1.中南大学湘雅医院国际医疗部 长沙 410008
2.中南大学湘雅医院老年医学科
3.中南大学湘雅医院中西医结合科
秦钦,女,在读硕士生
#钟广伟,男,博士,副教授,硕士生导师,主要研究方向:中医药衰老理论及高血压防治,Email:zgw7512@sina.com
纸质出版日期:2023-12-30,
收稿日期:2023-05-22,
移动端阅览
秦钦, 时拥月, 姚佳梅, 等. 天麻钩藤饮对自发性高血压大鼠p38 MAPK/HSP27通路影响的研究[J]. 北京中医药大学学报, 2023,46(12):1694-1705.
QIN Qin, SHI Yongyue, YAO Jiamei, et al. Study on the effect of
秦钦, 时拥月, 姚佳梅, 等. 天麻钩藤饮对自发性高血压大鼠p38 MAPK/HSP27通路影响的研究[J]. 北京中医药大学学报, 2023,46(12):1694-1705. DOI: 10.3969/j.issn.1006-2157.2023.12.010.
QIN Qin, SHI Yongyue, YAO Jiamei, et al. Study on the effect of
目的
2
探讨p38丝裂原活化蛋白激酶(p38 MAPK)/热休克蛋白27(HSP27)通路在天麻钩藤饮干预自发性高血压大鼠(SHR)中的作用机制。
方法
2
将30只SHR按随机数字表法分为模型组、缬沙坦组(0.03 g/kg)和天麻钩藤饮组(1 g/kg),每组各10只,WKY大鼠10只为正常组,缬沙坦组和天麻钩藤饮组大鼠分别给予相应药物灌胃,正常组与模型组大鼠灌胃等量生理盐水,连续8周。测量第0、14、28、42、56天各组大鼠的血压;给药结束后,观察各组大鼠的行为学变化;采用苏木精-伊红染色法观察血管形态结构变化,天狼星红染色法观察血管纤维化程度;采用电子显微镜观察细胞自噬超微结构;采用免疫荧光法检测血管组织中磷酸化p38 MAPK(p-p38 MAPK)和磷酸化HSP27(p-HSP27)的平均荧光强度;采用分光光度法检测血浆丙二醛(MDA)、超氧化物歧化酶(SOD)和血管组织抗超氧阴离子的含量;采用β-半乳糖苷酶(SA-β-gal)染色法观察血管衰老改变;采用实时荧光PCR法检测血管组织中p38 MAPK、MAP激酶活化蛋白激酶-2(MAPKAPK-2)、HSP27、自噬相关蛋白5(ATG5)及p62 mRNA表达;采用蛋白质印迹法检测血管组织中p-p38 MAPK、磷酸化MAPKAPK-2(p-MAPKAPK-2)、p-HSP27、ATG5及p62蛋白表达。
结果
2
与正常组比较,模型组大鼠血压和易激惹程度升高,旋转耐受时间缩短;血管中膜厚度(MT)和MT/血管内径(LD)的值增加,自噬小体数量减少,SA-β-gal阳性细胞率升高,MDA含量升高、SOD含量和抗超氧阴离子含量降低;p-p38 MAPK平均荧光强度升高,p-p38 MAPK、p-MAPKAPK-2蛋白及p62 mRNA和蛋白表达升高,p-HSP27平均荧光强度减少,HSP27、ATG5 mRNA和p-HSP27、ATG5蛋白表达下降(均
P
<
0.05)。与模型组比较,缬沙坦组和天麻钩藤饮组大鼠血压和易激惹程度下降、旋转耐受时间延长;MT、MT/LD降低,自噬小体数量增多,SA-β-gal阳性细胞率减少,MDA含量降低、SOD含量和抗超氧阴离子含量升高;p-p38 MAPK平均荧光强度下降,p-p38 MAPK、p-MAPKAPK-2蛋白及p62 mRNA和蛋白表达降低,p-HSPA27平均荧光强度升高,HSP27、ATG5 mRNA和p-HSP27、ATG5蛋白表达升高(均
P
<
0.05)。
结论
2
天麻钩藤饮能够延缓SHR的血管衰老,其机制可能是通过p38 MAPK/HSP27通路调节细胞自噬实现的。
Objective
2
We aimed to investigate the mechanism of action of
Tianma Gouteng
Decoction in the treatment of spontaneously hypertensive rats(SHR) by studying the p38 mitogen-activated protein kinase (p38 MAPK)/heat shock protein 27 (HSP27) pathway.
