Lingzhi and San-Miao-San with hyaluronic acid gel mitigate cartilage degeneration in anterior cruciate ligament transection induced osteoarthritis

Abstract

Objective: To investigate the mitigate efficacy of Chinese medicine Lingzhi (LZ) and San-Miao-San (SMS) combined with hyaluronic acid (HA)-gel in attenuating cartilage degeneration in traumatic osteoarthritis (OA).

Methods: The standardized surgery of anterior cruciate ligament transection (ACLT) was made from the medial compartment of right hind limbs of 8-week-old female SD rats and resulted in a traumatic OA. Rats (n ​= ​5/group) were treated once intra-articular injection of 50 ​μl HA-gel, 50 ​μl HA-gel+50 ​μg LZ-SMS, 50 ​μl of saline+50 ​μg LZ-SMS and null (ACLT group) respectively, except sham group. Limbs were harvested for μCT scan and histopathological staining 3-month post-treatment. Inflammatory cytokines from plasma and synovial fluid were detected using Immunology Multiplex Assay kit. The putative targets of active compounds in LZ-SMS and known therapeutic targets for OA were combined to construct protein-protein interaction network. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was adopted to predict the potential targets and signaling pathway of LZ-SMS in OA through the tool of DAVID Bioinformatics.

Results: In vivo, HA-gel ​+ ​LZ-SMS treatment resulted in a higher volume ratio of hyaline cartilage (HC)/calcified cartilage (CC) and HC/Sum (total volume of cartilage), compared to ACLT and HA-gel groups. In addition, histological results showed the elevated cartilage matrix, chondrogenic and osteoblastic signals in HA-gel ​+ ​LZ-SMS treatment. Treatment also significantly altered subchondral bone (SCB) structure including an increase in BV/TV, Tb.Th, BMD, Conn.Dn, Tb.N, and DA, as well as a significant decrease in Tb.Sp and Po(tot), which implied a protective effect on maintaining the stabilization of tibial SCB microstructure. Furthermore, there was also a down-regulated inflammatory cytokines and upregulated anti-inflammatory cytokine IL-10 in HA+LZ-SMS group. Finally, 64 shared targets from 37 active compounds in LZ-SMS related to the core genes for the development of OA. LZ-SMS has a putative role in regulating inflammatory circumstance through influencing the MAPK signaling pathway.

Conclusion: Our study elucidated a protective effect of HA-gel ​+ ​LZ-SMS in mitigating cartilage degradation and putative interaction with targets and signaling pathway for the development of traumatic OA.

The translational potential of this article: Our results provide a biological rationale for the use of LZ-SMS as a potential candidate for OA treatment.

Keywords: 3D, Three-dimensional; AC, Articular cartilage; ACLT, Anterior cruciate ligament transection; Acan, Aggrecan; Articular cartilage; BMD, Bone mineral density; BV/TV, Bone volume fraction; CC, Calcified cartilage; Conn.Dn, Connectivity density; DA, Degree of anisotropy; DL, Drug-likeness; ECM, Extracellular matrix; FDR, False discovery rate; GO, Gene ontology; HA, Hyaluronic acid; HC, Hyaline cartilage; Hyaluronic acid gel; KEGG, Kyoto Encyclopedia of Genes and Genomes; LZ-SMS, Lingzhi-San-Miao-San; Lingzhi and San-Miao-San; MZ, Middle zone area of articular cartilage; NC, Negative control; OA, Osteoarthritis; OB, Oral bioavailability; OMIM, Online Mendelian Inheritance in Man; Osteoarthritis; PPI, Protein–protein interaction; Po(tot), Total porosity; ROI, Region of Interest; SC, Superficial cartilage; SCB, Subchondral bone; SZ, Superficial zone of articular cartilage; Subchondral trabecular bone; Sum, Whole cartilage; TCM, Traditional Chinese medicine; TCMSP, Traditional Chinese Medicine Systems Pharmacology Database; Tb.N, Trabecular number; Tb.Pf, Trabecular bone pattern factor; Tb.Sp, Trabecular separation; Tb.Th, Trabecular thickness.

