毛蕊异黄酮对脊髓损伤大鼠神经元自噬及凋亡的影响

doi:10.11958/20231623

天津医药 ›› 2024, Vol. 52 ›› Issue (8): 798-803.doi: 10.11958/20231623

毛蕊异黄酮对脊髓损伤大鼠神经元自噬及凋亡的影响

李大强¹(), 李坚¹, 陆哲明², 曹阳¹^,^△()

1 锦州医科大学附属第一医院骨科（邮编121000）
2 普外科

收稿日期:2023-11-06 修回日期:2024-01-25 出版日期:2024-08-15 发布日期:2024-08-16
通讯作者: △E-mail：cyclcmh@sina.com
作者简介:李大强（1990），男，硕士在读，主要从事脊髓损伤基础方面研究。E-mail：lnyxylidaqiang@163.com
基金资助:
辽宁省教育厅科学研究面上项目(LJKZ0815)

Effects of calycosin on neuronal autophagy and apoptosis in rats with spinal cord injury

LI Daqiang¹(), LI Jian¹, LU Zheming², CAO Yang¹^,^△()

1 Department of Orthopedics
2 Department of General Surgery, the First Affiliated Hospital of Jinzhou Medical University, Jinzhou 121000, China

Received:2023-11-06 Revised:2024-01-25 Published:2024-08-15 Online:2024-08-16
Contact: △E-mail：cyclcmh@sina.com

李大强, 李坚, 陆哲明, 曹阳. 毛蕊异黄酮对脊髓损伤大鼠神经元自噬及凋亡的影响[J]. 天津医药, 2024, 52(8): 798-803.

LI Daqiang, LI Jian, LU Zheming, CAO Yang. Effects of calycosin on neuronal autophagy and apoptosis in rats with spinal cord injury[J]. Tianjin Medical Journal, 2024, 52(8): 798-803.

摘要/Abstract

摘要：

目的探讨毛蕊异黄酮（CAL）调控PI3K/Akt信号通路对脊髓损伤（SCI）大鼠神经元自噬及凋亡的影响及其作用机制。方法 32只成年SD大鼠随机分为假手术组（Sham组），模型组（SCI组）和CAL低、高剂量组，每组8只。采用改良Allen’s法建立大鼠中度SCI模型，术后CAL低、高剂量组立即分别予以20、40 mg/kg CAL腹腔注射，1次/d，连续7 d；Sham组和SCI组给予等量生理盐水。术后1、3和7 d，采用行为学评分（BBB）评估大鼠运动功能的恢复情况；术后7 d，尼氏染色检测大鼠脊髓前角运动神经元存活数量，Western blot检测p62、Beclin-1、微管相关蛋白1轻链3（LC3B）、磷脂酰肌醇-3激酶（PI3K）/蛋白激酶B（Akt）和活化型胱天蛋白酶-3（Cleaved-Caspase-3）、B细胞淋巴瘤-2（Bcl-2）、Bcl-2关联X蛋白（Bax）表达，免疫荧光染色检测大鼠脊髓前角神经元LC3B的表达。结果与Sham组相比，SCI组大鼠BBB评分、神经元存活数量、Bcl-2、Bcl-2/Bax、p-PI3K、p-PI3K/PI3K、p-Akt、p-Akt/Akt水平降低，p62、Beclin-1、LC3BⅡ、LC3BⅡ/Ⅰ、Cleaved-Caspase-3、Bax水平升高（P<0.05）；与SCI组相比，CAL低、高剂量组BBB评分、神经元存活数量、LC3BⅡ、LC3BⅡ/Ⅰ、Bcl-2、Bcl-2/Bax、p-PI3K、p-PI3K/PI3K、p-Akt、p-Akt/Akt水平升高，p62、Cleaved-Caspase-3、Bax水平降低（P<0.05）。结论 CAL可通过激活PI3K/Akt信号通路促进SCI大鼠神经元自噬，抑制其凋亡，进而发挥神经保护作用。

关键词: 毛蕊异黄酮, 脊髓损伤, 自噬, PI3K/Akt信号通路

Abstract:

Objective To explore the effect of calycosin (CAL) on neuronal autophagy and apoptosis in rats with spinal cord injury (SCI) by regulating PI3K/Akt signaling pathway and its mechanism. Methods A total of 32 male or female adult SD rats were randomly divided into the sham group, the SCI group, the CAL low (20 mg/kg) dose group and the CAL high (40 mg/kg) dose group with 8 rats in each group. The rat model of moderate SCI was established by modified Allen’s method. After successful modeling, rats were injected intraperitoneally immediately with different dosage of CAL or equal amount of saline once a day for 7 consecutive days. Basso, Beattie and Bresnahan (BBB) scores were used to evaluate the recovery of motor function of rats at 1, 3 and 7 d after surgery. At 7 d after surgery, Nissl staining was used to detect the surviving number of motor neurons in anterior horn of spinal cord. Western blot assay was used to assess expression levels of p62, Beclin-1, microtubule-associated protein 1 light chain 3 (LC3B), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway， Cleaved-Caspase-3, B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax) proteins. Immunofluorescence staining was used to measure the expression of LC3B in anterior neurons of spinal cord. Results Compared with the sham group, BBB scores, the surviving number of motor neurons and levels of Bcl-2, Bcl-2/Bax, p-PI3K, p-PI3K/PI3K, p-Akt and p-Akt/Akt were significantly decreased (P<0.05), and levels of p62, Beclin-1, LC3B Ⅱ, LC3B Ⅱ/Ⅰ, Cleaved-Caspase-3 and Bax were significantly increased in the SCI group (P<0.05). Compared with the SCI group, BBB scores, the survival of anterior horn motor neurons and levels of LC3B Ⅱ, LC3B Ⅱ/Ⅰ, Bcl-2, Bcl-2/Bax, p-PI3K, p-PI3K/PI3K, p-Akt and p-Akt/Akt were increased in the CAL low dose group and CAL high dose group, and levels of p62, Cleaved-Caspase-3 and Baxcould were significantly decreased (P<0.05). Conclusion CAL could promote autophagy and inhibit apoptosis of neurons through activating PI3K/Akt signaling pathway, thereby conferring a protective role following SCI in rats.

