Hansson Elisabeth, Skiöldebrand Eva
Department of Clinical Neuroscience, Institute of Neuroscience and Physiology, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
Department of Pathology, Institute of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Uppsala, Sweden.
Curr Res Pharmacol Drug Discov. 2021 Oct 19;2:100066. doi: 10.1016/j.crphar.2021.100066. eCollection 2021.
To treat osteoarthritic chondrocytes and thereby reduce the inflammation with a drug combination that primarily affects 5-HT- and ATP-evoked Ca signaling. In osteoarthritic chondrocytes, Ca signaling is elevated, resulting in increased production of ATP and inflammatory mediators. The expression of TLR4 and Na/K-ATPase was used to evaluate the inflammatory status of the cells.
Equine chondrocytes were collected from joints with mild structural osteoarthritic changes and cultured in monolayers. The cells were treated with a combination of bupivacaine (1 pM) and sildenafil (1 μM) in combination with vitamin D3 (100 nM). A high-throughput screening system, the Flexstation 3 microplate reader, was used to measure intra- and extracellular Ca signaling after exposure to 5-HT, glutamate, or ATP. Expression of inflammatory receptors was assessed by Western blotting.
Drug treatment substantially reduced 5-HT- and ATP-evoked intracellular Ca release and TLR4 expression compared to those in untreated chondrocytes. The combination of sildenafil, vitamin D3 together with metformin, as the ability to take up glucose is limited, increased Na/K-ATPase expression.
The combination of these three therapeutic substances at concentrations much lower than usually used, reduced expression of the inflammatory receptor TLR4 and increased the cell membrane enzyme Na/K-ATPase, which regulates cell volume and reduces increased intracellular Ca concentrations. These remarkable results indicate that this drug combination has disease-modifying osteoarthritis drug (DMOAD) properties and may be a new clinical therapy for osteoarthritis (OA).
用一种主要影响5-羟色胺（5-HT）和三磷酸腺苷（ATP）诱发的钙信号传导的药物组合来治疗骨关节炎软骨细胞，从而减轻炎症。在骨关节炎软骨细胞中，钙信号传导增强，导致ATP和炎症介质的产生增加。采用Toll样受体4（TLR4）和钠钾ATP酶的表达来评估细胞的炎症状态。
从结构有轻度骨关节炎改变的关节采集马软骨细胞，并进行单层培养。细胞用布比卡因（1皮摩尔）和西地那非（1微摩尔）与维生素D3（100纳摩尔）的组合进行处理。使用一种高通量筛选系统，即Flexstation 3微孔板读数仪，来测量暴露于5-HT、谷氨酸或ATP后细胞内和细胞外的钙信号传导。通过蛋白质印迹法评估炎症受体的表达。
与未处理的软骨细胞相比，药物处理显著降低了5-HT和ATP诱发的细胞内钙释放以及TLR4的表达。由于摄取葡萄糖的能力有限，西地那非、维生素D3与二甲双胍的组合增加了钠钾ATP酶的表达。
这三种治疗物质以远低于常用浓度的组合，降低了炎症受体TLR4的表达，并增加了调节细胞体积并降低细胞内钙浓度升高的细胞膜酶钠钾ATP酶。这些显著结果表明，这种药物组合具有改善病情的抗骨关节炎药物（DMOAD）特性，可能是骨关节炎（OA）的一种新的临床治疗方法。
