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1.北京中医药大学东直门医院 北京 100700
2.北京普仁医院
张轶斐,男,在读博士生
#刘伟敬,男,博士,研究员,主任医师,博士生导师,主要研究方向:中西医结合治疗肾脏病及内分泌疾病,E-mail:liuweijing-1977@hotmail.com.
纸质出版日期:2025-01-30,
网络出版日期:2024-12-27,
收稿日期:2024-09-23,
移动端阅览
张轶斐, 曹梓静, 张泽钰, 等. 益肾通络方调控TLR4/MyD88/NF-κB信号通路改善糖尿病肾病小鼠细胞焦亡的机制研究[J]. 北京中医药大学学报, 2025,48(1):21-33.
ZHANG YIFEI, CAO ZIJING, ZHANG ZEYU, et al. Mechanism of
张轶斐, 曹梓静, 张泽钰, 等. 益肾通络方调控TLR4/MyD88/NF-κB信号通路改善糖尿病肾病小鼠细胞焦亡的机制研究[J]. 北京中医药大学学报, 2025,48(1):21-33. DOI: 10.3969/j.issn.1006-2157.2025.01.004.
ZHANG YIFEI, CAO ZIJING, ZHANG ZEYU, et al. Mechanism of
目的
2
研究益肾通络方通过调控Toll样受体4(TLR4)/髓系分化初级反应蛋白质88(MyD88)/核转录因子-κB(NF-κB)信号通路,改善糖尿病肾病小鼠肾脏细胞焦亡的机制。
方法
2
采用随机数字表法将60只C57BL/6雄性小鼠分为对照组(10只)和干预组(50只),干预组采用链脲佐菌素(STZ)腹腔注射(50 mg/kg)构建糖尿病肾病小鼠模型。造模成功后,采用随机数字表法将干预组进一步分为模型组、司美格鲁肽组(40 μg/kg)和益肾通络方高(15.6 g/kg)、中(7.8 g/kg)、低(3.9 g/kg)剂量组。益肾通络方高、中、低剂量组灌胃相应剂量药物,司美格鲁肽组皮下注射司美格鲁肽注射液,对照组和模型组灌胃蒸馏水,连续12周。每4周监测各组小鼠随机血糖、生化法测定24 h尿蛋白含量;治疗后,采用生化法测定血清肌酐、尿素氮含量;称量小鼠肾脏质量,计算肾脏系数;苏木素-伊红、过碘酸-希夫、过碘酸六胺银和马松染色观察肾脏组织病理变化;酶联免疫吸附测定法检测尿液中β2微球蛋白(β2-MG)、中性粒细胞明胶酶相关脂质运载蛋白(NGAL)和肾损伤分子-1(KIM-1)水平;蛋白质印迹法和实时荧光PCR法检测肾脏组织中核苷酸结合结构域富含亮氨酸重复序列和含热蛋白结构域受体3(NLRP3)、胱天蛋白酶-1(Caspase-1)、焦孔素D(GSDMD)、白细胞介素-1β(IL-1β)和白细胞介素-18(IL-18)蛋白和mRNA表达;免疫组织化学法检测肾脏组织中TLR4、MyD88、NF-κB阳性染色面积占比。
结果
2
与对照组比较,模型组小鼠随机血糖,24 h尿蛋白,血清肌酐、尿素氮含量及肾脏系数均升高,尿液β2-MG、NGAL、KIM-1水平升高,肾组织NLRP3、Caspase-1、GSDMD、IL-1β和IL-18蛋白及mRNA表达升高,TLR4、MyD88、NF-κB蛋白阳性染色面积占比增多(
P
<
0.05),肾组织可见肾小球肥大等病理改变。与模型组比较,益肾通络方高剂量组治疗12周后随机血糖降低(
P
<
0.05);益肾通络方高、中剂量组小鼠24 h尿蛋白,血清肌酐、尿素氮含量及肾脏系数降低,尿液β2-MG、NGAL、KIM-1水平降低,肾组织NLRP3、Caspase-1、GSDMD、IL-1β和IL-18蛋白及mRNA表达降低,TLR4、MyD88、NF-κB蛋白染色面积占比减少(
P
<
0.05),肾组织病理损伤缓解。
结论
2
益肾通络方可能通过调控TLR4/MyD88/NF-κB信号通路,抑制肾脏细胞焦亡,从而减轻糖尿病肾病小鼠的肾小管间质损伤,发挥肾保护作用。
Objective
2
To investigate the mechanism of
Yishen Tongluo
Formula in ameliorating renal pyroptosis in diabetic nephropathy mice by regulating the toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway.
