Yellow pond turtle peptides ameliorate inflammation and cartilage damage toxicity in papain-induced KOA rats by inhibiting COX-2 and iNOS expressions

doi:10.21203/rs.3.rs-3123546/v1

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Yellow pond turtle peptides ameliorate inflammation and cartilage damage toxicity in papain-induced KOA rats by inhibiting COX-2 and iNOS expressions

https://doi.org/10.21203/rs.3.rs-3123546/v1

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Objective

The drugs in the treatment of knee osteoarthritis (KOA) have significant side effects and poor effectiveness. The plastron of yellow pond turtles is rich in small peptides. As such, it might inform potentially possible to uptake and enhance KOA drug utilization. Nevertheless, the cartilage protective activity of yellow pond turtle peptides (YPTP) in KOA and associated mechanism have not been reported.

Method

We explored KOA rat cartilage protection mediated by YPTP, and its underlying mechanisms using network pharmacology (NP), animal experimentation, and molecular docking (MD) analyses.

Results

Based on our NP data, COX-2 and iNOS were the primary target proteins of YPTP in KOA. Using an animal model, we revealed that YPTP strongly suppressed contents of COX-2, iNOS, and MMP-3 while upregulating COL II to alleviate inflammation and minimize cartilage damage. In addition, MD analysis further validated the 5 YPTP-derived peptides that interacted with COX-2 and iNOS via hydrogen bonding and hydrophobic interactions. Of note, the reduced YPTP dose employed in this investigation enhanced IL-1beta contents in synovial membranes, which requires further exploration.

Conclusion

YPTP ameliorated inflammation and cartilage damage in papain-induced KOA rats by suppressing both COX-2 and iNOS expressions.

peptide

nutrition

network pharmacology

cartilage damage

knee osteoarthritis

Knee osteoarthritis (KOA) is a persistent degenerative joint disease that is marked with pain, stiffness, dysfunction, and even deformity. It can severely lower quality of life of sufferers [1]. A common KOA symptom is articular cartilage degeneration and synovial inflammation [2]. KOA incidences are more prevalent among China's elderly population. In fact, among Chinese adults aged 60 years and older, KOA prevalence is as high as 21.5% and 42.8%, respectively [3]. Obese young adults also have an enhanced prevalence of KOA [4]. Common drugs for KOA treatment include nonsteroidal anti-inflammatory drugs (NSAIDs), acetaminophen, narcotics, and opioids, which relief pain and inflammation in patients [5]. Unfortunately, NSAIDs have severe gastrointestinal complications, and induce kidney and liver damage with long-term use. Acetaminophen is weak-acting, and has the potential to cause liver damage with long-term use. Moreover, narcotics and opioids usage is intricately linked to enhanced addiction risk, in addition to multiple adverse effects, such as, vomiting, sleepiness, and constipation [5]. Given these challenges, it is urgently critical to develop an alternative method for minimizing KOA progression that is both safe and robust.

In recent years, natural products have garnered much attention as an alternative for KOA mitigation. Numerous nutritional supplements are reported to produce a positive outcome on KOA. These include vitamin D, nerolidol, curcumin, chondroitin sulfate, and peptides [6–9]. Particularly, bioactive peptides, such as, small peptides and collagen peptides, are known to considerably slow down KOA progression [6, 8]. Furthermore, bioactive peptides are more efficiently absorbed and utilized by the human body than amino acid mixtures [10]. The yellow pond turtle (Mauremys mutica) plastron is a rich source of bioactive protein, which can be used to prepare yellow pond turtle peptides (YPTP) [11]. Given its potential as an anti-inflammatory source, there is some evidence that YPTP possesses special biological properties, such as, antioxidant, immune enhancing, and sleep promoting abilities [12, 13]. The Compendium of Materia Medica reported that the plastron of yellow pond turtle possesses antipyretic and anti-inflammatory properties, which can strengthen bone. Emerging evidences suggested that the ultrafine Carapax Trionycis powder alleviates bone damage and restores bone mineral density [14]. Based on these evidences, it can be speculated that YPTP may possess potential protective effects on KOA. Nevertheless, the cartilage protective activity of YPTP in KOA and associated mechanism have not been reported.

