuolization. Morphological abnormalities in dermal blood vessels included capillary loss; a thin and convoluted vessel wall dependent on the region; cytoplasm protruding towards lumens; and narrowed or slit-shaped lumens. Mitochondria of vascular endothelial cells swelled with loss of cristae, and the rough ER (rER) revealed luminal swelling and ribosomal detachment. Junctional complexes did not display apparent abnormalities. Mitochondria of pericytes and most dermal cells were swollen. Myelin-like bodies emerged as intra-mitochondrial dense deposits in involved epidermal or dermal cells. After the treatment with Kebai cream, the tissue revealed the uniform BM of the epithelium, and abundant hemidesmosomes and melanin granules. In the cytoplasm of basal cells and vascular endothelial cells, mitochondria displayed well-defined cristae and membrane integrity; rER displayed parallel rows and attached ribosomes. Vascular lumens were well-developed.10. No toxic and side effect was seen in the case group, while adermotrophia and skin stimulus can be observed in the control group, and no canceration was seen in both groups during follow up.11. The animal experiment showed that the inhibitory action can be seen in the treatment of Kebai cream with macro- and meta-dosage and the control group, while no obvious inhibitory action in the treatment of clobetasol propionate. There was no difference in the Kebai cream with macro- and meta-dosage group and the clobetasol propionate group (P>0.05). The better action of relieving itching (itching caused by 4-aminopyridine) can be seen in the treatment of Kebai cream with macro- and meta-dosage group. And the dose-effect relationship is great. There was no difference in the Kebai cream with macro-dosage group and the clobetasol propionate group (P >0.05).Conclusion1. VLS is a inflammatory skin disease, in which the dysequilibrium between oxidation and antioxidation can be seen. Hypoxia-ischemia (HI) may be involved in the pathogenesis of VLS.2. Kebai cream has a good clinical curative effect, and is an ideal medicine for theraphy of VLS.3. The mechanism of action of Kebai cream is to adjust the immune function, promote the balance between oxadation and antioxadation, improve the local hypoxia-ischemia microenvironment and the histomorphology of vulvar tissue.4. Follow up showed that Kebai cream can prevent canceration, and no toxic and side effect was found after long periods of administration.5. The pharmacodynamics showed that Kebai cream had better effect of antiinflammatory and relieving itching.