810nm弱激光对M1型骨髓源性巨噬细胞的作用及其机制

The effect of 810nm low level laser on M1 bone-marrow derived macrophage and its related mechanism

中文关键词:  弱激光治疗  炎症反应  M1型巨噬细胞

英文关键词:Low level laser therapy  Inflammation  M1 macrophage

中文摘要:

  【摘要】 目的：体外建立M1型骨髓源性巨噬细胞（bone marrow derived macrophage，BMDM）-弱激光照射模型，探讨弱激光照射对M1型BMDM炎症因子分泌的影响及其可能机制。方法：体外分离BALB/c小鼠BMDM，应用巨噬细胞集落刺激因子（macrophage colony-stimulating factor，M-CSF）条件性培养基培养，流式细胞术检测巨噬细胞标志物F4/80的表达，确定所获得细胞为纯度较高的BMDM。应用脂多糖（lipopolysaccharide，LPS）刺激24h诱导原代BMDM向M1型BMDM方向极化。将细胞随机分为弱激光治疗（low level laser therapy，LLLT）组和对照组，LLLT组采用810nm波长激光照射，光斑面积4.5cm2，照射功率密度2mW/cm2，照射持续440s，最终获得照射能量为4J，对照组置于暗箱，不进行照射。应用CCK8实验测定细胞活性，RT-PCR检测肿瘤坏死因子α（tumor necrosis factor-α，TNF-α）、白细胞介素1β（interlukin-1β，IL-1β）及诱导型一氧化氮合酶（inducible nitric oxide synthases，iNOS）的mRNA表达情况，ELISA检测TNF-α及IL-1β蛋白分泌，Western blot测定iNOS和M1型巨噬细胞极化的重要转录因子核因子-κB p65（nuclear factor-kappa B，NF-κB p65）的表达。组间比较采用独立样本t检验，P<0.05为差异有统计学意义。结果：LPS刺激后，使用弱激光照射的M1型BMDM细胞活性显著提高，升高至对照组的2.313±0.630倍（P<0.05）。LLLT组IL-1β mRNA表达为0.586±0.145，对照组为1.000±0.022，两组比较有统计学差异（P<0.05）；LLLT组TNF-α的mRNA表达为0.862±0.152，对照组为1.000±0.082，两组比较无统计学差异（P=0.082）；LLLT组iNOS的mRNA表达为0.720±0.039，对照组为1.000±0.024，两组比较有统计学差异（P<0.05）。对照组TNF-α为270.478±26.831pg/ml，LLLT组为209.365±5.600pg/ml，两组比较有统计学差异（P<0.05）；对照组的IL-1β为98.941±12.430pg/ml，LLLT组为77.076±2.023pg/ml，两组比较有统计学差异（P<0.05）。对照组的iNOS蛋白表达为1.005±0.075，LLLT组为0.210±0.010，两组比较有统计学差异（P<0.05）；对照组NF-κB p65蛋白表达为1.006±0.260，LLLT组为0.428±0.169，两组比较有统计学差异（P<0.05）。结论：810nm LLLT M1型BMDM可抑制其炎性因子IL-1β及iNOS的mRNA表达，下调炎性因子IL-1β及TNF-α的分泌，降低M1型BMDM表面标志物iNOS的表达，同时显著下调M1型巨噬细胞经典极化转录因子NF-κB p65的表达。810nm LLLT可调控M1型巨噬细胞的极化状态，抑制其炎性因子分泌，该调控作用和其下调M1型巨噬细胞经典极化转录因子NF-κB p65有明显相关性。

英文摘要:

  【Abstract】 Objectives: To investigate the effect of low level laser therapy on the secretion of inflammatory cytokines in M1 type cells and its possible mechanism by establishing a M1 type bone marrow-derived macrophages(BMDM)-low level laser irradiation model in vitro. Methods: BMDM of BALB/c mice were isolated and cultured in macrophage colony-stimulating factor (M-CSF) conditioned media. The flow cytometry was used to detect the expression of macrophage marker F4/80 to identify the culture results. The cells were stimulated for 24 hours with lipopolysaccharide(LPS) to induce M1 polarization. Cells were randomly divided into low level laser therapy(LLLT) group and control group. The LLLT group was irradiated with low level laser at 810nm wavelength, the area of light spot was 4.5cm2, the irradiation power density was 2.2mW/cm2, and the irradiation time was 440s. The total irradiation energy was 4J. The two groups were put into the dark boxes. But only the LLLT group received a laser irradiation. The cell viability was determined by Cell Counting Kit 8(CCK8) assay. The mRNA expression of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) and inducible nitric oxide synthase(iNOS) were detected by Real Time PCR(RT-PCR). The secretion of TNF-α and IL-1β protein was detected by ELISA. Western blot was used to determine the expression of iNOS and the expression of NF-κB p65, an important transcription factor of M1 macrophage signaling pathway. Student′s t test was chosen as the statistical method, P<0.05 was considered statistically significant. Results: After LPS stimulation, M1 BMDM was irradiated. CCK8 assay showed that compared with control group, the LLLT group could significantly increase the cell viability of BMDM to 2.313±0.630 folds of control group(P<0.05). RT-PCR results showed that compared with control group, the mRNA expression of IL-1β decreased from 1.000±0.022 in control group to 0.586±0.145 in LLLT group(P<0.05). The expression of TNF-α mRNA decreased from 1.000±0.082 in control group to 0.862±0.152 in LLLT group(P=0.082), the iNOS mRNA level also decreased from 1.00±0.024 in control group to 0.720±0.039 in LLLT group(P<0.05). ELISA showed that LLLT could down-regulate the secretion of TNF-α from 270.478±26.831pg/ml in control group to 209.365±5.600pg/ml in LLLT group(P<0.05). IL-1β was down-regulated from 98.941±12.430pg/ml in control group to 77.076±2.023pg/ml in LLLT group(P<0.05). Western bolt assay showed that, compared with control group, LLLT down-regulated the protein secretion of iNOS from 1.005±0.075 to 0.210±0.010(P<0.05). LLLT also significantly decreased NF-κB p65 expression, which decreased from 1.006±0.260 in control group to 0.428±0.169 in LLLT group(P<0.05). Conclusions: 810nm low level laser therapy can directly affect M1 BMDM, down-regulate the mRNA expression of IL-1β and iNOS and suppress the secretion of TNF-α and IL-1β. LLLT can also significantly decrease the expression of M1 macrophage surface marker iNOS and NF-κB p65, a classic transcription factor is involved in macrophage polarization. The results show that LLLT can regulate the polarization status of M1 macrophages and inhibit its inflammatory secretion, and this regulatory role has a strong correlation with the suprression on classic transcription factor NF-κB p65.