Network pharmacology (NP) is an emerging field that estimates potential targets and signaling networks. Herein, we employed NP analysis to predict targets of the YPTP-mediated protective activity in KOA. Subsequently, we administered self-prepared YPTP derived from the yellow pond turtle to papain-induced KOA rats to determine associated bioindicators for in vivo verification. Additionally, we examined the molecular interaction mechanism of YPTP and essential target proteins using molecular docking (MD). This investigation is highly significant to the scientific community, owing to its elucidation of the YPTP-based molecular mechanism in treating KOA, while discussing advantages of using YPTP to prevent KOA.

2.1 YPTP preparation and identification

We prepared YPTP (72.16%) in the laboratory [13]. In brief, freshly beaten turtle plastron underwent two 3 h boiling in 8-fold distilled water, prior to removal of the liposoluble components via petroleum ether extraction to extract total protein. Then, 1.2% alkaline protease (60 U/mg) was employed for total protein extract hydrolysis, then the solution underwent a 6 h heating at 55°C in a pH 7.5 NaOH solution to the enzymatic hydrolysis solution. Subsequently, the resulting solution was placed in an ultrafiltration system (Mw < 5 kDa) (Millipore Co., Bedford, MA, USA), and YPTP was retrieved via rotary evaporation concentration, followed by freeze-drying. YPTP sequence analysis was done with an HPLC-ESI-QqTOF-MS/MS system (AB SCIEX Co., Ltd, Shanghai, China). YPTP harbored Gla-His (JH), Lys-Asn-Gly-Pro (KNGP), Asn-Gln-Gly-Hyp-Pyr (NQGXB), Arg-Gly (RG), Trp-Gly-Pro-Gly-Pyr (WGPGB), Trp-Gly-Hyp-Pyr (WGXB), Trp-Asn-Ala-Arg-Trp-Pro-Gly-Gla (WNARWPGJ), Trp-Arg-Trp-Hyp-His-Thr-His-Asn-Trp (WRWXHTHNW), and Trp-Trp-Tyr-Trp-Arg-Hex (WWYWR-Hex) sequences, and the aforementioned peptide structures are provided in Fig. S1.

2.2 NP

2.2.1 Prediction of potential targets and analyses

The open access Swiss Target Prediction database (http://www.swisstargetprediction.ch/) was employed for relevant YPTP target candidate predictions using the “Homo sapiens” species designation [15]. In addition, KOA-associated target genes were identified using the GeneCards database (https://www.genecards.org/) with "score > 20" [16]. Subsequently, we generated a Venn diagram depicting YPTP- and KOA-associated targets using Jvenn (http://bioinfo.genotoul.fr/jvenn), and the target intersection was considered a potential YPTP target for KOA treatment.

2.2.2 YPTP-target-KOA and protein‒protein interaction (PPI) network and analysis

The identified YPTP-KOA targets from the Venn diagram were next plugged into Cytoscape 3.9.0. Following redundancy elimination, we generated the YPTP-target-KOA network. To identify the major YPTP targets in KOA, we imported the YPTP-KOA intersection targets into DAVID (https://david.ncifcrf.gov/) for KEGG pathway enrichment analysis. Additionally, the major target genes were also entered into the STRING database (https://string-db.org/) for generation of a PPI network. Following redundant data removal, PPI network visualization was conducted via Cytoscape 3.9.0. The PPI network topological features were assessed via the "Degree" of the Network Analyzer, and the target proteins with increased "Degree" were regarded as the major YPTP targets in KOA.

2.2.3 GO and KEGG pathway enrichment analyses

Using DAVID, we assessed common targets using following criteria: ease < 0.05, count ≥ 4, FDR < 0.01, and P < 0.05. The GO analysis was stratified into three categories: cellular component (CC), molecular function (MF), and biological process (BP). The 20 leading networks with -log(PValue) in the aforementioned categories were selected, and bubble charts were constructed using R 4.0.3.

2.3 Experimental validation

2.3.1 Animals

We obtained 32 male SD (SPF, 190–210 g) rats from the Animal Center for Disease Prevention and Control in Hubei Province (Certificate No. SCXK (Hubei) 2020-0019, Wuhan, China). All animal protocols strictly followed the recommendations of the People’s Republic of China (GB14925-2010) and the Center for Laboratory Animals, Huazhong Agricultural University (Permission No. HZAURA-2020-0010). The rats were housed at 22–25°C under 55 ± 10% humidity, and a 12 h light/ dark cycle, with ad libitum food and water (Permission No. SYXK (Hubei) 2020–0084).

