Chymotrypsin attenuates adjuvant-induced arthritis by downregulating TLR4, NF-κB, MMP-1, TNF-α, IL-1β, and IL-6 expression in Sprague–Dawley rats

Abstract

Objective

Rheumatoid arthritis (RA) is mainly characterized by synovial hyperplasia, angiogenesis, inflammatory cells infiltration. Chymotrypsin is a proteolytic enzyme with anti-inflammatory effects. The current project was intended to test the efficacy and mechanism of chymotrypsin in adjuvant-induced arthritis (AIA) rats to provide an experimental basis for the clinical application of chymotrypsin.

Methods

Sprague–Dawley rats were injected with complete Freund’s adjuvant (CFA) in the hind left paw pad to establish an AIA model. Forty rats were randomly divided into five groups (n = 8): blank; CFA model (model); low-dose chymotrypsin (CLD), 0.53 mg/kg; high-dose chymotrypsin (CHD), 1.06 mg/kg; piroxicam, 10 mg/kg. The treatments were performed in the subplantar region of the left hind paw from Day 8 (D8) to Day 28 after adjuvant injection. The body weight, paw diameter, swelling degree of paw, and arthritic score were measured on D0, D7, D14, D21, and D28. All animals were sacrificed on D29. Subsequently, the synovial tissue of the ankle joint of the rats was stained with HE to generate pathological sections for observation of the pathological changes of synovial tissue from the ankle joint. The protein levels of MMP-1, TNF-α, IL-1β, and IL-6 in the rats’ serum were determined by ELISA. Western blotting was used to detect the protein expression of TLR4 and NF-κB in the rat ankle tissue. The mRNA expression of TLR4, NF-κB, IL-1β, IL-6, and TNF-α in synovial tissue of the ankle joint was detected by RT-qPCR.

Results

The body weight of the rats in each group showed an increasing trend, and there was no significant difference in weight between the groups. CHD and piroxicam suppressed paw swelling and arthritic scores and decreased synovial hyperplasia, inflammatory cell infiltration, pannus formation, and bone destruction. Furthermore, the overproduction of MMP-1, TNF-α, IL-1β, and IL-6 in serum was remarkably attenuated in the chymotrypsin- and piroxicam-treated rats. The protein levels of TLR4 and NF-κB in the synovial tissue of the chymotrypsin group and the piroxicam group were significantly lower than those in the model group. Likewise, the rats treated with chymotrypsin and piroxicam had a substantial decline in the mRNA expression of TLR4, NF-κB, TNF-α, IL-1β, and IL-6 in synovial tissue.

Conclusions

Chymotrypsin alleviates the joint damage of AIA rats, probably by reducing the expression of MMP-1, TNF-α, IL-1β, and IL-6 through TLR4/NF-κB signaling pathway.

Keywords:

• Chymotrypsin
• rheumatoid arthritis
• TLR4/NF-κB signaling pathway
• MMP-1
• TNF-α
• IL-1β
• IL-6

Acknowledgments

The authors thank all people who contributed to this work.

Disclosure statement

Author Linlin Wang, Guangting Zeng, Jianqiang Li_, Huilan Li, Jia Luo, Zanling Zhang are employed by Qijun Tian. All authors declare no other competing interests.

Data availability statement

We can provide data for scientific research upon reasonable request.

Additional information

Funding

This study received funding from Hunan Jianqiao Pharmaceutical Co. Ltd of Hunan Province [No. 33020129148]. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. All authors declare no other competing interests.