Construction of OPEI vector for silencing TRAF6 to promote cartilage regeneration in inflammatory environment

doi:10.3969/j.issn.1674-8115.2022.07.002

Abstract

Abstract: Objective

·To construct a low toxicity and high-efficiency joint synovial siRNA transfection vector OPEI, and inhibit tumor necrosis factor receptor-associated factor 6 (TRAF6) to rescue the chondrogenic ability of bone marrow mesenchymal stem cells (BMSCs) under inflammatory conditions.

Methods

·The osteoarthritis (OA) models of SD rats (n=20) were established by medial meniscectomy of knee. Another sham operation group (n=10) was established, and the meniscus remained intact. The cartilage and synovium were collected 3 months after surgery. The expression of TRAF6 was detected by immunohistochemistry, and Western blotting was used to detect the expression of MMP13, TRAF6 and p-p65 in primary rat synovial cells induced by interleukin-1β (IL-1β). Further, the small molecule polyethylenimine (PEI) derivative OPEI was synthesized in anhydrous anaerobic environment, and the ability of OPEI to encapsulate siRNA was detected by agarose gel electrophoresis. The particle size and Zeta potential of OPEI/siRNA complex were measured by dynamic light scattering. The cytotoxicity of the formed complex to rat primary synovial cells was detected by MTT method. The effect of the complex on synovial cell apoptosis was analyzed by flow cytometry. The transfection efficiency of siRNA by OPEI in synovial cells in vivo and in vitro was detected by laser confocal technique and fluorescence microscopy. The proteoglycan content of chondrocyte matrix was detected by alcian blue staining.

Results

·Compared with the sham operation group, TRAF6 was highly expressed in synovium and cartilage of the rat OA models, and inhibition of TRAF6 could significantly reduce the expression of MMP13 and p-p65 in IL-1β-stimulated primary synovial cells. The siRNA transfection efficiency of OPEI in the rat primary synovial cells was as high as 99.33%. A large number of synovial cells ingested siRNA on the 3rd and the 7th day after injection of OPEI / Cy3-siRNA into rat knee joints. Two days after OPEI / siTRAF6 complex with different w/w ratios was transfected into rat primary synovial cells, the results of TRAF6 protein expression showed that the knockout efficiency of TRAF6 gene in the OPEI / siTRAF6 group was 49.05%, 74.61% and 83.18% respectively when the mass ratio was 3∶1, 4∶1 and 5∶1. After TRAF6 gene was silenced by OPEI/siTRAF6, compared with the control group (OPEI/siNC), alcian blue staining of chondrocytes was significantly enhanced under IL-1β stimulation.

Conclusion

·OPEI is a low toxicity and high efficiency siRNA transfection vector, silencing TRAF6 in synovial cells could promote OA cartilage regeneration.

Key words: OPEI, siRNA, synoviocytes, tumor necrosis factor receptor-associated factor 6 (TRAF6), bone marrow mesenchymal stem cells (BMSCs), cartilage regeneration, osteoarthritis (OA)

CLC Number:

LIU Hongqiang, LU Yanqing, GAO Yuxuan, WANG Yiyun, WANG Chuandong, ZHANG Xiaoling. Construction of OPEI vector for silencing TRAF6 to promote cartilage regeneration in inflammatory environment[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(7): 846-857.

Figures/Tables 7

Fig 1 Expression of TRAF6 in OA models and IL-1β-stimulated primary synovial cells

Fig 2 Characterization of OPEI/siRNA polyplex

Fig 3 Biocompatibility and cytotoxicity of OPEI and OPEI/siRNA polyplexes on rat primary synoviocytes

Fig 4 Cell transfection efficiency of OPEI/siRNA polyplexs in vitro and in vivo

Fig 5 TRAF6 knockdown efficiency of OPEI/siTRAF6 polyplexes in rat primary synoviocytes

Fig 6 Effect of TRAF6 knockdown on IL-1β-induced inflammation in rat primary synoviocytes

Fig 7 TRAF6 knockdown restores the chondrogenic ability of rBMSCs under IL-1β-induced inflammation.

TrendMD

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