目的探讨红景天苷对高糖所致原代培养神经元损伤是否具有保护作用及其机制。方法原代培养大鼠皮质神经元,分为对照组,50 m M高糖处理组,50μM红景天苷+50 m M高糖处理组,以及100μM红景天苷+50 m M高糖处理组。用免疫印迹检测cleaved caspase-3和脑源性神经营养因子(BDNF)的表达,用ELISA方法检测培养基中BDNF的含量。结果原代培养的皮质神经元纯度为95%以上。免疫印迹结果表明,与对照组比,高糖可诱导cleaved caspase 3的表达,表明高糖可以诱导神经元凋亡。与单纯高糖处理组相比,红景天苷预处理可显著降低cleaved caspase 3的表达。同时,高糖降低培养基中BDNF含量,而红景天苷可以抑制此作用。结论红景天苷通过调节BDNF的表达和分泌,降低cleaved caspase 3的表达,进而减轻高糖所致的神经元损伤。
This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA （shRNA） targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside （PAN）-induced podocyte injury. Two DNA sequences containing the small hairpin structure targeting TRPC6 were designed, synthesized and then inserted into the green fluorescence protein （GFP）-contained plasmids （pGC） to establish the plasmids pGCsi-TRPC6A and pGCsi-TRPC6B. Plasmids expressing scrambled shRNA were used as negative control and named pGCsi-NC. These plasmids were transfected into a conditionally immortalized murine podocyte cell line by using liposome. Flow cytometry was used to examine the transfection efficiency. TRPC6 mRNA and protein ex-pression levels were detected by RT-PCR and Western blotting. Cultured podocytes were divided into four groups： control group, PAN treatment group, PAN＋TRPC6 shRNA transfected group and PAN＋scrambled shRNA transfected group. The paracelluar permeability to BSA was evaluated by Millicell-PCF Inserts and cell viability was measured by the trypan blue assay. Immunofluorescent assay was used to observe the distribution of α-actinin-4 and α-tubulin. The results showed that the transfection efficiency of the shRNA expression vector was about 45%. Expression levels of TRPC6 mRNA and protein were downregulated after transfection with pGCsi-TRPC6A and pGCsi-TRPC6B. Knocking down TRPC6 gene could effectively reverse the PAN-induced increase in the paracelluar permeability to BSA. The distribution of α-actinin-4 and α-tubulin was disrupted after treatment with PAN, which was reversed by knocking down TRPC6 gene. It was concluded that knocking down TRPC6 gene could effectively prevent podocytes from the permeability increase induced by PAN, which may be related to the regulation of podocyte cytoskeleton.
