红景天苷通过抑制PI3K/AKT/mTOR信号通路对大鼠脓毒症急性肾损伤的保护作用

doi:10.3877/cma.j.issn.1674-6880.2024.03.003

目的

探讨红景天苷（SLDS）调控磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（AKT）/哺乳动物雷帕霉素靶蛋白（mTOR）信号通路对大鼠脓毒症急性肾损伤（SAKI）的保护作用。

方法

采用随机数字表法将40只Sprague-Dawley大鼠分为空白对照组（Control组）、假手术组（Sham组）、盲肠结扎穿孔术组（CLP组）和红景天苷预处理组（CLP + SLDS组），每组10只。评估各组大鼠肾组织病理损伤情况，并采用酶联免疫吸附测定（ELISA）法检测大鼠血清肌酐（Scr）、血浆中性粒细胞明胶酶相关脂蛋白（pNGAL）和血浆肾损伤分子1（pKIM-1）水平，同时检测血浆中白细胞介素1β（IL-1β）、肿瘤坏死因子α（TNF-α）、IL-4和IL-10的表达水平。采用TUNEL法观察肾组织细胞凋亡情况，实时荧光定量PCR（RT-qPCR）法检测含半胱氨酸的天冬氨酸蛋白水解酶3（Caspase-3）、B细胞淋巴瘤2相关X（Bax）和B细胞淋巴瘤2（Bcl-2）信使RNA（mRNA）表达水平。最后，采用western-blotting法检测磷酸化PI3K（p-PI3K）、磷酸化AKT（p-AKT）和磷酸化mTOR（p-mTOR）蛋白表达。

结果

4组大鼠肾组织病理损伤评分、Scr、pKIM-1、pNGAL、TNF-α、IL-1β、IL-4、IL-10、肾组织细胞凋亡数量、Caspase-3 mRNA、Bax mRNA、Bcl-2 mRNA及肾组织中p-PI3K、p-AKT和p-mTOR蛋白水平比较，差异均有统计学意义（F = 132.603、626.719、216.573、335.719、368.219、403.612、169.901、181.281、169.312、960.912、1 479.000、32.221、178.346、137.560、136.241，P均< 0.001）。与Sham组相比，CLP组大鼠肾组织病理损伤加重，Scr、pKIM-1、pNGAL、TNF-α、IL-1β、Caspase-3 mRNA、Bax mRNA以及p-PI3K、p-AKT和p-mTOR蛋白表达水平均明显升高，肾组织细胞凋亡数量显著增多，而IL-4、IL-10和Bcl-2 mRNA表达均降低（P均< 0.05）；与CLP组相比，CLP + SLDS组大鼠肾组织病理损伤减轻，Scr、pKIM-1、pNGAL、TNF-α、IL-1β、Caspase-3 mRNA、Bax mRNA以及p-PI3K、p-AKT和p-mTOR蛋白表达水平均显著降低，肾组织细胞凋亡数量显著减少，而IL-4、IL-10和Bcl-2 mRNA表达均增加（P均< 0.05）。

结论

SLDS通过抑制PI3K/AKT/mTOR信号通路对SAKI具有显著的保护作用。

关键词: 红景天苷, 脓毒症, 急性肾损伤, 磷脂酰肌醇3激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白, 信号通路

Objective

To explore the protective effect of salidroside (SLDS) on septic acute kidney injury (SAKI) in rats by regulating the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway.

Methods

Forty Sprague-Dawley rats were randomly divided into four groups, namely the control group, the sham group, the cecal ligation and perforation group (CLP group) and the SLDS pretreatment group (CLP + SLDS group), 10 rats in each group. We assessed the pathological damage of kidney tissue in each group, and used enzyme-linked immunosorbent assay (ELISA) to detect the levels of serum creatinine (Scr), plasma neutrophil gelatinase-associated lipocalin (pNGAL) and plasma kidney injury molecule 1 (pKIM-1) in rats. Meanwhile, we detected the expression levels of interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), IL-4 and IL-10 in plasma. We used the TUNEL method to observe renal cell apoptosis, real-time fluorescence quantitative PCR (RT-qPCR) method to detect cysteinyl aspartate specific proteinase-3 (Caspase-3), B-cell lymphoma-2 associated X (Bax) and B-cell lymphomato-2 (Bcl-2) messenger RNA (mRNA) expression, and western-blotting method to detect phosphorylated PI3K (p-PI3K), phosphorylated AKT (p-AKT) and phosphorylated mTOR (p-mTOR) protein expression.

