The Metabolic Characteristics And Protective Effects Mechanism Of DHEA In Rabbit Osteoarthritis

Osteoarthritis (OA) is the most common form of arthritis in human and constitutes a major cause of disability in the old people, It is a chronic arthritis characterized by articular cartilage degeneration and destruction diseases. Due to the mechanisms of OA remaining unclear, early treatment for OA is full of difficult. A number of drugs including nonsteroidal anti-inflammatory drugs (NSAIDs), glucosamine, steroids and hyaluronan (HA) were used for the treatment of early stage of OA. These drugs can alleviate the symptom of OA, but they can not reverse cartilage damage.Currently, there are many evidences supports that estrogen has beneficial effects for OA. And we also find that Dehydroepiandrosterone (DHEA) has such effects in previous studies. DHEA is a steroid hormone synthesized from cholesterol and pregnenolone in human and primate. Previous studies suggest that DHEA has protective effects against a variety of diseases, such as atherosclerosis, cancer, diabetes mellitus, obesity, ageing and arthritis, including rheumatoid arthritis.As a sex hormone precursor, DHEA can be transformed to testosterone and then to estradiol by the enzyme aromatase, which provoked our great interest. We’d like to investigate whether DHEA is directly relevant to OA or the protective effect of DHEA is contributed to estrogen.The present study included3parts. We first investigated the effects of DHEA on the expression of matrix metalloproteinase-13(MMP-13), tissue inhibitors of metalloproteinases-1(TIMP-1) and collagen type Ⅱ (Col-Ⅱ) in interleukin-1β (IL-1β)-induced rabbit chondrocytes. We found that DHEA inhibited the expression of MMP-13and increased the expression of TIMP-1and Col-Ⅱ in chondrocytes at mRNA level. Next, we investigated the metabolism of DHEA and whether the effects of DHEA are exerted via its conversion to estradiol, our results showed that articular chondrocytes express steroidogenesis-related enzyme genes such as3β-HSD、17β-HSD and P450, and that they were capable of locally synthesizing sex steroid hormones from DHEA, more important, the expression of MMP-13increased in the presence of letrozole and/or fulvestrant and expression of TIMP-1and Col-Ⅱ decreased. The in vivo study in rabbit model of OA induced by anterior cruciate ligament transection (ACLT) showed that injection of letrozole and/or fulvestrant30mins prior to injection of DHEA affected the protective effect of DHEA. These results demonstrate that the effects of DHEA may be mediated by its conversion to estradiol.Part1The effects of DHEA on MMP-13and TIMP-1expression in rabbit chondrocytesObjective:To investigate the effects of DHEA on MMP-13and TIMP-1expression in IL-1β-induced rabbit chondrocytes. Methods:Specimens were obtained from4weeks rabbits for chondrocyte culture. Rabbit chondrocytes were pre-treated with various concentration of DHEA, followed by stimulation with IL-1β. Expression levels of MMP-13, TIMP-1and Col-II mRNA were examined by real-time PCR.Results:DHEA inhibited the gene expression of MMP-13and increased the expression of TMP-1and Col-II in IL-1β-induced chondrocytes.Conclusions:These results demonstrate that DHEA exerts inhibitory effects on MMP-13expression in chondrocytes.Part2The metabolic characteristics anti-osteoarthritis effects of DHEA for rabbit chondrocytes in vitroObjective:To investigated the metabolism of DHEA and determine whether the effects of DHEA are exerted via its conversion to estradiol in rabbit chondrocytes. Methods:Chondrocytes stimulated with IL-1β were treated with DHEA, culture medium of0,0.5,1,2,4,6,8,12,24,36,48,60,72hours were collected to test the protein levels of DHEA, testosterone and estradiol by ELISA. Cells treated with IL-1β for24hours were collected to explore the expression of steroidogenesis-related enzyme such as3β-HSD、17β-HSD and P450by Western blot. Cells were divided into four groups, which were treated with the following drugs:group A received DHEA; group B received DHEA and letrozole; group C received DHEA and fulvestrant; and group D received DHEA, letrozole, and fulvestrant. Cells were then harvested for the analysis of MMP-13, TIMP-1and Col-Ⅱ levels.Results:Articular chondrocytes were capable of locally synthesizing sex steroid hormones from DHEA, they express steroidogenesis-related enzyme genes such as3β-HSD、17β-HSD and P450, and the expression of MMP-13increased in the presence of letrozole and/or fulvestrant and expression of TIMP-1and Col-Ⅱ decreased.This phenomenon was even more obviously when presented by both drugs.Conclusions:DHEA exert its protective effects in chondrocytes may be by its conversion to estradiol. This protection could be restrained by letrozole and/or fulvestrant. Part3The in vivo effects of DHEA in experimental osteoarthritis in rabbitsObjective:To investigated whether the effects of DHEA are exerted via its conversion to estradiol in a rabbit experimental model of osteoarthritis (OA).Methods:Thirty New Zealand rabbits underwent anterior cruciate ligament transection (ACLT) on right knee joint to induce OA and were randomly divided into five groups (n=6), based on the following treatments:Rabbits of Group A were injected with0.3ml of DHEA, rabbits in groups B and C were given DHEA after injection of letrozole or fulvestrant30min earlier; rabbits in group D were given both letrozole and fulvestrant30min prior to injection of DHEA, and rabbits in group E received the vehicle (DMSO) as a control. The injection was carried out Weekly in both knee four weeks after surgery. All rabbits in each group were sacrificed9weeks after the injection. The articular cartilage was collected for morphological, histological and genetic analysis of MMP-13, TIMP-1and Col-Ⅱ.Results:Assessed by the modified Mankin score, the DHEA-treated group showed less cartilage degradation as compared with cartilage co-injected with letrozole and/or fulvestrant. Gene expression of MMP-13increased in the presence of letrozole and/or fulvestrant and the expression of TIMP-1and Col-Ⅱ decreased. Letrozole and fulvestrant both affected the protective effect of DHEA.Conclusions:The effects of DHEA may be mediated by its conversion to estradiol.