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Effect of Bugu granules in a drug-containing serum on chondrocyte apoptosis and the Trx2 signaling pathway

Wirkung von Bugu-Granulat in einem arzneimittelhaltigen Serum auf die Chondrozytenapoptose und den Trx2-Signalweg

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Abstract

Traditional Chinese medicine for invigorating the kidney and promoting blood circulation is commonly prescribed for the treatment of osteoarthritis associated with kidney deficiency and blood stasis. However, the specific mechanisms of these medicines are still unclear. The present study aimed to evaluate the protective effects of Bugu granules against sodium nitroprusside-induced chondrocyte apoptosis and elucidate the underlying molecular mechanisms. Drug-containing serum was prepared by administering rats with Bugu granules and harvesting the serum. Chondrocytes were exposed to different dilutions of serum, and apoptosis assessed by flow cytometry after staining with annexin V‑FITC/PI. Flow cytometry showed that chondrocyte apoptosis increased significantly after incubation with 2 mol/L sodium nitroprusside for 24 h (t = −48.221, P = 0.000), and the apoptotic rate of chondrocytes decreased with increasing concentrations of drug-containing serum (F = 33.965, P = 0.000). Cellular levels of Trx2, ASK1, caspase‑3, and reactive oxygen species (ROS) were detected by enzyme-linked immunosorbent assay. The cellular content of Trx2 increased gradually with increasing concentrations of drug-containing serum (F = 2610.593, P = 0.000), while that of ASK1 (F = 2473.545, P = 0.000), caspase‑3 (F = 209.921, P = 0.000), and ROS (F = 1666.435, P = 0.000) all decreased significantly. The mRNA expression levels were analyzed by RT-qPCR, which revealed that expression levels of Trx2 and caspase‑3 mRNA increased and decreased significantly, respectively, following exposure to Bugu granules in the drug-containing serum (F = 6.974, P = 0.003 and F = 3.691, P = 0.191; respectively), but the expression of ASK1 mRNA was not significantly different between treatment groups (F = 1.784, P = 0.191). Taken together, these results support the hypothesis that the Trx2 signaling pathway is activated by Bugu granules, which in turn inhibits chondrocyte apoptosis. This may play a role in preventing the development of osteoarthritis.

Zusammenfassung

Traditionelle chinesische Arzneimittel für die Stärkung der Niere und Förderung des Blutkreislaufs werden häufig zur Behandlung einer Arthrose assoziiert mit Nierenstörung und Blutstase verschrieben. Die genauen Wirkmechanismen dieser Arzneimittel sind aber noch nicht bekannt. In der vorliegenden Studie sollten die protektiven Wirkungen von Bugu-Granulat bezüglich der Natriumnitroprussid-induzierten Chondrozytenapoptose untersucht und die zugrunde liegenden molekularen Mechanismen aufgeklärt werden. Arzneimittelhaltiges Serum wurde hergestellt, indem Bugu-Granulat in Ratten appliziert und danach das Serum gewonnen wurde. Chondrozyten wurden mit verschiedenen Verdünnungen des Serums versetzt; die Apoptose wurde durchflusszytometrisch nach Färbung mit Annexin V-FITC/PI bestimmt. Die Durchflusszytometrie ergab, dass die Chondrozytenapoptose nach Inkubation mit 2 mol/l Natriumnitroprussid für 24 h signifikant anstieg (t = −48,221; P = 0,000). Die Apoptoserate der Chondrozyten sank mit steigenden Konzentrationen des arzneimittelhaltigen Serums (F = 33,965; P = 0,000). Die zellulären Spiegel von Trx2, ASK1, Caspase‑3 und reaktiven Sauerstoffspezies (ROS) wurden mit einem „enzyme-linked immunosorbent assay“ bestimmt. Der zelluläre Trx2-Gehalt stieg schrittweise mit zunehmender Konzentration des arzneimittelhaltigen Serums (F = 2610,593, P = 0,000), während der von ASK1 (F = 2473,545, P = 0,000), Caspase‑3 (F = 209,921, P = 0,000) und ROS (F = 1666,435, P = 0,000) jeweils signifikant abnahm. Die mRNA-Expressions-Niveaus wurden mithilfe der RT-qPCR analysiert. Diese zeigte, dass die Expressionsniveaus von Trx2- und Caspase-3-mRNA nach Exposition gegenüber Bugu-Granulat im arzneimittelhaltigen Serum signifikant zu- bzw. abnahmen (F = 6,974, P = 0,003 bzw. F = 3,691, P = 0,191). Die Expression von ASK1-mRNA unterschied sich dagegen nicht signifikant zwischen den Behandlungsgruppen (F = 1,784, P = 0,191). In der Zusammenschau stützen diese Ergebnisse die Hypothese, dass der Trx2-Signalweg durch Bugu-Granulat aktiviert wird, was wiederum die Chondrozytenapoptose hemmt. Das könnte mit Blick auf die Prävention der Arthroseentwicklung von Bedeutung sein.

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Funding

This work was supported by the Fuzhou Science and Technology Plan Project of China (grant no. 2017-S-130-5) and Fujian Provincial Natural Science Foundation of China (grant no. 2016J01597).

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Authors and Affiliations

  1. Department of Orthopaedics, Fuzhou Second Hospital affiliated to Xiamen University, No. 47, Shang-teng Road, Cang-shan District, 350007, Fu Zhou, Fujian, China

    Guang-Shu Yu & Yan-Bin Lin

  2. Graduate School, Fujian University of Traditional Chinese Medicine, 350122, Fu Zhou, Fujian, China

    Hong-Bin Xu, Shou-Xiong Zhang & Jie-Hui Li

  3. Medical College, Xia Men University, 361102, Xia Men, Fujian, China

    Hai-Yang Wang

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  1. Guang-Shu Yu
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  2. Yan-Bin Lin
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  5. Jie-Hui Li
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Contributions

Guang-Shu Yu participated in the design of the study and performed the statistical analysis. Yan-Bin Lin conceived of the study, participated in its design and coordination and helped to draft the manuscript. Hong-Bin Xu participated in the literature search and data integration. Shou-Xiong Zhang, Jie-Hui Li and Hai-Yang Wang participated in the experiment and statistical analysis of data. All authors read and approved the final manuscript.

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Correspondence to Yan-Bin Lin.

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Conflict of interest

Guang-Shu Yu, Yan-Bin Lin, Hong-Bin Xu, Shou-Xiong Zhang, Jie-Hui Li and Hai-Yang Wang declare that they have no competing interests.

For this article no studies with human participants were performed by any of the authors. All studies performed were in accordance with the ethical standards indicated in each case. The present study was approved by the Animal Care and Use Committee of the Fuzhou Second Hospital Affiliated to Xiamen University.

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U. Müller-Ladner, Bad Nauheim

U. Lange, Bad Nauheim

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The datasets used during the present study are available from the corresponding author on reasonable request.

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Yu, GS., Lin, YB., Xu, HB. et al. Effect of Bugu granules in a drug-containing serum on chondrocyte apoptosis and the Trx2 signaling pathway. Z Rheumatol 79, 304–311 (2020). https://doi.org/10.1007/s00393-019-00688-z

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  • DOI: https://doi.org/10.1007/s00393-019-00688-z

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