2.3.2 Experimental design

The process diagram was provided in Fig. S2. In all, 32 rats were arbitrarily separated into 4 groups (n = 8 per group) as follows: normal; KOA, oral saline administration; LYPTP, receiving 75 mg/kg/d YPTP supplement; and HYPTP, receiving 150 mg/kg/d YPTP supplement. The KOA models were induced on days 14, 17, and 20 in the KOA, LYPTP, and HYPTP groups. Induction was performed via 0.15 mL administration of a 2:1 mixture of 4% papain and 0.3 mol/L L-cysteine into the left knee's intra-articular region. In contrast, 0.15 mL saline was intraarticularly administered in normal rats [17, 18]. L-cysteine and papain were acquired from Guangzhou Saiguo Biotech Co., Ltd (Guangzhou, China). From day 14 since injection, the left knee joint diameter was determined using a Vernier caliper, and the body weight was recorded throughout the experiment. Prior to the intra-articular administration, all rats were intraperitoneally anesthetized. For this experiment, we used pre-experimental YPTP doses.

A KOA model can develop after 2-weeks of treatment with 4% papain and 0.3 mol/L L-cysteine [17, 18]. On day 38, rats were given euthanasia, before extraction of retro-orbital blood samples, which then underwent centrifugation, followed by supernatant storage at − 80°C till further analyses. We also sliced off both patellar surfaces to expose the femoral condyle. Subsequently, 0.5 mL saline was administered into the left knee joint, then aspirated to retrieve synovial fluid, which was then stored at − 80°C. Next, the synovium, along with the patella, was quickly sheared off and covered in 4% paraformaldehyde. The femoral and tibial distal portions of the knee joints were also transected, prior to fixation in 4% paraformaldehyde. To determine animal health, we also extracted and weighed the rat livers and spleens, and the organ index was computed as listed below:

$$\text{O}\text{r}\text{g}\text{a}\text{n} \text{i}\text{n}\text{d}\text{e}\text{x} \left(\text{m}\text{g}/\text{g}\right)=\frac{\text{w}\text{e}\text{i}\text{g}\text{h}\text{t} \text{o}\text{f} \text{o}\text{r}\text{g}\text{a}\text{n} \left(\text{m}\text{g}\right)}{\text{b}\text{o}\text{d}\text{y} \text{m}\text{a}\text{s}\text{s} \left(\text{g}\right)}\times 100\%$$

2.3.3 Histology

To further elucidate how YPTP affects the meniscus and cartilagenous structures of rats, we conducted histological analysis. Intact knee joints underwent a 2-month decalcification at room temperature (RT) in 10% EDTA, which removed the synovium. The synovium was stripped, before embedding in paraffin. Upon decalcification, knee joints were longitudinally sliced, prior to paraffin embedding. Subsequently, mid-sagittal sections (4 µm) were hematoxylin and eosin (H&E) to assess meniscus and articular cartilaginous structures using an optical microscope, toluidine blue (TB) stained to examine the expression of proteoglycan.

2.3.4 Modified Mankin score (MMS)

MMS (Table S1) was computed to assess cartilage damage, chondrocyte count, proteoglycan staining status, and tideline integrity according to the H&E and TB staining [19]. An elevated score represented more severe KOA.

2.3.5 Determination of parameters in serum and synovial fluid

Corresponding ELISA kits for collagen type II C-telopeptide (CTX-II), cartilage oligomeric matrix protein (COMP), prostaglandin E2 (PGE2), interleukin-1beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) were acquired from Shanghai Huding Biological Technology Co., Ltd. (Shanghai, China). The nitric oxide (NO) assay kit was acquired from Nanjing Jiancheng Biotechnology Co., Ltd. (Nanjing, China). Following associated directions, we assessed the YPTP-mediated regulation of KOA inflammation using serum and synovial fluid samples. We next quantified the serum CTX-II, COMP, and proinflammatory cytokines (IL-6, NO, TNF-α, PGE2, and IL-1β), as well as synovial fluid-based proinflammatory cytokines (IL-1β, IL-6, and TNF-α) using the Bio-Rad assay kit.

