Objective: To construction the telmisartan/collagen/polycaprolactone (PCL) nerve conduit and assess its effect on repairing sciatic nerve defect in rats.
Methods: The 60% collagen/hexafluoroisopropanol (HFIP) solution and 40% PCL/HFIP solution were prepared and mixed (collagen/PCL solution). Then the 0, 5, 10, and 20 mg of telmisartan were mixed with the 10 mL collagen/PCL solution, respectively. Telmisartan/collagen/PCL nerve conduits were fabricated via high voltage electrospinning technology. The structure of nerve conduit before and after crosslinking was observed by using scanning electron microscope (SEM). The drug release efficiency was detected by in vitro sustained release method. RAW264.7 cells were cultured with lipopolysaccharide to induce inflammation, and then co-cultured with nerve conduits loaded with different concentrations of telmisartan for 24 hours. The mRNA expressions of inducible nitric oxide synthase (iNOS) and Arginase 1 (Arg-1) were detected by using real-time fluorescence quantitative PCR. Forty adult Wistar rats were randomly divided into 4 groups ( n=10). After preparing 15-mm-long sciatic nerve defect, the defect was repaired by cross-linked nerve conduits loaded with 0, 5, 10, and 20 mg telmisartan in groups A, B, C, and D, respectively. After operation, the general condition of rats was observed after operation; the sciatic function index (SFI) was tested; the bridging between the nerve conduit and sciatic nerve, and the integrity of nerve conduit were observed; the tissue growth in nerve conduit and material degradation were observed by HE staining; the expressions of CD86 (M1 macrophage marker), CD206 (M2 macrophage marker), myelin basic protein (MBP), and myelin protein 0 (P0) in new tissues were also observed by immunohistochemical staining; the expressions of neurofilament 200 (NF-200) and S-100β in new tissues were assessed by immunofluorescence staining.
Results: The general observation showed that the inner diameter of the nerve conduit was 1.8 mm and the outer diameter was 2.0 mm. After cross-linking by genipin, the nanofiber became thicker and denser. The drug release test showed that the telmisartan loaded nerve conduit could be released gradually. With the increase of telmisartan content in nerve conduit, the iNOS mRNA expression decreased and the Arg-1 mRNA expression increased; and the differences between 20 mg group and other groups were significant ( P<0.05). In vivo experiment showed that all animals in each group survived until the completion of the experiment. The SFI was significantly higher in groups C and D than in groups A and B at different time points ( P<0.05) and in group D than in group C at 6 months after operation ( P<0.05). HE staining showed that there were significantly more new tissues in the middle of the nerve conduit in group D after operation than in other groups. Immunohistochemical staining showed that CD86 and CD206 stainings were positive in each group at 1 month after operation, among which group D had the lowest positive rate of CD86 and the highest positive rate of CD206, and there were significant differences in the positive rate of CD206 between group D and groups A, B, and C ( P<0.05); the MBP and P0 stainings were positive in groups C and D at 6 months, and the positive rate in group D was significantly higher than that in group C ( P<0.05). Immunofluorescence staining showed that the NF-200 and S-100β expressions in group D were significantly higher than those in other groups.
Conclusion: Telmisartan/collagen/PLC nerve conduit can promote the sciatic nerve defect repair in rats through promoting the polarization of M1 macrophages to M2 macrophages, and the nerve conduit loaded with20 mg telmisartan has the most significant effect.
目的: 构建替米沙坦/胶原蛋白/聚己内酯（polycaprolactone，PCL）神经导管，观察其修复大鼠坐骨神经缺损的效果。.
方法: 制备质量浓度为60%的胶原蛋白/六氟异丙醇混合液、40%PCL/六氟异丙醇溶液并混匀后，分别将0、5、10、20 mg替米沙坦溶于10 mL混合液。利用高压静电纺丝技术构建替米沙坦/胶原蛋白/PCL神经导管；扫描电镜观察京尼平交联前后神经导管结构；体外缓释方法检测药物释放率。取RAW264.7细胞与脂多糖培养使其致炎后，与负载不同浓度替米沙坦的神经导管共培养24 h， 实时荧光定量PCR检测诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）和精氨酸酶 1（Arginase 1，Arg-1）mRNA的表达。 将40只成年Wistar大鼠随机分成A、B、C、D组（ n=10），制备坐骨神经15 mm长缺损后，分别以交联后负载0、5、10、20 mg替米沙坦的神经导管桥接修复缺损。术后观察大鼠一般情况，检测坐骨神经运动功能指数（sciatic functional index，SFI），大体观察神经导管与坐骨神经桥接情况以及导管完整性，HE染色观察神经导管内组织生长和材料降解情况，免疫组织化学染色观察新生组织中M1型巨噬细胞标志分子CD86和M2型巨噬细胞标志分子CD206、髓磷脂碱基蛋白（myelin basic protein，MBP）和髓磷脂蛋白0（myelin protein 0，P0）的表达，免疫荧光染色观察新生组织中神经丝蛋白200（neurofilament 200，NF200）和S-100β表达。.
结果: 大体观察示制备的神经导管内径为 1.8 mm、外径2.0 mm，呈白色；交联后纳米纤维变粗，结构更致密。药物缓释检测示神经导管负载的替米沙坦能实现缓释效应。实时荧光定量PCR检测示，随着替米沙坦浓度增加，iNOS mRNA相对表达量下调，Arg-1 mRNA相对表达量上调，其中20 mg组与其他各组比较差异均有统计学意义（ P<0.05）。动物体内实验观测示，术后各组大鼠均存活至实验完成。术后各时间点，C、D 组SFI均高于A、B 组（ P<0.05），且6个月时D组高于C组（ P<0.05）。HE染色示术后D组神经导管中段新生组织明显多于其他各组。免疫组织化学染色示，各组术后1个月时CD86及CD206均呈阳性，其中D组CD86阳性率最低、CD206阳性率最高，其中CD206差异有统计学意义（ P<0.05）；6个月时仅C、D组MBP和P0染色呈阳性，且D组阳性率高于C组（ P<0.05）。免疫荧光染色观察示D组NF-200和S-100β表达明显强于其他各组。.
结论: 替米沙坦/胶原蛋白/PCL神经导管通过促进M1型巨噬细胞向M2型巨噬细胞极化，促进大鼠坐骨神经缺损修复，其中负载20 mg替米沙坦的神经导管促进效果最显著。.
Keywords: Telmisartan; collagen; macrophage polarization; nerve regeneration; polycaprolactone; rat.