蛋白激酶Cβ抑制剂LY333531对糖尿病大鼠造影剂肾病的保护作用

doi:10.3877/cma.j.issn.1674-6880.2020.06.003

目的

探讨蛋白激酶Cβ（PKCβ）抑制剂LY333531对造影剂诱导的糖尿病大鼠急性肾损伤的保护作用及其机制。

方法

将40只大鼠分为健康对照组（C组）、糖尿病组（D组）、糖尿病造影剂组（DC组）以及糖尿病+ Y333531 +造影剂组（DCL组），每组各10只。对D组、DC组及DCL组大鼠腹腔注射2%链脲佐菌素（60 mg / kg）建立糖尿病模型；对DCL组大鼠给予LY333531灌胃预处理（10 mg·kg^-1·d^-1），持续14 d；对DC组与DCL组大鼠进行尾静脉注射76%复方泛影葡胺（10 ml / kg），建立造影剂肾病模型。苏木素-伊红（HE）染色观察各组大鼠肾组织病理变化；检测各组大鼠血糖、血肌酐、尿微量白蛋白（mAlb）和N-乙酰-β-D-葡萄糖苷酶（NAG）水平；采用实时荧光定量PCR法检测转化生长因子β1（TGFβ1）、Smad3、Smad7、Bax、Caspase3和Bcl2的信使RNA（mRNA）表达水平；采用Western-blotting检测TGFβ1、Bax、Caspase3、Bcl2蛋白及磷酸化PKCβ（pPKCβ） / PKCβ、磷酸化p38（p-p38） / p38的比值。

结果

HE结果显示，C组大鼠肾小球、肾小管结构正常；D组大鼠肾组织内肾小球及肾小管结构形态较为完整，间质周围极少量炎症细胞；DC组肾小管上皮细胞脱落、周围炎症细胞浸润明显；DCL组大鼠肾小管上皮细胞脱落程度明显降低，肾小管及肾小球周围间质少量炎症细胞浸润。各组大鼠间血糖、血肌酐、尿mAlb、尿NAG水平、TGFβ1蛋白及其mRNA、Smad3 mRNA、Smad7 mRNA、Bax蛋白及其mRNA、Caspase3蛋白及其mRNA、Bcl2蛋白及其mRNA表达水平、pPKCβ / PKCβ和p-p38 / p38比值间比较，差异均有统计学意义（F = 67.976，P < 0.001；F = 27.155，P < 0.001；F = 41.201，P < 0.001；F = 59.635，P < 0.001；F = 21.073，P < 0.001；F = 28.365，P < 0.001；F = 15.215，P < 0.001；F = 36.273，P < 0.001；F = 14.489，P < 0.001；F = 23.172，P < 0.001；F = 17.103，P < 0.001；F = 29.916，P < 0.001；F = 12.026，P < 0.001；F = 13.368，P < 0.001；F = 6.126，P = 0.002；F = 6.434，P = 0.002）。进一步两两比较发现，D组、DC组以及DCL组大鼠的血糖水平较C组大鼠均明显升高（P均< 0.05）；与DC组大鼠比较，DCL组大鼠的的血肌酐、尿mAlb、尿NAG水平、TGFβ1蛋白及其mRNA、Smad3 mRNA、Bax蛋白及其mRNA、Caspase3蛋白及其mRNA、pPKCβ / PKCβ和p-p38 / p38比值均显著降低，而Smad7 mRNA、Bcl2蛋白及其mRNA表达水平均显著升高（P均< 0.05）。

结论

PKCβ抑制剂LY333531能通过抑制PKCβ-TGFβ-p38-Caspase3通路改善造影剂诱导的糖尿病大鼠急性肾损伤。

关键词: 糖尿病, 造影剂肾病, 蛋白激酶C, 大鼠

Objective

To explore the protective effect of protein kinase Cβ (PKCβ) inhibitor LY333531 in diabetic rats with contrast-induced acute kidney injury and its mechanism.

