27-羥基膽固醇與肥胖、停經和根尖病變發展之關聯：作用機轉及臨床治療潛能

Abstract

根尖病變是一種由微生物感染根管系統所誘發的免疫發炎反應，根尖骨吸收為臨床重要表徵。參與根尖病變發展的細胞和發炎介質與引發骨質疏鬆的成員相似，而停經造成的雌激素（17β-estradiol, E2）缺乏、及肥胖合併高膽固醇血症皆是骨質疏鬆的高危險因子。27-羥基膽固醇（27-hydroxycholesterol, 27-HC），一種內生性選擇性雌激素受體調節物（Selective estrogen receptor modulator, SERMs），扮演引渡者串聯起肥胖、停經與骨質疏鬆之病理關聯。Raloxifene作為第二代SERM，廣泛用於治療停經後婦女之骨質疏鬆，然而與根尖組織相關研究尚未明朗。因此本研究目的為驗證膽固醇、E2及27-HC三者於根尖病變發展之關係，及raloxifene臨床應用之潛能。實驗以巨噬細胞株（J774）分別在膽固醇、E2及27-HC刺激下，以ELISA檢測27-HC濃度；西方墨點法分析CYP27A1及iNOS的表現量。25隻6週大雌性Sprague-Dawley大鼠進行根尖病變誘導實驗，分別給予卵巢切除（Ovariectomy, OVX）、高脂飲食（High fat diet, HFD）及raloxifene，實驗分為五組：ND、ND/OVX、HFD、HFD/OVX及HFD/OVX/Ralo（1 mg/kg/d），以傳統根尖片及微米級電腦斷層掃描（micro-CT）監測根尖病變發展；透過ELISA評估病灶中27-HC濃度及血液中膽固醇濃度。研究結果顯示J774中，膽固醇會刺激粒腺體內CYP27A1表現以促進27-HC生成，然而當27-HC至一定濃度時會產生回饋抑制CYP27A1的表現。給予E2和raloxifene皆會抑制27-HC對iNOS的誘導作用。動物實驗方面，血液中膽固醇濃度與體重增加百分比有正相關。在影像分析中，肥胖與停經會加劇根尖病變的病程，而raloxifene可減緩病灶的發展。總結而言，膽固醇會刺激巨噬細胞生成27-HC，進而啟動發炎反應；而停經導致E2的抗發炎作用喪失，因而加劇根尖病變發展。Raloxifene可逆轉E2缺乏的影響，抑制發炎相關介質，減弱根尖病變中的發炎性骨吸收，暗示著raloxifene於臨床應用的潛能。

Apical lesion is an immune inflammatory response originated from microbial infection of the root canal system. Resorption of periapical bone is a clinically cardinal sign. Most cells and inflammatory mediators involved in the pathogenesis of periapical lesions also participate in that of osteoporosis. Estrogen (17β-estradiol, E2) deprivation by menopause, and obesity comorbidity with hypercholesterolemia are both high risk factors for osteoporosis. 27-hydroxycholesterol (27-HC), an endogenous selective estrogen receptor modulator (SERM), links the pathogenesis of obesity, menopause and osteoporosis. Raloxifene, a second generation SERM, is widely used in treatment of osteoporosis in postmenopausal women. However, the research about raloxifene and periapical tissue is still no clear. Therefore, the purpose of this study is to verify the relationship between cholesterol, E2 and 27-HC in the propagation of apical lesions, and the therapeutic potential of raloxifene. Macrophage cell line (J774) is treated with cholesterol, E2 and 27-HC separately. The concentration of 27-HC is detected by ELISA kit. Western blot is used to analyze CYP27A1 and iNOS expression. Twenty-five female Sprague–Dawley rats at 6 weeks of age are subjected to apical lesion induction, and are received OVX, HFD, or raloxifene respectively. The experiment is divided into ?ve groups: ND, ND/OVX, HFD, HFD/OVX, and HFD/OVX/Ralo (1 mg/kg/d). Progression of the apical lesion is monitored by conventional radiography and microcomputed tomography. The concentration of 27-HC in apical lesion and serum cholesterol are assessed by ELISA kit. The results indicate that cholesterol enhances the formation of 27-HC by increasing CYP27A1 expression in J774 mitochondria. Interestingly, 27-HC shows feedback inhibition on CYP27A1 expression when it reaches a certain concentration. Estrogen and raloxifene both attenuate the induction effect of 27-HC on iNOS expression. In animal studies, the level of serum cholesterol has a positive correlation with the percentage of weight gain. Moreover, the image analysis shows that obesity and menopause aggravate the propagation of apical lesions; by contrast, raloxifene alleviates the progression of the disease. In summary, cholesterol stimulates macrophages to produce 27-HC, which in turn initiates an inflammatory response. Besides, loss of the anti-inflammatory effect of estrogen due to menopause exacerbates the development of apical lesions. Raloxifene reverses the effect of estrogen deficiency, inhibits inflammatory mediators, and suppresses the progression of inflammatory bone resorption in periapical lesions. The above results imply that raloxifene gives light on a novel therapeutic strategy in apical periodontitis.