2.3.6 Determination of joint structure parameters using immunohistochemistry (IHC)

Using IHC, we assessed the YPTP-mediated regulation of various inflammation-related protein contents in the rat synovium and cartilage. The synovium-based inflammation-related proteins (IL-1β, TNF-α, COX-2, and iNOS), as well as the articular cartilage repair-related (COL II and MMP-3) protein expressions were evaluated using IHC. Once we deparaffinized and rehydrated paraffin sections, we conducted antigen retrieval by incubating the specimen in citric acid (pH 6.0) antigen retrieval buffer. Subsequently, we placed it in PBS (pH 7.4), prior to a 30 min blocking in 3% BSA, overnight incubation in primary antibody at 4°C, a 50 min incubation in secondary antibody at RT, PBS rinses, DAB staining, and finally a 3 min hematoxylin counterstaining. Visualization was performed under a microscope following dehydration and mounting.

2.4 MD

For molecular modeling and docking computations, we employed the Discovery Studio 2018. This investigation examined the association between YPTP and key targets. Since the YPTP-derived JH, NQGXB, RG, WGPGB, and WGXB possessed an enzyme-resistant structure [20], we selected the aforementioned 5 peptides for MD analysis. Ibuprofen, a frequently employed KOA mitigating drug, served as a positive control [21, 22]. The 2D structures of 5 peptide ligands and ibuprofen were drawn using ChemDraw 19.0, prior to the conversion to 3D structures using Discovery Studio 2018. COX-2 and iNOS were selected as receptors for MD analysis. The COX-2 (PDB ID 3NT1) and iNOS (PDB ID 2ORO) crystal structures were retrieved from the RCSB protein database (http://www.rcsb.org/pdb) [23]. Prior to MD analysis, the receptor proteins (COX-2 and iNOS) were prepared for docking by removing ligands and water and introducing hydrogen [23]. The COX-2 active site was identified as Arg216, Glu140, and Tyr134, with x: -10.044, y: -54.22, z: -24.09 [21]. The iNOS active site was identified as Arg193, Ile195, and Met368, with x: -10.044, y: -54.22, z: -24.09 [24]. Lastly, CDOCKER, a semiflexible docking mode, was employed for MD analysis in the CHARMM force field. The optimal binding pose was the protein‒ligand complex conformation with the highest scores of -CDOCKER energy and -CDOCKER interaction energy, and the resulting data was visually analyzed using Discovery Studio 2018.

2.5 Statistical analysis

Data are provided as means ± standard deviations (SD). Inter-group comparisons employed one-way analysis of variance (ANOVA). P < 0.05 was set as the significance standard. SPSS was employed for all data analyses.

3.1 Screening of potential genes of YPTP for KOA mitigation

In all, 230 YPTP- and 1918 KOA-related targets were identified using Swiss and GeneCards, respectively. Then, 122 overlapping targets were screened using Jvenn (Fig. 1A). We combined the 118 targets for YPTP and KOA and removed redundancy to form the YPTP-target-KOA network using Cytoscape 3.9.0 (Fig. 1B). As depicted in Fig. 1C, the following genes exhibited degrees > 5: TNF, FPR2, APP, AGTR2, AGTR1, STAT3, NFKBIA, BDKRB2, PTGS2, NOS2, and so on. The most highly connected gene was TNF, which indicated that this gene possessed certain essential functions.

3.2 Generation and analysis of the YPTP-target-KOA and PPI network

We screened and identified 20 major targets from the 5 leading KEGG networks that overlapped with the YPTP-target-KOA network, and entered them into STRING to generate a PPI network. Individual node topological features within the network were computed via Network Analyzer in the Cytoscape 3.9.0. In Fig. 1D, nodes with a stronger blue color indicate a higher degree value. PTGS2 (COX-2) exhibited the maximum degree (28) of connectivity, followed by MMP9, STAT3, TNF, MAPK8, MMP2, CASP8, EGFR, MMP1, CASP3, GSK3B, IKBKB, MMP3, BIRC2, AGTR1, NOS2, and so on. Therefore, PTGS2 (COX-2) was a major potential target that represented the YPTP-mediated effect on KOA.

