根皮素对2型糖尿病肾病小鼠的肾保护作用及机制

王兴红; 马永超; 孙缦利; 李超敏; 李海霞

doi:10.13386/j.issn1002-0306.2022120211

根皮素对2型糖尿病肾病小鼠的肾保护作用及机制

doi: 10.13386/j.issn1002-0306.2022120211

王兴红^{1, 2,},
马永超^{1, 2, ,},
孙缦利^{1, 2},
李超敏^{1, 2},
李海霞³

1.
漯河医学高等专科学校，河南漯河 462000
2.
河南省特医食品工程技术研究中心，河南漯河 462000
3.
新疆医科大学第一附属医院，新疆乌鲁木齐 830054

基金项目: 河南省高等学校重点科研项目（20B310007，22B550010）；河南省重点研发与推广专项（科技攻关）项目（232102310501）；河南省科技发展计划项目（科技攻关）（1721023610312）；河南省高等学校青年骨干教师培养计划资助项目（2018GGJS258）

详细信息

作者简介:
王兴红（1981−），女，硕士，副教授，研究方向：糖尿病肾病发病机制与防治，E-mail：xinghong0124@163.com

通讯作者:
马永超（1976−），男，博士，副教授，研究方向：炎症与肿瘤发生，E-mail：13839527381@163.com

中图分类号: TS201.4
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出版历程
- 收稿日期: 2022-12-28
- 网络出版日期: 2023-04-05
- 刊出日期: 2023-06-01

Renal-protection Effect and the Potential Mechanism of Phloretin in Mice with Type-2 Diabetic Nephropathy

WANG Xinghong^{1, 2
,},
MA Yongchao^{1, 2
, ,},
SUN Manli^{1, 2},
LI Chaomin^{1, 2},
LI Haixia³

1.
Luohe Medical College, Luohe 462000, China
2.
Henan Special Medical Food Engineering Technology Research Center, Luohe 462000, China
3.
The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, China

摘要

摘要: 为探究根皮素对2型糖尿病肾病小鼠肾损伤的分子保护作用及潜在机制，将小鼠随机分为正常对照组、糖尿病模型组、阳性药物组（二甲双胍500 mg/kg）、根皮素低、中、高剂量组（100、200、400 mg/kg·d）。造模成功后，各组小鼠灌胃给予相应药物，12周后，观察肾脏肥大指数、肾脏病理形态学变化，测定血尿素氮（BUN）、肌酐（Scr）和尿β2-微球蛋白（β2-MG）、血清白细胞介素-1β（IL-1β）和白细胞介素-18（IL-18）等指标，并检测肾组织丙二醛（MDA）含量、谷胱甘肽过氧化物酶（GSH-Px）活性和转录因子E2相关因子2（Nrf2）/血红素加氧酶-1（HO-1）/核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）通路相关蛋白表达。结果显示：与模型组比较，根皮素中、高剂量组小鼠肾脏肥大指数极显著减小（P<0.01）；HE染色和Masson染色见肾小球体积明显减小、系膜增生明显减少，肾小球和肾间质纤维化明显减轻；血BUN、Scr、IL-1β、IL-18和肾组织MDA含量、α-平滑肌肌动蛋白（α-SMA）、胶原蛋白Ι（Collagen Ι）、NLRP3、IL-1β和Gasdermin D（GSDMD）蛋白表达显著降低（P<0.05或P<0.01）；肾组织GSH-Px活性、Nrf2、HO-1、钙粘附蛋白-E（E-cadherin）蛋白表达显著升高（P<0.05）。结果表明：根皮素能保护2型糖尿病肾病小鼠肾功能改善肾纤维化，可能与调控Nrf2/HO-1/NLRP3通路改善氧化应激与炎症反应有关。
- 糖尿病肾病 /
- 根皮素 /
- 转录因子E2相关因子2/血红素加氧酶-1/核苷酸结合寡聚化结构域样受体蛋白3通路 /
- 氧化应激 /
- 炎症
Abstract: To study the protective effect of phloretin against renal injury in mice with type-2 diabetic nephropathy and determine its potential mechanism at the molecular level, mice were randomized into a normal control group, diabetes model group, positive drug group (metformin, 500 mg/kg), and low-, medium-, and high-dose phloretin groups (100, 200, and 400 mg/kg·d, respectively). After successful modeling, mice in each group were administered the corresponding drugs via the intragastric route. Kidney hypertrophy index and renal pathomorphological changes were observed after 12 weeks. Blood urea nitrogen (BUN), serum creatinine (Scr), urine β2-microglobulin, serum interleukin (IL)-1β and IL-18 levels were determined. Malondialdehyde (MDA) level, glutathione peroxidase (GSH-Px) activity, and nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)/nucleotide binding oligomerization domain-like receptor protein 3 (NLRP3) pathway-related protein expression in renal tissues were also determined. Compared with the model group, the medium- and high-dose phloretin groups showed a significant decrease in the kidney hypertrophy index of mice (P<0.01). An obvious reduction in glomerular volume was determined using Hematoxylin and Eosin staining and Masson staining, and a significant decrease in mesangial proliferation and an apparent alleviation in glomerular and renal interstitial fibrosis were noted. Significant reductions in BUN, Scr, IL-1β, and IL-18 levels in the blood, in MDA levels in renal tissues, and in the protein expression of α-smooth muscle actin, Collagen I, NLRP3, IL-1β, and Gasdermin D were noted (P<0.05 or P<0.01). A significant increase in GSH-Px activity and in the expression of Nrf2, HO-1, and E-cadherin in renal tissues (P<0.05) was also founded. The results indicated that phloretin could protect renal function and alleviate renal fibrosis in mice with type-2 diabetic nephropathy. Its mechanism may be related to the regulation of the Nrf2/HO-1/NLRP3 pathway to improve oxidative stress and alleviate the inflammatory response.
- diabetic nephropathy /
- phloretin /
- Nrf2/HO-1/NLRP3 pathway /
- oxidative stress /
- inflammation