Results

There were significant differences in the pathological damage score, Scr, pKIM-1, pNGAL, TNF-α, IL-1β, IL-4, IL-4, IL-10, number of apoptosis, Caspase-3 mRNA, Bax mRNA, Bcl-2 mRNA, p-PI3K protein, p-AKT protein and p-mTOR protein in kidney tissue among the four groups (F = 132.603, 626.719, 216.573, 335.719, 368.219, 403.612, 169.901, 181.281, 169.312, 960.912, 1 479.000, 32.221, 178.346, 137.560, 136.241; all P < 0.001). Compared with the sham group, the pathological damage of kidney tissue was worsened, the levels of Scr, pKIM-1, pNGAL, TNF-α, IL-1β, Caspase-3 mRNA, Bax mRNA, p-PI3K protein, p-AKT protein and p-mTOR protein in kidney tissue and the number of renal cell apoptosis were significantly increased, and the levels of IL-4, IL-10 and Bcl-2 mRNA were decreased in the CLP group (all P < 0.05). Compared with the CLP group, the pathological damage of kidney tissue was reduced, the levels of Scr, pKIM-1, pNGAL, TNF-α, IL-1β, Caspase-3 mRNA, Bax mRNA, p-PI3K protein, p-AKT protein and p-mTOR protein in kidney tissue and the number of renal cell apoptosis were significant decreased, and the levels of IL-4, IL-10 and Bcl-2 mRNA were increased in the CLP + SLDS group (all P < 0.05).

Conclusion

SLDS has a significant protective effect on SAKI by inhibiting the PI3K/AKT/mTOR signaling pathway.

Key words: Salidroside, Sepsis, Acute kidney injury, Phosphatidylinositol 3 kinase/protein kinase B/mammalian target of rapamycin, Signaling pathway

图1 各组大鼠肾组织损伤的病理改变注：CLP.盲肠结扎穿孔术；SLDS.红景天苷；Control组为空白对照组；Sham组为假手术组；CLP组为盲肠结扎穿孔术组；CLP + SLDS组为红景天苷预处理组；a图为Control组，b图为Sham组，两图均可见肾小球包膜完整，肾小管结构清晰，间质未见炎症细胞；c图为CLP组，可见肾小管包膜破裂，肾小管内皮细胞脱落，结构紊乱，间质炎症细胞浸润；d图为CLP + SLDS组，可见肾小管包膜相对完整，肾小管结构清晰，间质少量炎症细胞浸润（苏木素-伊红染色　× 400）

表1 各组大鼠肾组织病理损伤评分及肾脏功能变化（

± s）

组别	只数	肾组织病理损伤评分（分）	Scr（μmol/L）	pKIM-1（μg/L）	pNGAL（μg/L）
Control组	10	0.20 ± 0.16	21.2 ± 1.1	12.2 ± 0.4	13.0 ± 0.4
Sham组	10	0.40 ± 0.16	19.8 ± 1.1	12.0 ± 0.4	12.5 ± 0.4
CLP组	10	3.80 ± 0.13^a	183.3 ± 3.7^a	37.7 ± 1.4^a	77.2 ± 2.6^a
CLP + SLDS组	10	2.40 ± 0.16^b	107.3 ± 4.9^b	24.3 ± 0.6^b	32.6 ± 1.0^b
F值		132.603	626.719	216.573	335.719
P值		< 0.001	< 0.001	< 0.001	< 0.001

表1 各组大鼠肾组织病理损伤评分及肾脏功能变化（

± s）

组别	只数	肾组织病理损伤评分（分）	Scr（μmol/L）	pKIM-1（μg/L）	pNGAL（μg/L）
Control组	10	0.20 ± 0.16	21.2 ± 1.1	12.2 ± 0.4	13.0 ± 0.4
Sham组	10	0.40 ± 0.16	19.8 ± 1.1	12.0 ± 0.4	12.5 ± 0.4
CLP组	10	3.80 ± 0.13^a	183.3 ± 3.7^a	37.7 ± 1.4^a	77.2 ± 2.6^a
CLP + SLDS组	10	2.40 ± 0.16^b	107.3 ± 4.9^b	24.3 ± 0.6^b	32.6 ± 1.0^b
F值		132.603	626.719	216.573	335.719
P值		< 0.001	< 0.001	< 0.001	< 0.001

表2 各组大鼠血浆炎症因子变化（ng/L，

± s）

组别	只数	TNF-α	IL-1β	IL-4	IL-10
Control组	10	12.5 ± 0.4	11.7 ± 0.4	48.2 ± 0.8	42.2 ± 0.7
Sham组	10	12.7 ± 0.4	14.6 ± 0.5	48.5 ± 0.8	42.8 ± 0.6
CLP组	10	36.0 ± 0.8^a	41.5 ± 0.8^a	26.3 ± 0.8^a	22.3 ± 0.8^a
CLP + SLDS组	10	24.3 ± 0.6^b	28.1 ± 1.0^b	40.4 ± 0.7^b	33.7 ± 0.6^b
F值		368.219	403.612	169.901	181.281
P值		< 0.001	< 0.001	< 0.001	< 0.001

表2 各组大鼠血浆炎症因子变化（ng/L，

± s）

组别	只数	TNF-α	IL-1β	IL-4	IL-10
Control组	10	12.5 ± 0.4	11.7 ± 0.4	48.2 ± 0.8	42.2 ± 0.7
Sham组	10	12.7 ± 0.4	14.6 ± 0.5	48.5 ± 0.8	42.8 ± 0.6
CLP组	10	36.0 ± 0.8^a	41.5 ± 0.8^a	26.3 ± 0.8^a	22.3 ± 0.8^a
CLP + SLDS组	10	24.3 ± 0.6^b	28.1 ± 1.0^b	40.4 ± 0.7^b	33.7 ± 0.6^b
F值		368.219	403.612	169.901	181.281
P值		< 0.001	< 0.001	< 0.001	< 0.001