3.3 GO and KEGG pathway enrichment analyses

To elucidate the function and associated networks of the 118 targets, 485 GO items and 87 KEGG networks were identified using the DAVID database (Table S2 & S3). Bubble plots of the 20 leading BP, CC, MF, and KEGG pathways with -log(PValue) values were generated with R 4.0.3, as depicted in Fig. 1E-H. In plots, a stronger red color indicates a higher enrichment degree, and a stronger possibility of being a drug target. Based on our GO data, enrichment was mostly observed in 348 BP terms, namely, proteolysis (GO:0006508), inflammatory response (GO:0006954), and collagen catabolic process (GO:0030574); 49 CC terms, namely, integral component of plasma membrane (GO:0005887), proteinaceous extracellular matrix (GO:0005578), and protein complex (GO:0043234); and 88 MF terms, namely, protein binding (GO:0005515), serine-type endopeptidase activity (GO:0004252), and metalloendopeptidase activity (GO:0004222). These data suggested that YPTP potentially modulated KOA using inflammation and cartilage metabolism [25, 26]. In addition, as depicted in Table S4, the first 5 ranking enriched KEGG networks were the NF-κB (hsa04064), TNF (hsa04668), HIF-1 (hsa04066), PI3K-AKT (hsa04151), and cancer-related axes (hsa05200).

3.4 The YPTP-mediated modulation of KOA severity

KOA severity was assessed using two biomarkers CTX-II and COMP [27]. As depicted in Fig. 2A and B, KOA rats exhibited markedly enhanced CTX-II and COMP contents, compared to normal rats, thereby indicating that the KOA rat model was successfully established. Based on our evaluation of the CTX-II and COMP contents, the KOA severity was as follows: KOA rats > LYPTP rats > HYPTP rats > normal rats. Taken together, our combined data suggested that KOA severity can be inversely associated with YPTP therapy (LYPTP or HYPTP).

3.5 The YPTP-based effects on KOA rat health and knee joint swelling

As depicted in Fig. S3A, the KOA rats exhibited no marked difference in body weight or organ index, compared to the other rats. This indicated that YPTP had little effect on rat health. As illustrated in Fig. 2C, on days 7 and 13, the knee joint diameters of the different rat groups were not obviously different. Following 31 days of KOA induction, the YPTP rats demonstrated a marked decline in knee joint diameter, relative to the KOA rats (P < 0.05). Meanwhile, the AUCs reduced by 57.8% in LYPTP rats, and by 97.50% in HYPTP rats, relative to the KOA rats (Fig. 2D). This indicated that both LYPTP and HYPTP strongly suppressed knee joint swelling (P < 0.05).

3.6 The YPTP-mediated regulation of articular cartilage damage in KOA rats

As depicted in Fig. S3B, in normal rats, the cartilage surface appeared smooth and glossy, whereas, KOA rats exhibited severe cartilage damage and slight swelling of the joints. In contrast to the KOA rats, the LYPTP and HYPTP rats displayed slight cartilage surface damage. As shown in Fig. 2E, the TB and H&E staining revealed strong focal cartilage defects and reduced proteoglycans in KOA versus normal rats. Based on our histological morphological analysis, the KOA rats possessed rough, localized stenosis and adhesions on the surface of articular cartilage. The proteoglycan content and cartilage surface damage were markedly diminished in the LYPTP and HYPTP rats following YPTP treatment. Additionally, the HYPTP rat exhibited smoother cartilage surfaces, better arrangement of cells, and a more integrated tide line, compared to the LYPTP rats. KOA severity was also examined using MMS. As depicted in Fig. 2F, in normal (0.08 ± 0.07) and HYPTP (3.27 ± 1.40) rats, the MMS was drastically reduced, relative to the KOA (11.00 ± 1.50) and LYPTP (9.26 ± 1.39) rats. Together, these results were consistent with data from KOA biomarkers and knee joint swelling. These evidences suggest that YPTP strongly reduces articular cartilage damage.