Methods
2
Sixty C57BL/6 male mice were randomly divided into control (10 mice) and intervention groups (50 mice) using random number table method. The diabetes nephropathy model was established by intraperitoneally injecting streptozotocin(50 mg/kg). After modeling
the intervention group was further divided into model
semaglutide (40 μg/kg)
and high-
medium-
and low-dose
Yishen Tongluo
Formula groups (15.6
7.8
and 3.9 g/kg
respectively) using random number table method. The high-
medium-
and low-dose
Yishen Tongluo
Formula groups were administered corresponding doses of medication by gavage
the semaglutide group received a subcutaneous injection of sema
glutide injection
and the control group and model groups were administered distilled water by gavage for 12 consecutive weeks. Random blood glucose levels of mice in each group were monitored
and the 24-h urinary protein content was measured using biochemical method every 4 weeks; after treatment
the serum creatinine and urea nitrogen levels were measured using biochemical method. The weight of the kidneys was measured
and the renal index was calculated. Hematoxylin and eosin
periodic acid-Schiff
periodic Schiff-methenamine
and Masson staining were used to observe the pathological changes in renal tissue. An enzyme-linked immunosorbent assay was used to detect urinary β2-microglobulin (β2-MG)
neutrophil gelatinase-associated lipocalin (NGAL)
and kidney injury molecule-1 (KIM-1) levels. Western blotting and real-time fluorescence PCR were used to detect the relative protein and mRNA expression levels of nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 (NLRP3)
Caspase-1
gasdermin D (GSDMD)
interleukin-1β (IL-1β)
and interleukin-18 (IL-18) in renal tissue. Immunohistochemistry was used to detect the proportion of protein staining area of the TLR4
MyD88
and NF-κB in renal tissue.
Results
2
Compared with the control group
the random blood glucose
24-h urinary protein
serum creatinine
urea nitrogen
and renal index of the model group increased
and the urine β2-MG
NGAL
and KIM-1 levels increased. The relative protein and mRNA expression levels of NLRP3
Caspase-1
GSDMD
IL-1β
and IL-18 in renal tissue increased
and the proportion of TLR4
MyD88
and NF-κB protein positive staining areas increased (
P
<
0.05). Pathological changes such as glomerular hypertrophy were observed in the renal tissue of the model group. Compared with the model group
the
Yishen Tongluo
Formula high-dose group showed a decrease in random blood glucose after 12 weeks of treatment (
P
<
0.05). The
Yishen Tongluo
Formula high- and medium-dose groups showed a decrease in 24-h urinary protein
creatinine
urea nitrogen
and renal index
as well as decreased β2-MG
NGAL
and KIM-1 levels. NLRP3
Caspase-1
GSDMD
IL-1 β
and IL-18 relative protein and mRNA expression levels were also reduced
and the proportion of TLR4
MyD88
and NF-κB protein positive staining areas was reduced (
P
<
0.05). Pathological damage to renal tissue was ameliorated.
Conclusion
2
Yishen Tongluo
Formula may exert protective renal effects by inhibiting renal pyroptosis and alleviating tubular interstitial injury in diabetic nephropathy mice by regulating the TLR4/MyD88/NF-κB signaling pathway.
糖尿病肾病益肾通络方细胞焦亡Toll样受体4/髓样分化因子88/核转录因子-κB信号通路小鼠
diabetes nephropathyYishen Tongluo Formulapyroptosistoll-like receptor 4/myeloid differentiation primary response protein 88/nuclear factor-κB signaling pathwaymice
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