图2 各组大鼠肾组织细胞凋亡情况注：CLP.盲肠结扎穿孔术；SLDS.红景天苷；DAPI. 4',6-二脒基-2-苯基吲哚；Merg.融合；Control组为空白对照组；Sham组为假手术组；CLP组为盲肠结扎穿孔术组；CLP + SLDS组为红景天苷预处理组；Control组及Sham组均可见少量绿色荧光细胞即肾小管上皮细胞；CLP组可见大量绿色荧光细胞；CLP + SLDS组可见少量绿色荧光细胞（TUNEL法　× 400）

表3 各组大鼠肾组织细胞凋亡和凋亡基因表达（

± s）

组别	只数	细胞凋亡数量（个）	Caspase-3 mRNA	Bax mRNA	Bcl-2 mRNA
Control组	10	5.7 ± 1.1	0.100 ± 0.007	0.100 ± 0.004	0.424 ± 0.017
Sham组	10	5.9 ± 1.2	0.132 ± 0.007	0.124 ± 0.004	0.438 ± 0.019
CLP组	10	37.0 ± 6.2^a	0.576 ± 0.010^a	0.531 ± 0.008^a	0.261 ± 0.013^a
CLP + SLDS组	10	12.8 ± 3.9^b	0.414 ± 0.009^b	0.378 ± 0.007^b	0.342 ± 0.008^b
F值		169.312	960.912	1 479.000	32.221
P值		< 0.001	< 0.001	< 0.001	< 0.001

表3 各组大鼠肾组织细胞凋亡和凋亡基因表达（

± s）

组别	只数	细胞凋亡数量（个）	Caspase-3 mRNA	Bax mRNA	Bcl-2 mRNA
Control组	10	5.7 ± 1.1	0.100 ± 0.007	0.100 ± 0.004	0.424 ± 0.017
Sham组	10	5.9 ± 1.2	0.132 ± 0.007	0.124 ± 0.004	0.438 ± 0.019
CLP组	10	37.0 ± 6.2^a	0.576 ± 0.010^a	0.531 ± 0.008^a	0.261 ± 0.013^a
CLP + SLDS组	10	12.8 ± 3.9^b	0.414 ± 0.009^b	0.378 ± 0.007^b	0.342 ± 0.008^b
F值		169.312	960.912	1 479.000	32.221
P值		< 0.001	< 0.001	< 0.001	< 0.001

图3 各组大鼠肾组织p-PI3K、p-AKT、p-mTOR蛋白表达（n = 10）注：p-PI3K.磷酸化磷脂酰肌醇3激酶；p-AKT.磷酸化蛋白激酶B；p-mTOR.磷酸化哺乳动物雷帕霉素靶蛋白；GAPDH.甘油醛-3-磷酸脱氢酶；CLP.盲肠结扎穿孔术；SLDS.红景天苷；Control组为空白对照组；Sham组为假手术组；CLP组为盲肠结扎穿孔术组；CLP + SLDS组为红景天苷预处理组

表4 各组大鼠肾组织p-PI3K、p-AKT和p-mTOR蛋白表达（

± s）

组别	只数	p-PI3K	p-AKT	p-mTOR
Control组	10	0.354 ± 0.011	0.230 ± 0.005	0.315 ± 0.008
Sham组	10	0.369 ± 0.009	0.238 ± 0.006	0.312 ± 0.012
CLP组	10	0.746 ± 0.022^a	0.665 ± 0.028^a	0.627 ± 0.017^a
CLP + SLDS组	10	0.587 ± 0.013^b	0.416 ± 0.019^b	0.373 ± 0.011^b
F值		178.346	137.560	136.241
P值		< 0.001	< 0.001	< 0.001

表4 各组大鼠肾组织p-PI3K、p-AKT和p-mTOR蛋白表达（

± s）

组别	只数	p-PI3K	p-AKT	p-mTOR
Control组	10	0.354 ± 0.011	0.230 ± 0.005	0.315 ± 0.008
Sham组	10	0.369 ± 0.009	0.238 ± 0.006	0.312 ± 0.012
CLP组	10	0.746 ± 0.022^a	0.665 ± 0.028^a	0.627 ± 0.017^a
CLP + SLDS组	10	0.587 ± 0.013^b	0.416 ± 0.019^b	0.373 ± 0.011^b
F值		178.346	137.560	136.241
P值		< 0.001	< 0.001	< 0.001

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Protective effect of salidroside on septic acute kidney injury in rats by inhibiting phosphatidylinositol 3 kinase/protein kinase B/mammalian target of rapamycin signal pathway

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Protective effect of salidroside on septic acute kidney injury in rats by inhibiting phosphatidylinositol 3 kinase/protein kinase B/mammalian target of rapamycin signal pathway