3.7 The YPTP-mediated regulation of systemic inflammation and joint inflammation

We observed significant differences in serum proinflammatory cytokines (IL-6, NO, TNF-α, PGE2, and IL-1β) between KOA and normal rats (Fig. 3A-E). The LYPTP and HYPTP rats displayed drastically reduced contents of the aforementioned proinflammatory cytokines, relative to the KOA rats (P < 0.05), thereby suggesting that YPTP suppresses systemic inflammation. Additionally, our assessment of the proinflammatory cytokine contents in rat synovial fluid (IL-1β, IL-6, and TNF-α) (Fig. 3F-H) revealed that the IL-1β, IL-6, and TNF-α levels followed the order of KOA > LYPTP > HYPTP > normal rats (P < 0.05). Based on these evidences, YPTP has the potential to relieve knee inflammatory reactions in KOA rats.

3.8 The YPTP-mediated regulation of inflammatory protein expressions in the synovium

Using IHC (Fig. 4A-H), we demonstrated that the inflammatory protein contents (IL-1β, TNF-α, COX-2, and iNOS) in the synovium of KOA rats were considerably higher than normal rats. In contrast, HYPTP treatment drastically reduced the IL-1β and TNF-α protein expressions, compared to the normal rats (P < 0.05), LYPTP treatment hardly reduced the IL-1β and TNF-α protein expressions, compared to the normal rats. Similarly, both LYPTP and HYPTP treatment strongly reduced COX-2 and iNOS protein expressions (P < 0.05). Given these evidences, it is likely that YPTP mediates its protective effects on the knee joint via reduction of COX-2 and iNOS contents, and suppression of synovial inflammation.

3.9 The YPTP-mediated regulation of articular cartilage repair in KOA rats

To elucidate the YPTP-mediated regulation of rat articular cartilage repair-associated proteins, we examined the COL II and MMP-3 expressions in vivo using IHC (Fig. 4I-L). Relative to the KOA rats, the COL II expression in LYPTP and HYPTP rats rose by 43.75% and 77.88%, respectively, whereas, the MMP-3 expression diminished by 24.63% and 80.91%, respectively. Together, these data indicated that YPTP alleviates papain-induced articular cartilage structural damage in KOA rats by potentially suppressing COL II expression, while simultaneously enhancing MMP-3 expression.

3.10 MD analysis

To explore a theoretical basis for the inhibitory activity and potential molecular interactions between peptides and key targets, we selected 5 peptides (namely, JH, WGPGB, RG, WGXB, and NQGXB) and ibuprofen as ligands, whereas, COX-2 and iNOS were chosen as receptors for MD analysis using CDOCKER. MD analysis establishes binding affinity scores using the score ligand pose module [23]. Based on our analysis of the 5 peptides and positive ibuprofen control (Table S5), The -CDOCKER energy scores, along with the -CDOCKER interaction energy scores, followed the order of NQGXB > JH > WGPGB > RG > WGXB > ibuprofen. Generally, increased scores (-CDOCKER energy and -CDOCKER interaction energy) indicate stronger binding affinity and stability. Using this criterion, the 5 aforementioned peptides demonstrated strong and stable interactions with COX-2 and iNOS [28]. Figure 5 and Table S5 illustrate the 3D and 2D interactions of the 5 peptides with COX-2 and iNOS and associated residues. As depicted in Fig. 5, the 5 peptides penetrated deep within the receptor docking site, and the protein‒ligand complexes were primarily stabilized using hydrogen bonds and hydrophobic interactions. JH, NQGXB, RG, WGPGB, WGXB, and ibuprofen formed 3, 8, 7, 5, 9, and 3 hydrogen bonds with COX-2, respectively. Hydrophobic interactions only formed between WGPGB and COX-2. In addition, van der Waals forces were formed within the COX-2 and ligands complexes, except for RG and ibuprofen. In terms of iNOS, JH, NQGXB, RG, WGPGB, WGXB, and iNOS formed 8, 5, 5, 5, 4, and 3 hydrogen bonds, respectively. JH, WGPGB, WGXB, and ibuprofen formed 1, 3, 8, and 1 hydrophobic interaction with iNOS, respectively. Based on these analyses, the aforementioned 5 peptides easily interacted with COX-2 and iNOS receptors, and may elicit potential protective activity on KOA.

