基于PI3K/AKT信号通路探讨益气养阴活血利水方抑制早期糖尿病视网膜病变大鼠微血管周细胞凋亡的作用机制

Mechanism of Yiqi Yangyin Huoxue Lishui Formula in inhibiting microvascular pericyte apoptosis in rats with early diabetic retinopathy

目的观察益气养阴活血利水方对早期糖尿病视网膜病变(diabetic retinopathy,DR)大鼠视网膜微血管周细胞中磷酸肌醇3-激酶蛋白(phosphatidylinositol 3-kiases,PI3K)/丝氨酸/苏氨酸蛋白激酶(phospho-alpha serine/threonine-protein kinase,AKT)信号通路相关因子表达的影响,探讨益气养阴活血利水方抑制早期DR视网膜微血管周细胞凋亡的作用机制。方法138只雄性SPF级SD大鼠随机分成空白组(等体积蒸馏水),模型组,羟苯磺酸钙组[150 mg/(kg·d)],益气养阴活血利水方低剂量组[6.2 g/(kg·d)]、中剂量组[12.4 g/(kg·d)]、高剂量组[24.8 g/(kg·d)],每组23只。除空白组外,各组均采用链佐脲菌素腹腔注射并维持10周高血糖状态,诱导早期DR大鼠模型。造模成功后给药4周,取大鼠眼球进行检测。采用HE染色观察视网膜病理变化,采用TUNEL染色检测大鼠视网膜微血管周细胞凋亡,采用免疫组织化学法及RT-PCR法检测PI3K、AKT、Bcl-2相关X蛋白(Bcl2-associated X protein,Bax)、胱天蛋白酶-8(cysteine aspartic acid specific protease-8,Caspase-8)、胱天蛋白酶-3(cysteine aspartic acid specific protease-3,Caspase-3)、细胞凋亡死亡激动剂BID蛋白(apoptotic death agonist BID protein,Bid)在视网膜微血管周细胞中的蛋白和mRNA表达水平。结果与空白组比较,模型组大鼠血糖显著升高(P<0.01),体质量显著降低(P<0.01),视网膜层次结构不清,各层细胞排列疏松,可见大量凋亡细胞,视网膜中PI3K、AKT mRNA表达水平下降(P<0.01),视网膜细胞凋亡数及Bax、Caspase-8、Caspase-3、Bid mRNA表达水平明显升高(P<0.05)。与模型组比较,益气养阴活血利水方中剂量、高剂量组大鼠血糖均降低(P<0.01);羟苯磺酸钙组、益气养阴活血利水方各剂量组大鼠体质量均升高(P<0.01);益气养阴活血利水方各剂量组大鼠视网膜组织层次结构较清楚,细胞排列相对紧密,视网膜微血管周细胞凋亡数下降(P<0.05);益气养阴活血利水方高剂量组大鼠视网膜微血管周细胞中PI3K、AKT蛋白和mRNA表达水平均明显升高(P<0.01),Bax、Caspase-8、Caspase-3、Bid蛋白和mRNA表达水平均明显下降(P<0.05)。结论益气养阴活血利水方可能通过调控PI3K/AKT信号通路,上调PI3K、AKT,下调Bax、Caspase-8、Caspase-3、Bid的表达,抑制早期DR大鼠视网膜微血管周细胞的凋亡,从而发挥其对早期DR视网膜微血管周细胞凋亡的保护作用。

Objective To observe the effects of Yiqi Yangyin Huoxue Lishui Formula(YQYYHXLSF)on the expressions of phosphoinositol 3-kinases(PI3K)/phospho-alpha serine/threonine-protein kinase(AKT)signaling pathway-related factors in retinal microvascular pericytes of rats with early diabetic retinopathy(DR),and to investigate the mechanism of YQYYHXLSF in inhibiting apoptosis of retinal microvascular pericytes in early DR.Methods A total of 138 male SPF SD rats were randomized into blank(equal-volume distilled water),model,calcium dobesilate[150 mg/(kg·d)],low-dose[6.2 g/(kg·d)],medium-dose[12.4 g/(kg·d)],and high-dose[24.8 g/(kg·d)]YQYYHXLSF groups,with 23 rats in each group.Except for the blank group,all groups were injected intraperitoneally with streptozotocin and maintained a hyperglycemic state for 10 weeks to induce early DR rat models.After successful modeling,the eyeballs of rats were tested after 4 weeks of administration.HE staining was used to observe the pathological changes of retina,and TUNEL staining was used to check the apoptosis of retinal microvascular pericytes.Immunohistochemistry and RT-PCR were taken to examine the protein and mRNA expression levels of PI3K,AKT,Bcl2 associated X protein(Bax),cysteine aspartic acid specific protease-8(Caspase-8),cysteine aspartic acid specific protease-3(Caspase-3)and apoptotic death agonist BID protein(Bid)in retinal perivascular pericytes.Results Compared with blank group,the rats in model group showed a significant increase in blood glucose(P<0.01),a significant decrease in body mass(P<0.01),unclear retinal hierarchy,loose cell arrangement in each layer with a large number of apoptotic cells visible.The mRNA expression levels of PI3K and AKT in retina were lower(P<0.01),while the number of apoptotic cells of retinal cells and the mRNA expression levels of Bax,Caspase-8,Caspase-3,and Bid were significantly higher(P<0.05).Compared with model group,the medium-dose and high-dose YQYYHXLSF groups showed a decrease in the blood glucose levels of rats(P<0.01);the calcium dobesilate group and all the YQYYHXLSF groups showed an increase in the body mass of rats(P<0.01);all the YQYYHXLSF groups manifested clear retinal tissue hierarchical structure of rats,relatively close cell arrangement,and decreased the number of apoptotic cells of retinal microvascular pericytes(P<0.05);and the protein and mRNA expression levels of PI3K and AKT in retinal microvascular pericytes of rats in high-dose YQYYHXLSF group were significantly higher(P<0.01),the protein and mRNA expression levels of Bax,Caspase-8,Caspase-3,and Bid were significantly lower(P<0.05).Conclusion YQYYHXLSF may inhibit the apoptosis of retinal microvascular pericytes in early DR rats by regulating the PI3K/AKT signaling pathways,including upregulating the expressions of PI3K and AKT and downregulating those of Bax,Caspase-8,Caspase-3,and Bid,thus exert its protective effect.