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图 1 根皮素对各组小鼠肾脏病理学的影响（HE染色，200×）

Figure 1. Effect of phloretin on renal pathology in mouse (HE staining, 200×)

注：A：正常对照组；B：糖尿病模型组；C：二甲双胍组；D、E、F：根皮素低、中、高剂量组；图2~图7同。

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图 2 根皮素对各组小鼠肾脏病理学改变的影响（Masson染色，400×）

Figure 2. Effect of phloretin on pathological changes of kidney in mouse (Masson stain, 400×)

注：与正常对照组比较：^▲P<0.05，^▲▲P<0.01；与糖尿病模型组比较：^*P<0.05，^**P<0.01；图3~图7，表1~表2同。

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图 3 根皮素对各组小鼠肾脏α-SMA蛋白表达的影响（免疫组化SP法，400×）

Figure 3. Effect of phloretin on α- SMA protein expression in mouse kidney (immunohistochemical SP method, 400×)

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图 4 根皮素对各组小鼠肾脏Collagen Ι蛋白表达的影响（免疫组化SP法，400×）

Figure 4. Effect of phloretin on Collagen Ι protein expression in mouse kidney (immunohistochemical SP method, 400×)

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图 5 根皮素对各组小鼠肾脏E-cadherin蛋白表达的影响（免疫组化SP法，400×）

Figure 5. Effect of phloretin on E-cadherin protein expression in mouse kidney (immunohistochemical SP method, 400×)

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图 6 根皮素对各组小鼠肾脏Nrf2、HO-1蛋白表达的影响

Figure 6. Effect of phloretin on Nrf2 and HO-1 protein expression in mouse kidney

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图 7 根皮素对各组小鼠肾脏NLRP3、IL-1β和GSDMD蛋白表达的影响

Figure 7. Effect of phloretin on NLRP3, IL-1β and GSDMD protein expression in mouse kidney

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表 1 根皮素对各组小鼠MDA、GSH-Px、BUN和Scr含量的影响

Table 1. Effect of phloretin on MDA, GSH-Px, BUN and Scr content of mice in each group

组别	剂量（mg/kg）	MDA（μmol/L）	GSH-Px（U/mL）	BUN（mmol/L）	Scr（μmol/L）
正常对照组组	−	11.13±1.86	175.88±19.89	6.99±0.56	42.2±4.0
糖尿病模型组	−	21.21±4.03^▲▲	138.13±12.12^▲▲	16.23±1.42^▲	61.7±7.6^▲
二甲双胍组	500	14.87±2.43^**	151.22±12.78^*	11.87±0.87^**	51.8±5.1^**
根皮素低剂量组	100	20.71±2.86	139.32±13.76	15.34±1.02	60.0±7.1
根皮素中剂量组	200	14.91±2.66^**	159.31±14.89^*	11.04±0.99^**	50.9±5.5^**
根皮素高剂量组	400	13.88±2.01^**	167.88±16.99^*	10.23±0.98^**	49.8±4.9^**

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表 2 根皮素对各组小鼠血清IL-1β、IL-18、肾肥大指数和尿β2-MG含量的影响

Table 2. Effect of phloretin on serum IL-1β, IL-18, renal hypertrophy index and urine β2-MG content of mice in each group

组别	剂量（mg/kg）	IL-1β（pg/mL）	IL-18（ng/mL）	肾脏肥大指数（×10⁻³）	β2-MG（mg/24 h）
正常对照组	−	2.24±0.14	35.13±2.01	2.6±0.5	15.67±2.25
糖尿病模型组	−	5.67±0.24^▲	98.77±6.14^▲	6.7±0.6^▲	42.46±8.67^▲
二甲双胍组	500	3.36±0.14^**	52.718±4.89^**	3.8±0.1^**	24.11±7.34^**
根皮素低剂量组	100	5.54±0.25	91.67±6.57	6.1±0.5	44.21±8.35
根皮素中剂量组	200	3.34±0.15^**	52.718±4.87^**	3.8±0.3^**	24.87±7.75^**
根皮素高剂量组	400	3.21±0.12^**	49.49±4.77^**	3.7±0.2^**	23.23±6.99^**

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