The Effects of LPS With or Without Growth Factors on S16 Rat Schwann Cell Proliferation

As a preliminary study, the immortalized S16 rat Schwann cell line was cultured with different combinations of lipopolysaccharide (LPS, a cell wall immunostimulatory component found in Gram-negative bacteria) and growth factors (the neuronal mitogen heregulin and the artificial plant extract forskolin) to determine the effects of the different treatment combinations on cell proliferation. The cells received one of the following treatments: N2 control media, 12.5 ng/mL of heregulin (H), 2 uM of forskolin (F), or heregulin plus forskolin (H+F), and 0 ,5, 50, or 500 ng/mL of LPS, for 1, 3, 12, or 24 hours. The CyQUANT MTT Cell Viability Assay Kit (Thermo Fisher Scientific) was used to perform the proliferation assay. Optical density was measured as an indicator of cell proliferation, with a high optical density representing high proliferation and a low optical density representing low proliferation. It was hypothesized that, for all time points, cells treated with LPS plus growth factors would experience more proliferation than cells treated with LPS only.

The results only partially support the hypothesis because there were some cases where the LPS + growth factor treatments had less proliferation than the LPS only (N2) treatments (i.e., H + 5 or 50 ng/mL of LPS for 24 hours). One of the most important findings is that between the treatments used, it appears as though either 5 or 50 ng/mL of LPS combined with both heregulin and forskolin for 24 hours results in the highest optical density, and therefore the most Schwann cell proliferation.

It also appears as though Schwann cells can tolerate higher concentrations of LPS for shorter periods of time and lower concentrations of LPS for longer periods of time. This indicates that, eventually, higher doses of LPS may become too toxic for the Schwann cells to handle, inhibiting proliferation, and causing cell death. Also, considering Schwann cells treated with LPS for 24 hours had generally higher optical densities than cells treated with LPS for 1, 3, and 12 hours, there may be some period of recovery between 12 and 24 hours.

When looking at each individual growth factor treatment, the results suggest that during exposure to particular concentrations of LPS for 24 hours, Schwann cells grown in the presence of forskolin, with or without heregulin, experience an increase in proliferation, whereas Schwann cells grown in the presence of heregulin experience a decrease in proliferation. These findings suggest that, when Schwann cells are stimulated with LPS, heregulin and forskolin, alone, activate two very distinct pathways to initiate opposite responses, with heregulin hindering cell division and forskolin promoting cell division. However, when heregulin and forskolin are combined, the forskolin-activated cAMP pathway may overcompensate by promoting even more cell division to offset the decrease in cell division initiated by the heregulin pathway.

本研究作为一项预实验，将永生化S16大鼠雪旺细胞系（immortalized S16 rat Schwann cell line）接种于脂多糖（lipopolysaccharide, LPS，革兰氏阴性菌细胞壁所含的免疫刺激成分）与生长因子[包含神经元丝裂原调蛋白（heregulin）及人工植物提取物福司柯林（forskolin）]的不同组合培养基中，以明确不同处理组合对细胞增殖的影响。细胞接受以下任一处理方案：N2对照培养基、12.5 ng/mL调蛋白（heregulin, H）、2 μM福司柯林（forskolin, F）、调蛋白联合福司柯林（H+F），以及浓度分别为0、5、50、500 ng/mL的LPS，处理时长设置为1、3、12或24小时。本实验采用CyQUANT MTT细胞活力检测试剂盒（Thermo Fisher Scientific）开展增殖检测，以光密度值作为细胞增殖水平的评价指标：光密度值越高，代表细胞增殖能力越强；反之则增殖能力越弱。本研究提出假设：在所有检测时间点，联合使用LPS与生长因子处理的细胞，其增殖水平均高于仅用LPS处理的细胞。

实验结果仅部分支持该假设：存在部分LPS联合生长因子处理组的增殖水平低于仅用LPS（N2对照）处理组的情况，例如处理24小时的H+5 ng/mL或H+50 ng/mL LPS组。本研究最重要的发现之一为：在所有受试处理方案中，浓度为5或50 ng/mL的LPS联合调蛋白与福司柯林共同处理细胞24小时时，光密度值最高，对应雪旺细胞的增殖能力最强。

此外，雪旺细胞对高浓度LPS的耐受时长较短，而对低浓度LPS的耐受时长更长。该结果提示：随着时间推移，更高剂量的LPS会对雪旺细胞产生过度毒性，抑制细胞增殖并诱导细胞死亡。此外，经LPS处理24小时的雪旺细胞光密度值普遍高于处理1、3及12小时的细胞，提示细胞在12至24小时之间可能存在一段恢复阶段。

针对单一生长因子处理组的分析结果显示：在特定浓度LPS处理24小时的条件下，添加福司柯林（无论是否联合调蛋白）培养的雪旺细胞增殖能力均有所提升，而仅添加调蛋白培养的雪旺细胞增殖能力则出现下降。上述结果提示：当雪旺细胞受到LPS刺激时，调蛋白与福司柯林可分别激活两条截然不同的信号通路，引发相反的生物学效应——调蛋白会抑制细胞分裂，而福司柯林则促进细胞分裂。当二者联合使用时，福司柯林激活的环磷酸腺苷（cAMP）通路可通过过度激活细胞分裂，代偿调蛋白通路介导的细胞分裂抑制效应。