Study On Mechanism Of RL-QN15 Inhibiting Inflammatory Response In Knee Osteoarthritis

Objective(s):Knee Osteoarthritis(KOA)is a joint disease caused by a number of factors,mainly manifested by fibrosis of the articular cartilage,cartilage cracks,ulcers and loss.The etiology is unknown,and its occurrence is related to age,obesity,inflammation,trauma and genetic factors,among which inflammation plays an extremely important role in the occurrence and development of KOA,and many pathophysiological processes are related to it.Currently,KOA cannot be completely cured,so it is becoming a hot research topic to find treatments that can reduce inflammation and even reverse cartilage destruction.It has been confirmed that polypeptide molecules can help regulate and maintain the physiological activities of the body,with low toxicity,few adverse reactions,easy to be absorbed by the human body,and have significant effects on human cardiovascular system diseases,endocrine system diseases,psychiatric system diseases,rheumatic immune diseases and cancer.Previous studies have shown that natural active polypeptide RL-QN15 has anti-inflammatory and wound healing effects.The purpose of this study was to investigate whether the application of the novel active peptide RL-QN15 can inhibit the inflammatory response of osteoarthritis at the animal and cellular levels,and to study the regulatory mechanism of the inflammatory response of osteoarthritis,so as to lay a foundation for the development of the novel active peptide RL-QN15 as a novel therapeutic candidate for inhibiting the inflammatory response of osteoarthritis.Knee Osteoarthritis(KOA)is a joint disease caused by a number of factors,mainly manifested by fibrosis of the articular cartilage,cartilage cracks,ulcers and loss.The etiology is unknown,and its occurrence is related to age,obesity,inflammation,trauma and genetic factors,among which inflammation plays an extremely important role in the occurrence and development of KOA,and many pathophysiological processes are related to it.Currently,KOA cannot be completely cured,so it is becoming a hot research topic to find treatments that can reduce inflammation and even reverse cartilage destruction.It has been confirmed that polypeptide molecules can help regulate and maintain the physiological activities of the body,with low toxicity,few adverse reactions,easy to be absorbed by the human body,and have significant effects on human cardiovascular system diseases,endocrine system diseases,psychiatric system diseases,rheumatic immune diseases and cancer.Previous studies have shown that natural active polypeptide RL-QN15 has antiinflammatory and wound healing effects.The purpose of this study was to investigate whether the application of the novel active peptide RL-QN15 can inhibit the inflammatory response of osteoarthritis at the animal and cellular levels,and to study the regulatory mechanism of the inflammatory response of osteoarthritis,so as to lay a foundation for the development of the novel active peptide RL-QN15 as a novel therapeutic candidate for inhibiting the inflammatory response of osteoarthritis.Methods:1.An animal model of knee osteoarthritis in SD rats was established.Intra-articular injection of RN-QN15(1 nM/50 μL,10 nM/50 μL)was performed once a week for four weeks to explore the anti-inflammatory effect of RN-QN15 on KOA;2.Micro-MRI was used to determine the water content of articular cartilage during the course of knee osteoarthritis in rats,and to evaluate the maintenance effect of RNQN15 on the structure and function of articular cartilage matrix during the occurrence and development of KOA;3.Micro-CT was used to observe the trabecular structure of knee subchondral bone in rats,and to evaluate the protective effect of RN-QN15 on knee subchondral bone during the occurrence and development of KOA;4.Gross staining,HE staining and Safranin O/Fast green staining were used to observe the damage degree of inflammation reactions on various joint structures,and evaluate the inflammation inhibition effect of RN-QN15 on KOA staining;5.Immunohistochemical staining was used to detect type Ⅱ collagen,Matrix Metaloproteinase-3(MMP-3),A Disintegrin And Metalloproteinase Domain in articular cartilage With Thrombospondin motifs-4,ADAMTS-4)expression level,to evaluate the protective effect of RN-QN15 on the extracellular matrix of articular cartilage;6.Human chondroma cells(SW1353)were cultured in vitro,and the effects of RNQN15 on the activity and proliferation of SW1353 were detected by MTS method;7.The cell inflammation model of KOA was established through IL-1β induction,and the expression levels of SW1353 ADAMTS-4 and MMP-3 were detected by Western Blotting,further confirming the protective effect of RN-QN15 on articular cartilage in vitro.To explore the mechanism of RN-QN15 inhibiting inflammatory response in osteoarthritis.Results:1.Micro-MR showed that RL-QN15 could help maintain water content in articular cartilage;2.It was found by Micro-CT that RL-QN15 had a protective effect on the structure of subchondral bone;3.It was found that RL-QN15 could reduce the damage of knee joint of SD rats caused by osteoarthritis(the optimal concentration was 10 nM,50μL)by gross morphological observation,HE staining and saffron solid green staining,which showed that it could reduce articular cartilage degeneration and synovial hyperplasia and improve subchondral bone injury;4.Immunohistochemical staining showed that the expression of type II collagen was up-regulated and the expression of ADAMTS-4 and MMP-3 was down-regulated after treatment with RL-QN15;5.In vitro culture of SW1353,it was found that the proliferation activity of SW1353 was enhanced after treatment with different concentrations of RL-QN15;6.Western Blotting results showed that the expressions of ADAMTS-4 and MMP-3 were significantly down-regulated after the action of RL-QN15.Conclusion(s):RL-QN15 can help maintain the integrity of cartilage extracellular matrix network structure and the stability of bone trabecular structure.RL-QN15 could antagonize the damage of bone,cartilage and synovium caused by inflammatory response in osteoarthritis model(the optimal concentration was 10 nM,50μL).RL-QN15 could promote the proliferation of SW1353(the optimal concentration was 10 nM).The protective effect of RL-QN15 on cartilage may be achieved by decreasing the expression of ADAMTS-4 and MMP-3 and increasing the expression of type Ⅱ collagen.This study demonstrated the value of RL-QN15 in the field of inhibiting the inflammatory response of KOA,increased the research object of drug treatment of KOA,provided a new possibility for the application of peptide drugs,laid the foundation for novel therapeutic candidates of KOA,and provided a new idea for clinical treatment and theoretical basis for it.