文献：A thermosensitive, reactive oxygen species-responsive, MR409-encapsulated hydrogel ameliorates disc degeneration in rats by inhibiting the secretory autophagy pathway

文献链接：

作者：Qiangqiang Zheng, Haotian Shen, Zongrui Tong, Linxiang Cheng, Yuzi Xu, Zhiyun Feng, Shiyao Liao, Xiaojian Hu, Zongyou Pan, Zhengwei Mao and Yue Wang

相关产品：PLGA-PEG-PLGA（聚乳酸-羟基乙酸共聚物-聚乙二醇-聚乳酸-羟基乙酸共聚物）

原文摘要：

Lumbar disc degeneration is a common cause of chronic low back pain and an important contributor to various degenerative lumbar spinal disorders. However, currently there is currently no effective therapeutic strategy for treating disc degeneration. The pro-inflammatory cytokine interleukin-1β (IL-1β) mediates disc degeneration by inducing apoptotic death of nucleus pulposus (NP) cells and degradation of the NP extracellular matrix. Here, we confirmed that extracellular secretion of IL-1β via secretory autophagy contributes to disc degeneration, and demonstrate that a thermosensitive reactive oxygen species (ROS)-responsive hydrogel loaded with a synthetic growth hormone-releasing hormone analog (MR409) can protect against needle puncture-induced disc degeneration in rats.

Methods: The expression levels of proteins related to secretory autophagy such as tripartite motif-containing 16 (TRIM16) and microtubule-associated protein light chain 3B (LC3B) were examined in human and rat disc tissues by histology and immunofluorescence. The effects of TRIM16 expression level on IL-1β secretion were examined in THP-1 cells transfected with TRIM16 plasmid or siRNA using ELISA, immunofluorescence, and immunoblotting. The in vitro effects of MR409 on IL-1β were examined in THP-1 cells and primary rat NP cells using ELISA, immunofluorescence, immunoblotting, and qRT-PCR. Further, MR409 was subcutaneously administered to aged mice to test its efficacy against disc degeneration using immunofluorescence, X-ray, micro-CT, and histology. To achieve controllable MR409 release for intradiscal use, MR409 was encapsulated in an injectable ROS-responsive thermosensitive hydrogel. Viscosity, rheological properties, release profile,and biocompatibility were evaluated. Thereafter, therapeutic efficacy was assessed in a needle puncture-induced rat model of disc degeneration at 8 and 12 weeks post-operation using X-ray, magnetic resonance (MR) imaging, histological analysis, and immunofluorescence.

Results: Secretory autophagy-related proteins TRIM16 and LC3B were robustly upregulated in degenerated discs of both human and rat. Moreover, while upregulation of TRIM16 facilitated, and knockdown of TRIM16 suppressed, secretory autophagy-mediated IL-1β secretion from THP-1 cells under oxidative stress, MR409 inhibited ROS-induced secretory autophagy and IL-1β secretion by THP-1 cells as well as IL-1β-induced pro-inflammatory and pro-catabolic effects in rat NP cells. Daily subcutaneous injection of MR409 inhibited secretory autophagy and ameliorated age-related disc degeneration in mice. The newly developed ROS-responsive MR409-encapsulated hydrogel provided a reliable delivery system for controlled MR409 release, and intradiscal application effectively suppressed secretory autophagy and needle puncture-induced disc degeneration in rats.   

Conclusion: Secretory autophagy and associated IL-1β secretion contribute to the pathogenesis of disc degeneration, and MR409 can effectively inhibit this pathway. The ROS-responsive thermosensitive hydrogel encapsulated with MR409 is a potentially efficacious treatment for disc degeneration.

PLGA-PEG-PLG，可注射水A：PLGA 即聚乳酸 - 羟基乙酸共聚物（Poly (lactic - co - glycolic acid)），是由乳酸（Lactic Acid）和羟基乙酸（Glycolic Acid）聚合而成。乳酸有 L - 乳酸和 D - 乳酸两种异构体。PEG 是聚乙二醇（Polyethylene Glycol），它是一种线性聚合物，分子链中含有大量的醚键（-O-），这使得 PEG 具有良好的亲水性。PEG 的分子量可以有多种选择，不同分子量的 PEG 会影响整个共聚物的性质。基于PLGA-PEG-PLGA 的性能凝胶Mr409囊泡的合成如下：

图：合成示意

可注射水凝胶Mr409囊泡的制备：

含有囊泡/MR409的PLGA-PEG-PLGA溶液在生理条件下在一定温度下显示了溶液到凝胶的转变。使用RS6000应力控制流变仪测试热敏水凝胶的流变学行为。将PLGA-PEGPLGA溶解在低温的氯化钠水溶液中。在一个小的平板和一个较大的基板之间的间隙中加入一个样品。在应变下，设置温度升高速率，角频率。添加或不添加过氧化氢测定复合水凝胶中荧光标记MR409类似物的释放谱。将含有囊泡/MR409类似物的水凝胶样品密封在透析盒中，并在一定温度下浸泡在含有PBS或PBS2/H2O2的烧杯中。用紫外吸收法定量各时间点释放的MR409模拟物的浓度。

图：粘度变化曲线

结论：

该文献成功地制备出了一种基于PLGA-PEG-PLGA（聚乳酸-乙二醇-聚乳酸）合成的创新型可注射水凝胶，命名为Mr409囊泡。这种新开发的水凝胶具有ros（活性氧）响应特性，为有效地封装并控制MR409的释放提供了一个高度可靠的传递系统。