基于lncRNA /Hedgehog信号通路表达探讨骨关节炎中自噬对软骨细胞凋亡的影响

朱毅琳; 彭潇; 张贵福; 龙惠南

doi:10.12259/j.issn.2095-610X.S20250605

基于lncRNA /Hedgehog信号通路表达探讨骨关节炎中自噬对软骨细胞凋亡的影响

doi: 10.12259/j.issn.2095-610X.S20250605

1.
长沙市中医医院/长沙市第八医院骨伤科，湖南长沙　410000
2.
广州市荔湾区骨伤科医院，广东广州　510000

基金项目: 湖南省中医药管理局科研项目（C2024032）；长沙市中医药科研课题（B202301）

详细信息

作者简介:
朱毅琳（1981～），男，广东茂名人，医学学士，副主任医师，主要从事脊柱骨折及相关疾病，退行性骨关节病，创伤骨科研究工作

通讯作者:
朱毅琳，E-mail：fifafifa765@163.com

中图分类号: R684
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出版历程
- 收稿日期: 2024-11-29
- 刊出日期: 2025-06-25

Effects of Autophagy on Chondrocyte Apoptosis in Osteoarthritis: An Investigation Based on lncRNA/Hedgehog Signaling Pathway Expression

1.
Orthopedics Department, Changsha Traditional Chinese Medicine Hospital /Changsha Eighth Hospital，Changsha Hunan 410000
2.
Orthopedics Department Hospital of Liwan District，Guangzhou Guangdong 510000，China

摘要

摘要: 目的探究骨关节炎中lncRNA /Hedgehog信号通路介导的自噬对软骨细胞功能的影响。方法在OA软骨细胞（SW1353）建立LPS诱导的软骨细胞炎症模型，并通过胶原II免疫荧光染色和甲苯胺蓝染色进行鉴定，分为Normal组、LPS组、LPS/lncRNA HHIP-AS1 抑制剂组和LPS/Scr组，RT-qPCR检测lncRNA HHIP- as1和HHIP在软骨细胞中的表达，蛋白印迹HHIP、Gli1、Gli2、LC3B-I ab、p62表达，TUNEL染色和流式细胞术（FC）检测细胞凋亡，免疫荧光法（IFA）检测自噬LC3B表达。结果用LPS处理SW1细胞，与正常软骨细胞相比，LPS诱导后，软骨细胞体积增大，胞浆内空泡增多，细胞核体积增大，部分细胞形态不规则，数量相对减少，甲苯胺蓝染色和II型胶原免疫组化染色减少，LPS刺激会诱导细胞死亡和自噬，lncRNA HHIP- as1和HHIP上调（P < 0.05），Hedgehog信号通路HHIP、Gli1和Gli2的关键分子持续上调（P < 0.05），LPS处理的软骨细胞有明显的凋亡（P < 0.05），LC3B（绿色）积累，LC3B生物合成和加工升高（LC3B I和II水平升高），p62降解升高（P < 0.05），lncRNA HHIP-AS1抑制剂降低LPS诱导的OA软骨细胞凋亡（细胞凋亡率降低）和自噬（降低了LPS处理的软骨细胞的自噬率，LC3B生物合成和加工减少（LC3B I和II水平降低），p62降解减弱），差异有统计学意义（P < 0.05）。结论 LncRNA HHIP-AS1可能通过调控Hedgehog信号通路抑制LPS诱导的OA软骨细胞凋亡和自噬。
- 骨关节炎 /
- lncRNA HHIP-AS1 /
- Hedgehog /
- 自噬 /
- 软骨细胞
Abstract: Objective To investigate the effects of lncRNA/Hedgehog signaling pathway-mediated autophagy on chondrocyte function in osteoarthritis （OA）. Methods Established an LPS-induced inflammatory chondrocyte model in OA chondrocytes （SW1353）, and identified it through collagen II immunofluorescence staining and toluidine blue staining, dividing the groups into Normal, LPS, LPS/lncRNA HHIP-AS1 inhibitor, and LPS/Scr groups. RT-qPCR was used to detect lncRNA HHIP-AS1 and HHIP expression in chondrocytes, Western blot was used to assess HHIP, Gli1, Gli2, LC3B-I/II, and p62 protein expression, TUNEL staining and flow cytometry （FC） were used to detect cell apoptosis, and immunofluorescence assay （IFA） was used to detect autophagy LC3B expression. Results When SW1 cells were treated with LPS, compared with normal chondrocytes, after LPS induction, the volume of chondrocytes increased, the number of vacuoli in the cytoplasm increased, the volume of the nucleus increased, the morphology of some cells was irregular, and the number relatively decreased. Toluidine blue staining and type II collagen immunohistochemical staining decreased. LPS stimulation would induce cell death and autophagy. lncRNA HIP-AS1 and HHIP were upregulated （P < 0.05）, the key molecules of the Hedgehog signaling pathway HHIP, Gli1 and Gli2 were continuously upregulated （P < 0.05）, chondrocytes treated with LPS showed obvious apoptosis （P < 0.05）, and LC3B （green） accumulated. The biosynthesis and processing of LC3B increased （the levels of LC3B I and II increased）, the degradation of p62 increased （P < 0.05）, and the lncRNA HIP-AS1 inhibitor reduced LPS-induced apoptosis of OA chondrocytes （decreased apoptosis rate） and autophagy （decreased autophagy rate of chondrocytes treated with LPS）. The biosynthesis and processing of LC3B decreased （the levels of LC3B I and II decreased）, and the degradation of p62 weakened）, and the difference was statistically significant （P < 0.05）. Conclusion The lncRNA HHIP-AS1 may inhibit LPS-induced OA chondrocyte apoptosis and autophagy by regulating the Hedgehog signaling pathway.
- Osteoarthritis /
- lncRNA HIP-AS1 /
- Hedgehog /
- Autophagy /
- Chondrocyte