YPTP, a naturally active yellow pond turtle plastron-derived peptide possesses significant alleviation of osteoarthritis value, as well as pivotal physiological functions, namely, antioxidant, immunity-boosting, and sleep-promoting activities [13, 29]. However, till date, there is little known about the YPTP-mediated anti-inflammatory or cartilage protective activities. Hence, herein, we explored the cartilage protective activity of YPTP on KOA and associated mechanisms using NP and animal models. NP analysis revealed that YPTP strongly ameliorated inflammation and cartilage damage in KOA by suppressing COX-2 and iNOS contents. Next, we generated a YPTP-target-KOA network to better predict YPTP target genes. Based on our analysis, YPTP potentially inhibited TNF [30], FPR2 [31], APP [32], AGTR2, AGTR1 [33], STAT3 [34], NFKBIA [35], BDKRB2, PTGS2, and NOS2 [36]. Of note, although the aforementioned YPTP target genes are not the same as the icariin target genes, which treat OA, and include NOS3, NFKBIA, AKT, MAPK, and so on ^[25], the key genes for both diseases are primarily involved in inflammation and chondrocyte regulation. Among them, NOS2 (iNOS) is intricately linked to COX-2 content and the NF-κB pathway [37]. KEGG enrichment analysis revealed that YPTP alleviated KOA via several signaling networks, such as, NF-κB, TNF, HIF-1, PI3K-Akt, and cancer-associated networks. The NF-κB axis, which integrates all aforementioned pathways, is critical for KOA inflammation. Studies revealed that NF-κB activation stimulates target proteins COX-2 and iNOS expressions, which result in additional cartilage damage [38]. Curcumin ameliorates KOA by suppressing NF-κB activation prior to COX-2 and iNOS inhibition [9]. Similarly, oleic acid mitigates inflammation by reducing COX-2 and iNOS contents [39]. Given these evidences, it is highly likely that YPTP ameliorates KOA by suppressing COX-2 and iNOS expressions. Herein, our conclusion was further validated using animal experiments and MD technology.

Using animal models and NP, we revealed that YPTP strongly alleviated inflammation by suppressing the COX-2, iNOS, IL-1β, IL-6, TNF-α, NO, PGE2, and MMP-3 gene expressions. In addition, it enhanced COL II expression to minimize cartilage damage. Prior investigations revealed that KOA is marked with synovial inflammation and cartilage destruction, and inflammation and inflammatory cytokines heavily contribute to KOA pathophysiology [40]. Potential inflammation-related target pathways are presented in Fig. 6. During KOA progression, under stimulated conditions, inflammatory cytokines activate NF-κB, which upregulates COX-2 and iNOS expressions, and induces expressions of other proinflammatory cytokines, namely, IL-6, TNF-α, NO, and PGE2 [41]. Moreover, excess expression of the aforementioned proinflammatory cytokines induce upregulation of metalloproteinase-3 (MMP-3) [42]. MMP-3 strongly mediates cartilage matrix and COL II degradation, which, in turn, destroys the cartilage [43]. This may be one of the mechanisms of YPTP-mediated cartilage protection. In terms of the peptide segment, it may be possible, in future investigations, to properly formulate this substance with other substances in order to increase drug utilization while achieving a joint-protective effect.

Several small peptides harboring glycine, proline, glutamine, histidine, and tyrosine residues exhibit anti-enzymatic activities, and can be absorbed into the circulation by means of special transporters [20, 44]. The 5 peptides identified in this investigation contained proline, histidine, glutamine, glycine, and tryptophan, which possess anti-enzymatic properties, and the potential to target COX-2 inhibition. Thus, these 5 peptides were selected for MD analysis. Based on our results, the 5 peptides strongly interacted with COX-2 and iNOS proteins. The peptide-protein complex interactions occur primarily through hydrogen bonding and hydrophobic associations. Our result validated our conclusions from NP and animal model analysis, thus providing a theoretical basis for the possibility of targeted therapy. Furthermore, there are reports that COX-2-based intervention is highly beneficial in suppressing KOA development and progression [5]. NSAIDs that inhibit COX have emerged as robust therapeutic agents for KOA, however, COX has at least two isomers, COX-1 and COX-2 [45]. COX-1 is functional in normal cells, and COX-1 inhibition produces the main side effects of NSAIDs. Hence, development of COX-2-targeted inhibition compounds may be crucial to KOA therapy.