Methods

Totally 40 rats were randomly divided into a control group (C group), a diabetic group (D group), a diabetes with contrast-induced nephropathy group (DC group) and a diabetes with Y333531 and contrast-induced nephropathy group (DCL group), 10 rats in each group. The rats in the D, DC and DCL groups were injected intraperitoneally with 2% streptozotocin (60 mg / kg) to induce a diabetes model. Then the rats in the DCL group were pretreated with LY333531 (10 mg·kg^-1·d^-1) by intragastric administration for 14 d; the rats in the DC and DCL groups were injected with 76% diatrizoate (10 mL / kg) by tail vein to establish a model of contrast-induced nephropathy. The pathological changes in kidney were observed by hematoxylin-eosin (HE) staining. The levels of blood glucose, serum creatinine, urine microalbumin (mAlb) and urine N-acetyl-β-D-glucosidase (NAG) were detected. The messenger RNA (mRNA) levels of transforming growth factor beta1 (TGFβ1), Smad3, Smad7, Bax, Caspase3 and Bcl2 were examined by real-time fluorescence quantitative PCR. The protein levels of TGFβ1, Bax, Caspase3 and Bcl2 were detected by Western-blotting, as well as the phospho-PKCβ (pPKCβ) / PKCβ and phospho-p38 (p-p38) / p38.

Results

According to HE, the glomeruli and renal tubules in the group C were structurally normal; the glomeruli and renal tubules in the group D were relatively structurally complete with few inflammatory cells around the interstitium; the epithelial cells of renal tubules in the DC group were exfoliated and the inflammatory cells obviously infiltrated around renal tubules; in the DCL group, the degree of epithelial cell shedding in renal tubules was significantly reduced, and a small amount of inflammatory cells infiltrated in the interstitium around renal tubules and glomeruli. The blood glucose, serum creatinine, urine mAlb, urine NAG, TGFβ1 protein and its mRNA, Smad3 mRNA, Smad7 mRNA, Bax protein and its mRNA, Caspase3 protein and its mRNA, Bcl2 protein and its mRNA, pPKCβ / PKCβ and p-p38 / p38 all showed significant differences among the four groups (F = 67.976, P < 0.001; F = 27.155, P < 0.001; F = 41.201, P < 0.001; F = 59.635, P < 0.001; F = 21.073, P < 0.001; F = 28.365, P < 0.001; F = 15.215, P < 0.001; F = 36.273, P < 0.001; F = 14.489, P < 0.001; F = 23.172, P < 0.001; F = 17.103, P < 0.001; F = 29.916, P < 0.001; F = 12.026, P < 0.001; F = 13.368, P < 0.001; F = 6.126, P = 0.002; F = 6.434, P = 0.002). Further pairwise comparison revealed that the levels of blood glucose in the D, DC and DCL groups were significantly higher than those in the C group (all P < 0.05). As compared with the DC group, the serum creatinine, urine mAlb, urine NAG, TGFβ1 protein and its mRNA, Smad3 mRNA, Bax protein and its mRNA, Caspase3 protein and its mRNA, pPKCβ / PKCβ and p-p38 / p38 decreased markedly, while the Smad7 mRNA, Bcl2 protein and its mRNA increased obviously in the DCL group (all P < 0.05).

Conclusion

The PKCβ inhibitor LY333531 can improve contrast-induced acute kidney injury in diabetic rats by inhibiting the PKCβ-TGFβ-p38-Caspase3 pathway.

Key words: Diabetes mellitus, Contrast-induced nephropathy, Protein kinase C, Rats

图1 各组小鼠肾脏组织病理变化（HE染色　× 20）

图2 各组大鼠血糖、血肌酐、尿mAlb以及尿NAG水平的比较

图3 各组大鼠间TGFβ1蛋白及其mRNA、Smad3 mRNA及Smad7 mRNA表达水平的比较

图4 各组大鼠凋亡相关蛋白及基因表达水平的比较

图5 各组大鼠间pPKCβ / PKCβ和p-p38 / p38比值的比较

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Protective effect of protein kinase Cβ inhibitor LY333531 in diabetic rats with contrast-induced nephropathy

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Protective effect of protein kinase Cβ inhibitor LY333531 in diabetic rats with contrast-induced nephropathy