HTML全文

图 1 lncRNA HHIP-AS1和HHIP在LPS诱导的小鼠OA软骨细胞中表达

A：倒置显微镜下观察细胞形态（200×）；B：甲苯胺蓝染色观察细胞形态（100×）；C：免疫细胞化学检测骨关节软骨细胞标志物胶原蛋白 II的表达（200×）；D～E：lncRNA HHIP-AS1和HHIP表达；与Normal组比较，^**P < 0.01；^***P < 0.001。

Figure 1. Expression of lncRNA HHIP-AS1 and HHIP in LPS-induced mouse OA chondrocytes

下载: 全尺寸图片幻灯片

图 2 lncRNA HHIP-AS1对Hedgehog信号通路的影响

A：蛋白质印迹检测Hedgehog信号通路；B：蛋白印迹计量分析Hedgehog表达；C：蛋白印迹计量分析Gli1表达；D：蛋白印迹计量分析Gli2表达；与Normal组比较，^***P < 0.001；与LPS组比较，^###P < 0.001。

Figure 2. Effects of lncRNA HHIP-AS1 on the Hedgehog signaling pathway

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图 3 lncRNA HHIP-AS1/Hedgehog信号通路对LPS诱导的OA软骨细胞凋亡的影响

A：TUNEL染色检测LPS处理的软骨细胞死细胞数量（×100）；B：使用FC来评估死亡细胞数量；每个图的右上象限表示早期凋亡细胞。

Figure 3. Impact of lncRNA HHIP-AS1/Hedgehog signaling pathway on apoptosis of LPS-induced OA chondrocytes

下载: 全尺寸图片幻灯片

图 4 lncRNA HHIP-AS1/Hedgehog信号通路对LPS诱导的OA软骨细胞自噬的影响

A：通过IFA（×400）评估GFP-LC3B的细胞位置；B：蛋白质印迹检测自噬表达；C：蛋白质印迹计量分析LC3B表达；D：蛋白质印迹计量分析LC3B表达；与Normal组比较，^***P < 0.05；与LPS组比较，^###P < 0.05。