Importantly, the dose of YPTP employed in our investigations stimulated IL-1beta contents in synovial membranes. This indicated that low glabella-derived peptide doses may instead promote inflammation, which remains to be explored and verified in future experiments. The underlying mechanisms behind yellow pond turtle-derived monopeptide absorption and metabolism and their inhibition of COX-2 and iNOS in vitro requires further exploration in the near future.

In conclusion, herein, we adopted methods of NP-based prediction, animal in vivo validation, and MD analysis to explore the potential cartilage protective mechanisms of YPTP on KOA. Our findings suggested that YPTP ameliorated inflammation and cartilage damage in papain-induced KOA rats by suppressing expression of key targets COX-2 and iNOS. This paper might inform potentially possible to uptake and enhance KOA drug utilization.

AUC	area under the curve;
BP	biological processes;
CC	cellular components;
COLLII	type II collagen;
COMP	cartilage oligomeric matrix protein;
COX-2	cyclooxygenase-2;
CTX-II	collagen type II C-telopeptide;
H&E	hematoxylin and eosin staining;
IL-1β	interleukin-1β;
iNOS	inducible nitric oxide synthase;
KOA	knee osteoarthritis;
MD	molecular docking;
MF	molecular functions;
MMP-3	metalloproteinase-3;
MMS	modified mankin score;
NF-κB	nuclear factor kappa B;
NO	nitric oxide;
NP	network pharmacology;
PGE2	prostaglandin E2;
PPI	protein-protein interaction;
TB	toluidine blue staining;
TNF-α	tumor necrosis factor alpha;
YPTP	yellow pond turtle peptides

Data Availability

The dataset used to support the findings of this study are available from the corresponding author upon request.

Authors’ Contributions

Jia-xing Yan: Conceptualization, Methodology, Investigation, Formal analysis, Writing - original draft. Zuo-an Li: Writing - review & editing, Funding acquisition. Lan-jie Feng: Validation, Formal analysis. Lu-hong Shen: Validation, Resources. Jiu-liang Zhang: Writing - review & editing, Project administration, Funding acquisition.

Conflicts of Interest

There are no conflicts of financial and personal relationships in this work.

Acknowledgements

This research is supported by National Key R&D Program from Ministry of Science and Technology of the People’s Republic of China (No. 2021YFE0194000) and the Fundamental Research Funds for the Central Universities of China (No. 2662018PY022) and the Natural Science Foundation of Fujian Province (No. 2017J01174).

Animal Ethics

The protocol was reviewed and accepted by the Tab of Animal Experimental Ethical Inspection of Laboratory Animal Centre, Huazhong Agriculture University, ID number HZAURA-2020-0010.

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Yellow pond turtle peptides ameliorate inflammation and cartilage damage toxicity in papain-induced KOA rats by inhibiting COX-2 and iNOS expressions

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Abstract

Objective

Method

Results

Conclusion

Figures

1. Introduction

2. Materials and methods

2.1 YPTP preparation and identification

2.2 NP

2.2.1 Prediction of potential targets and analyses

2.2.2 YPTP-target-KOA and protein‒protein interaction (PPI) network and analysis

2.2.3 GO and KEGG pathway enrichment analyses

2.3 Experimental validation

2.3.1 Animals

2.3.2 Experimental design

2.3.3 Histology

2.3.4 Modified Mankin score (MMS)

2.3.5 Determination of parameters in serum and synovial fluid

2.3.6 Determination of joint structure parameters using immunohistochemistry (IHC)

2.4 MD

2.5 Statistical analysis

3. Results

3.1 Screening of potential genes of YPTP for KOA mitigation

3.2 Generation and analysis of the YPTP-target-KOA and PPI network

3.3 GO and KEGG pathway enrichment analyses

3.4 The YPTP-mediated modulation of KOA severity

3.5 The YPTP-based effects on KOA rat health and knee joint swelling

3.6 The YPTP-mediated regulation of articular cartilage damage in KOA rats

3.7 The YPTP-mediated regulation of systemic inflammation and joint inflammation

3.8 The YPTP-mediated regulation of inflammatory protein expressions in the synovium

3.9 The YPTP-mediated regulation of articular cartilage repair in KOA rats

3.10 MD analysis

4. Discussion

Abbreviations

Declarations

Data Availability

Authors’ Contributions

Conflicts of Interest

Acknowledgements

Animal Ethics

References

Additional Declarations

Supplementary Files

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