Figure 1

Histological images and quantitative analysis of rat tibial plateau in different groups. (A) The representative images of toluidine blue, tartrazine and fast green stained the tibial medial articular cartilage in different treatment groups. Light color stained superficial zone of articular cartilage (SZ) pointed by the red arrow and by the red dash line separated area were shown in ACLT and HA groups. The black and yellow bidirectional arrows pointed the middle zone of articular cartilage (MZ) and thickness of hyaline cartilage (HC) and calcified cartilage (CC) in ACLT and LZ-SMS groups, respectively. Scale bar, 50 ​μm. (B–E) The Bar charts are shown the quantitative analyzed volume ratios at different zone of cartilage in each group tibial plateau. They are volume ratios of SZ/Sum (total volume of cartilage), HC/Sum, CC/Sum and HC/CC. Differences among treatments are compared by one-way ANOVA (n ​= ​5/group, ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.0001, ACLT vs. other groups). Wilcoxon test is used to assess the differences between two different treatments (n ​= ​5/group, p ​> ​0.05, HA+LZ-SMS vs. other groups). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 2

Immunohistochemistry images and quantitative analysis of chondrogenic and osteoblastic signals in different groups. (A) The representative images of BMP2+ and TGF-β1+ chondrogenic cells and RANKL+ ​osteoblasts are shown at articular cartilage and SCB plate area in different groups, respectively. Scale bar, 50 ​μm. The Bar charts are shown the quantitative analysis of the number of (B) BMP2+ and (C) TGF-β1+ cells per cartilage area (mm²), and (D) RANKL+osteoblasts per SCB plate area (mm²) in each group, respectively. Differences among treatments are compared by one-way ANOVA (n ​= ​5/group, ^#p ​< ​0.05, ^##p ​< ​0.01, ACLT vs. other groups). Wilcoxon test is used to assess the differences between two different treatments (n ​= ​5/group,∗p ​< ​0.05, HA+LZ-SMS vs. other groups).

Figure 3

Anti-inflammatory LZ-SMS suppress pro-inflammatory cytokines in plasma and synovial fluid. The Bar charts are shown the levels of (A) plasma and (B) synovial fluid cytokines including IL-12p70, IL-17A, TNF-α, INF-γ, IL-6 and IL-10 from different treated groups. Differences among treatments are compared by one-way ANOVA (n ​= ​5/group, ^#p ​< ​0.05, ^##p ​< ​0.01, ACLT vs. other groups). Wilcoxon test is used to assess the differences between two different treatments (n ​= ​5/group,∗p ​< ​0.05, HA+LZ-SMS vs. other groups).

Figure 4

Micro-architecture of subchondral bone in the ACLT induced OA rats. (A) 3-view micro-CT images of the right hind knee joint of rats with different treatment. Blue arrow pointed blue rectangle is shown the region of interest (ROI) in Sham group. Blue arrow pointed bone erosion is shown in ACLT group. Red arrow pointed bone sclerosis at distal medial tibial SCB plate is shown in LZ-SMS and ACLT groups. Green arrow pointed SCB cysts are shown in ACLT and HA groups. (B) 3D micro-CT reconstruction images of the ROI under the loadbearing area of medial tibial SCB from rats with different treatment. (C) The Bar charts are shown with the mean values of detailed micro-architectural parameters of medial tibial SCB. Differences among each parameter are compared by one-way ANOVA (n ​= ​5/group, ^#p ​< ​0.05, ^##p ​< ​0.01, ^###p ​< ​0.0001, ACLT vs. other groups). Wilcoxon test is used to assess the differences between two different treatments (n ​= ​5/group, ∗p ​< ​0.05, ∗∗p ​< ​0.01; ∗∗∗p ​< ​0.0001, HA+LZ-SMS vs. other groups). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 5

Active compound of LZ-SMS target-OA and protein–protein interaction (PPI) network analysis. (A) Venn diagram of drug-disease targets. The cyan circle represents 2496 known therapeutic targets for the treatment of OA. The red circle represents 108 known drug-targets of LZ-SMS. Totally 64 shared targets were shown between LZ-SMS and OA. (B) Active compound-target-disease network analysis. The blue rectangle represents active compound from LZ-SMS. The green oval represents the shared targets between LZ-SMS and OA. (C) The PPI analysis of 64 overlapping targets of LZ-SMS and OA. The nodes get larger with increasing degree. Edges: PPI between shared targets and their interactive partners. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 6

Gene Ontology (GO) enrichment and KEGG pathways analysis for 64 drug-disease targets. (A) The top 30 significant GO enrichment analysis. The red circle represents the gene count of each biological process. (B) The top 30 significant KEGG pathways. The blue rectangle shows the MAPK signaling pathway. The bigger of circles, the more targets are. The darker of color, the more significant is. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)