Key words: calycosin, spinal cord injury, autophagy, PI3K/Akt signaling pathway

中图分类号:

R651.2，R285.6

图/表 9

表1 各组大鼠SCI后BBB评分比较（分，$\bar{x} \pm s$）

Tab.1 Comparison of neurological function scores after SCI between the four groups of rats

组别	n	1 d	3 d	7 d
Sham组	6	21.00±0.00	21.00±0.00	21.00±0.00
SCI组	6	0.44±0.18^a	1.83±0.75^a	3.94±0.77^a
CAL低剂量组	6	0.56±0.27^a	2.89±0.66^b	6.17±1.05^b
CAL高剂量组	6	0.67±0.42^a	3.95±0.88^b	8.61±1.18^bc
F		0.778	11.372^＊＊	31.740^＊＊

图1 各组脊髓前角组织尼氏染色（×200）

Fig.1 Nissl staining for anterior horn of spinal cord in each group (×200)

表2 各组大鼠SCI后p62、Beclin-1和LC3B蛋白表达的比较（$\bar{x} \pm s$）

Tab.2 Comparison of p62, Beclin-1 and LC3B protein expression levels after SCI between the four groups of rats

组别	n	p62/ β-actin	Beclin-1/ β-actin	LC3BⅡ/Ⅰ	LC3BⅡ/ β-actin
Sham组	4	0.49±0.10	0.28±0.05	0.49±0.05	0.47±0.07
SCI组	4	1.03±0.11^a	0.54±0.09^a	0.81±0.08^a	0.87±0.10^a
CAL低剂量组	4	0.70±0.09^b	0.63±0.05	1.06±0.06^b	1.10±0.10^b
CAL高剂量组	4	0.39±0.11^bc	0.94±0.11^bc	1.26±0.03^bc	1.30±0.15^bc
F		30.722^＊＊	47.307^＊＊	148.901^＊＊	42.684^＊＊

图2 Western blot检测各组大鼠SCI后脊髓组织p62、Beclin-1和LC3B蛋白表达 A：Sham组；B：SCI组；C：CAL低剂量组；D：CAL高剂量组。

Fig.2 Expression levels of p62, Beclin-1 and LC3B following SCI in each group detected by Western blot assay

图3 各组脊髓前角组织免疫荧光染色（×100）

Fig.3 Immunofluorescent staining of anterior horn of spinal cord in each group (×100)

表3 各组大鼠SCI后Bax、Cleaved-Caspase-3 和 Bcl-2蛋白表达的比较（n=4，$\bar{x} \pm s$）

Tab.3 Comparison of Bax, Cleaved-Caspase-3 and Bcl-2 protein expression levels between the four groups of rats

组别	Bax/ β-actin	Cleaved-Caspase-3/ β-actin	Bcl-2/ β-actin	Bcl-2/ Bax
Sham组	0.43±0.07	0.53±0.08	0.91±0.15	2.14±0.38
SCI组	0.92±0.08^a	0.85±0.14^a	0.49±0.06^a	0.54±0.09^a
CAL低剂量组	0.67±0.15^b	0.61±0.05^b	0.74±0.10^b	1.24±0.34^b
CAL高剂量组	0.46±0.10^bc	0.47±0.03^b	1.06±0.10^bc	2.25±0.36^bc
F	19.059^＊＊	16.447^＊＊	19.748^＊＊	26.671^＊＊

图4 Western blot检测各组大鼠SCI后脊髓组织Bax、Cleaved-Ccaspase-3和Bcl-2蛋白表达 A：Sham组；B：SCI组；C：CAL低剂量组；D：CAL高剂量组。

Fig.4 Expression levels of Bax, Cleaved-Caspase-3 and Bcl-2 following SCI in each group detected by Western blot assay

表4 各组大鼠SCI后p-PI3K和p-Akt蛋白表达的比较（n=4，$\bar{x} \pm s$）

Tab.4 Comparison of p-PI3K and p-Akt protein expression levels between the four groups of rats

组别	p-PI3K/PI3K	p-PI3K/β-actin	p-Akt/Akt	p-Akt/β-actin
Sham组	0.87±0.10	0.80±0.10	0.81±0.11	0.70±0.11
SCI组	0.69±0.12^a	0.59±0.11^a	0.63±0.15^a	0.57±0.14^a
CAL低剂量组	0.90±0.15^b	0.93±0.16^b	0.89±0.08^b	0.78±0.18^b
CAL高剂量组	0.98±0.11^b	1.06±0.20^b	0.96±0.17^b	0.95±0.20^b
F	4.220^＊	7.210^＊＊	4.534^＊	3.978^＊

图5 Western blot检测各组大鼠SCI后脊髓组织p-PI3K和p-Akt蛋白表达 A：Sham组；B：SCI组；C：CAL低剂量组；D：CAL高剂量组。

Fig.5 Expression levels of p-PI3K and p-Akt following SCI in each group detected by Western blot assay

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毛蕊异黄酮对脊髓损伤大鼠神经元自噬及凋亡的影响

Effects of calycosin on neuronal autophagy and apoptosis in rats with spinal cord injury

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