Figure 4. Effects of lncRNA HHIP-AS1/Hedgehog signaling pathway on autophagy of LPS-induced OA chondrocytes

下载: 全尺寸图片幻灯片

表 1 lncRNA HHIP-AS1和HHIP在LPS诱导的小鼠OA软骨细胞中表达（$\bar x \pm s $）

Table 1. Expression of lncRNA HHIP-AS1 and HHIP in LPS-induced mouse OA chondrocytes （$\bar x \pm s $）

分组	lncRNA HHIP-AS1	HHIP mRNA
Normal组（n=9）	1.070±0.370	0.890±0.320
LPS组（n=9）	2.650±0.360	2.250±0.390
t	9.180	8.080
P	<0.001^*	<0.001^*
^*P < 0.05。

下载: 导出CSV

表 2 lncRNA HHIP-AS1对Hedgehog信号通路的影响（$\bar x \pm s $）

Table 2. Effects of lncRNA HHIP-AS1 on the Hedgehog signaling pathway（$\bar x \pm s $）

分组	HHIP/GAPDH	Gli1/GAPDH	Gli2/GAPDH
Normal组（n=9）	0.530±0.170	0.390±0.120	0.490±0.110
LPS组（n=9）	1.350±0.260^Δ	1.010±0.140^Δ	1.140±0.130^Δ
LPS/HHIP-AS1 inhibitor（n=9）	0.270±0.050	0.400±0.100	0.620±0.150
LPS/Scr（n=9）	1.110±0.160^#	1.000±0.150^#	1.060±0.190^#
F	98.520	45.140	32.75
P	<0.001^*	<0.001^*	<0.001^*
^*P < 0.05；与Normal组比较，^ΔP < 0.05；与LPS组比较，^#P < 0.05。

下载: 导出CSV

表 3 lncRNA HHIP-AS1/Hedgehog信号通路对LPS诱导的OA软骨细胞凋亡的影响（$\bar x \pm s $）

Table 3. Impact of lncRNA HHIP-AS1/Hedgehog signaling pathway on apoptosis of LPS-induced OA chondrocytes （$\bar x \pm s $）

分组	TUNEL染色阳性细胞（%）	凋亡率（%）
Normal组（n=9）	5.080±1.010	7.450±1.110
LPS组（n=9）	19.120±1.960^Δ	22.210±2.120^Δ
LPS/HHIP-AS1 inhibitor（n=9）	4.210±1.040	6.410±2.010
LPS/Scr（n=9）	20.130±1.660^#	23.340±2.110^#
F	289.120	245.090
P	<0.001^*	<0.001^*
与Normal组比较，^ΔP < 0.05；与LPS组比较，^#P < 0.05；^*P < 0.05。

下载: 导出CSV

表 4 lncRNA HHIP-AS1/Hedgehog信号通路对LPS诱导的OA软骨细胞自噬的影响（$\bar x \pm s $）

Table 4. Effects of lncRNA HHIP-AS1/Hedgehog signaling pathway on autophagy of LPS-induced OA chondrocytes （$\bar x \pm s $）

分组	LC3B/GAPDH	p62/GAPDH
Normal组（n=9）	0.380±0.090	1.440±0.150
LPS组（n=9）	1.320±0.110^Δ	0.310±0.120^Δ
LPS/HHIP-AS1 inhibitor（n=9）	0.510±0.130	1.410±0.110
LPS/Scr（n=9）	1.430±0.160^#	0.390±0.140^#
F	294.83	198.48
P	0.001^*	0.001^*
与Normal组比较，^ΔP < 0.05；与LPS组比较，^#P < 0.05；^*P < 0.05。

下载: 导出CSV

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