Methods
2
Using the random number table method
30 SHR were divided into the model group
the Valsartan group (0.03 g/kg)
and the
Tianma Gouteng
Decoction group (1 g/kg)
10 rats per group. Additionally
10 Wistar-Kyoto (WKY) rats were assigned to the normal group. Rats in the Valsartan group and the
Tianma Gouteng
Decoction group were given corresponding drugs
while rats in the normal group and the model group were administered an equivalent amount of saline orally for 8 consecutive weeks. Blood pressure was recorded on days 0
14
28
42
and 56. Following treatment completion
behavioral changes were observed. Hematoxylin-eosin staining was used to observe the changes in vascular morphology and structure. Masson staining was used to observe the degree of vascular fibrosis. The autophagy ultrastrucure were examined by electron microscopy. An immunofluorescence assay was conducted and the average fluorescence intensities of phosphorylated p38 MAPK (p-p38 MAPK) and phosphorylated HSP27 (p-HSP27) in vascular tissue were calculated. Plasma malondialdehyde (MDA) content
plasma superoxide dismutase (SOD) content
and vascular tissue anti-superoxide anion content were measured by spectrophotometry. Vascular aging was evaluated through a senescence-associated β-galactosidase (SA-β-gal) staining method. The mRNA expression levels of p38 MAPK
MAPK-activated protein kinase-2 (MAPKAPK-2)
HSP27
autophagy-associated protein 5 (ATG5)
and p62 in vascular tissue were determined by real-time PCR. The protein expression levels of p-p38 MAPK
p-MAPKAPK-2
p-HSP27
ATG5
and p62 in vascular tissue were determined by Western blotting.
Results
2
Compared with the normal group
the blood pressure and irritability were increased
the rotational tolerance time was decreased
the thickness of vascular media (MT) and the ratio of vascular media thickness to vascular inner diameter (MT/LD) were significantly increased
the number of autophagosomes was decreased
the proportion of SA-β-gal positive cells was increased
the content of MDA was increased
the content of SOD and anti-superoxide anion were decreased
the average fluorescence intensity of p-p38 MAPK was increased
the protein expression levels of p-p38 MAPK and p-MAPKAPK-2 were increased
the mRNA and protein levels of p62 were increased
the average fluorescence intensity of p-HSP27 was decreased
and the mRNA expression level of HSP27 and the protein expression levels of p-HSP27
and the mRNA and protein levels of ATG5 were decreased in the model group (
P
<
0.05). Compared with the model group
the blood pressure and irritability were decreased
the rotational tolerance time was increased
MT and MT/LD were decreased
the number of autophagosomes was increased
the proportion of SA-β-gal positive cells was decreased
the content of MDA was decreased
the content of SOD and anti-superoxide anion were increased
the average fluorescence intensity of p-p38 MAPK was decreased
the protein levels of p-p38 MAPK and p-MAPKAPK-2 were decreased
the mRNA and protein levels of p62 were decreased
and the average fluorescence intensity of p-HSP27
the mRNA expression level of HSP27 and the protein expression levels of p-HSP27
and the mRNA and protein expression levels of ATG5 were increased in the Valsartan group and the
Tianma Gouteng
Decoction group (
P
<
0.05).
Conclusion
2
Tianma Gouteng
Decoction can delay vascular aging in a spontaneous hypertensive rat model
possibly by regulating autophagy through the p38 MAPK/HSP27 pathway.
天麻钩藤饮高血压p38丝裂原活化蛋白激酶/热休克蛋白27信号通路细胞自噬大鼠
Tianma Gouteng Decoctionhypertensionp38 mitogen-activated protein kinase/heat shock protein 27 pathwayautophagyrats
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