当归补血汤加味方通过调控神经节细胞ER stress改善糖尿病视网膜病变的机制研究

Microvascular and neural dysfunction are hallmarks of Diabetic retinopathy (DR), in which neural dysfunction may occur earlier and exacerbate microvascular dysfunction. So far, most treatments for DR are targeting microvascular dysfunction, however, with limited effects indicating that more attention for neural dysfunction are required. Based on Chinese traditional medicine theory, we proposed and proved that an aqueous extract of Radix Astragali, Angelica sinensis, and Panax notoginseng (RRP) was protective in preventing vascular lesions in DR. Of note, our preliminary data suggested that RRP could prevent not only vascular lesions but also retinal ganglion cell (RGC) lesions in DR, together with ER stress inhibition. Inhibition of ER stress could ameliorate hypoxia induced RGC apoptosis. These results indicated that RRP could possibly ameliorate DR progress by preventing ER stress mediated RGC lesions. Thus, we proposed a further investigation on the underlying molecular mechanisms mediating ER stress effect on RGC apoptosis as well as the molecular targets of RRP treatment in STZ induced type 1 diabetic animal model and hypoxia stimulated RGC-5 culture model. This study will help to elucidate the mechanisms and therapy potentials of RRP application on DR.

早期糖尿病视网膜病变包括神经病变与血管病变，且神经病变可能早于并加剧血管病变的发生。然而现有的治疗方式大多靶向血管病变且疗效有限。从“气血亏虚，血瘀络阻”的病机角度出发，我们发现当归补血汤加味方（RRP）可有效缓解早期糖尿病视网膜的血管病变。基于“多靶向，多机制”的复方特点，RRP能否改善神经病变从而更早更全面地缓解该疾病呢？预实验发现：RRP可缓解糖尿病视网膜神经节细胞（RGC）损伤及其内质网应激(ER stress)的激活；抑制ER stress可缓解缺氧引起的RGC凋亡，提示RRP可能通过抑制ER stress介导的RGC损伤改善糖尿病视网膜病变。本课题计划运用I型糖尿病大鼠模型和RGC-5细胞缺氧诱导模型深入阐明ER stress介导RGC凋亡的上下游分子机制及RRP的作用靶点，为早期糖尿病视网膜病变治疗和RRP的运用提供新的科学依据。

我们在前期工作中发现，当归补血汤加味方可有效缓解糖尿病大鼠视网膜的血管和神经病变，其中，Muller细胞的异常激活可被有效缓解，提示当归补血汤加味方可能通过靶向Muller缓解糖尿病视网膜病变的发生。在本研究中，我们采用Muller细胞系Mio-M1构建了体外损伤模型。通过模拟高糖和低氧的病理环境我们发现，低氧可显著促进Mio-M1的凋亡水平以及异常高表达血管新生因子和促炎因子；在此基础上，我们进一步对当归补血汤加味方含药血清进行了高效液相质谱分析，并对低氧诱导的Mio-M1细胞进行预处理，发现当归补血汤加味方含药血清预处理可有效缓解低氧诱导引起的Mio-M1细胞凋亡，但是对其高表达血管新生因子和促炎因子并无影响；通过加入内质网应激阻断剂TUDCA和激动剂TM，我们发现，阻断内质网应激可有效缓解低氧诱导的Mio-M1凋亡，而激活内质网应激可直接引起Mio-M1凋亡，该结果提示内质网应激介导了低氧诱导的Mio-M1凋亡；结合Westen Blot检测我们发现内质网应激通路之一ATF4/CHOP可被低氧诱导激活，而当归补血汤加味方含药血清可有效缓解ATF4/CHOP的激活，提示ATF4/CHOP介导了当归补血汤加味方对低氧诱导的Mio-M1的保护作用。综上，当归补血汤加味方可通过缓解内质网应激的ATF4/CHOP通路缓解低氧诱导的视网膜Muller细胞损伤。该研究提示在糖尿病视网膜病变过程中，长期的缺氧（如增殖期糖尿病视网膜病变）可能会造成Muller细胞的凋亡。该研究亦为当归补血汤加味方在治疗糖尿病视网膜病变中的作用提供了新的理论和实验依据。