"VSports在线直播" Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. VSports app下载.

Https

The site is secure V体育官网. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. .

. 2021 Feb;11(2):435-445.
doi: 10.1002/2211-5463.13064. Epub 2020 Dec 31.

PLCγ1 inhibition combined with inhibition of apoptosis and necroptosis increases cartilage matrix synthesis in IL-1β-treated rat chondrocytes

Affiliations

PLCγ1 inhibition combined with inhibition of apoptosis and necroptosis increases cartilage matrix synthesis in IL-1β-treated rat chondrocytes

Xiaolei Chen et al. FEBS Open Bio. 2021 Feb.

Abstract

Osteoarthritis (OA) is an age-related, chronic degenerative disease. With the increasing median age of the population, this disease has become an important public health problem VSports手机版. New, disease-modifying therapies are needed. A potential novel molecular target is phospholipase Cγ1 (PLCγ1), a critical enzyme with important functions including calcium signaling regulation and cell proliferation. In rat chondrocytes treated with IL-1β (20 ng·mL-1 for 36 h), inhibition of PLCγ1 with U73122 (2 μm for 12 h) increased levels and expression of the cartilage matrix components Collagen2 and Aggrecan. This beneficial effect of PLCγ1 inhibition was counteracted by increased chondrocyte apoptosis and necroptosis, increased cell death, and increase levels of ROS, all potentially negative for OA. Combined treatment of IL-1β + U73122-treated chondrocytes with inhibitors of apoptosis (Z-VAD, 10 μm) and necroptosis (Nec-1, 30 μm) enhanced the increases in levels and expression of Collagen2 and Aggrecan, and prevented the increases in cell death and ROS levels. These results suggest that PLCγ1 inhibition may be a viable approach for an OA therapy, if combined with targeted inhibition of chondrocyte apoptosis and necroptosis. .

Keywords: PLCγ1; U73122; apoptosis; necroptosis; osteoarthritis. V体育安卓版.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
Inhibition of PLCγ1 with U73122 increased Collagen2 and Aggrecan levels in IL‐1β‐treated rat chondrocytes. Rat chondrocytes were treated with IL‐1β at 10, 20, and 40 ng·mL−1 for 36 h. Protein levels of Collagen2 and Aggrecan were analyzed by Western blotting (A). Chondrocytes pretreated with IL‐1β (20 ng·mL−1 for 36 h) were treated with the PLCγ1 inhibitor U73122 at 1, 2, 4, and 6 μm for 12 h. Total and phosphorylated protein levels of PLCγ1, Collagen2, and Aggrecan were analyzed by Western blotting (B). In addition, chondrocytes pretreated with IL‐1β (20 ng·mL−1 for 36 h) were treated with U73122 (2 μm for 12 h), and mRNA levels of Collagen2 and Aggrecan were analyzed by RT‐PCR (C). β‐Actin was used as the control for Western blotting, and GAPDH was used as the control for RT‐PCR. Values are means and standard deviations, the error bars represent SD. One‐way ANOVA with the Dunnett test was used to calculate P values. These results are representative of at least three independent experiments in each experiment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig. 2
Fig. 2
U73122‐induced apoptosis and necroptosis in IL‐1β‐treated rat chondrocytes, and the effect of combined treatment with the apoptosis and necroptosis inhibitors Z‐VAD and Nec‐1, respectively. Rat chondrocytes pretreated with IL‐1β (20 ng·mL−1 for 36 h) were treated with U73122 (2 μm for 12 h). Cell proliferation (A), percentage of dead cells (B), and ROS level (C) were measured. Protein levels of apoptosis and necroptosis indexes Bcl‐2, Bax, P53, pro/cleaved‐caspase3, RIP1, and RIP3 were analyzed by Western blotting (D). Chondrocytes pretreated by IL‐1β (20 ng·mL−1 for 36 h) and U73122 (2 μm for 12 h) were treated with Z‐VAD (10 μm for 12 h) or/and Nec‐1 (30 μm for 12 h). Cell proliferation (E), percentage of dead cells (F), and ROS level (G) were measured, and protein levels of apoptosis and necroptosis indexes were analyzed by Western blotting (H). The results of ROS were normalized, means of the first group in Fig. 2C,G were used as the control for ROS. β‐Actin was used as the control for Western blotting. Values are means and standard deviations, the error bars represent SD. One‐way ANOVA with the Dunnett test was used to calculate P values. These results are representative of at least three independent experiments in each experiment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig. 3
Fig. 3
U73122 combined with apoptosis and necroptosis inhibitors increased Collagen2 and Aggrecan levels in IL‐1β‐treated rat chondrocytes. Chondrocytes pretreated by IL‐1β (20 ng·mL−1 for 36 h) and U73122 (2 μm for 12 h) were treated with Z‐VAD (10 μm for 12 h) or/and Nec‐1 (30 μm for 12 h). Protein (A) and mRNA (B) levels of Collagen2 and Aggrecan were analyzed by Western blotting and RT‐PCR, respectively. β‐Actin was used as the control for Western blotting, and GAPDH was used as the control for RT‐PCR. Values are means and standard deviations, the error bars represent SD. One‐way ANOVA with the Dunnett test was used to calculate P values. These results are representative of at least three independent experiments in each experiment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig. 4
Fig. 4
Molecular mechanism of PLCγ1 inhibition combined with inhibition of apoptosis and necroptosis increases cartilage matrix synthesis in IL‐1β‐treated rat chondrocytes. Inhibition of PLCγ1 phosphorylation by U73122 improved cartilage matrix synthesis in IL‐1β‐treated rat chondrocytes. However, it also increased cell apoptosis and necroptosis, which reduced the effect on cartilage matrix synthesis. Inhibition of programmed cell death by simultaneous treatment with the apoptosis and the necroptosis inhibitor increased chondrocyte proliferation and further enhanced cartilage matrix synthesis.

References

    1. Malfait AM (2016) Osteoarthritis year in review 2015: biology. Osteoarthr Cartil 24, 21–26. - PMC (VSports注册入口) - PubMed
    1. Busija L, Bridgett L, Williams SRM, Osborne RH, Buchbinder R, March L and Fransen M (2010) Osteoarthritis. Best Pract Res Clin Rheumatol 24, 757–768. - "VSports在线直播" PubMed
    1. Barnett R (2018) Osteoarthritis. Lancet 391, 1985. - PubMed
    1. Glyn‐Jones S, Palmer AJR, Agricola R, Price AJ, Vincent TL, Weinans H and Carr AJ (2015) Osteoarthritis. Lancet 386, 376–387. - PubMed
    1. Accadbled F, Vial J and Sales de Gauzy J (2018) Osteochondritis dissecans of the knee. Orthop Traumatol Surg Res 104, S97–S105. - PubMed

MeSH terms