The Molecular Mechanism Investigation of HBP-A Slows Down Meniscus Hypertrophy and Mineralisation by the Damage Mechanical Model

Abstract

HBP-A is the main active component of a traditional Chinese medicine Huaizhen Yanggan Capsule, for the remarkable treatment of knee osteoarthritis (KOA). This study aimed to elucidate the ameliorative effect of HBP-A on meniscus hypertrophy and mineralisation in KOA and the molecular mechanism of its action. An Hartley guinea pig model of KOA that underwent anterior cruciate ligament transection (ACLT) and a model of rat primary meniscus fibrochondrocytes (PMFs) were used to investigate the ameliorative effect of HBP-A on meniscal hypertrophy and calcification and its signal transduction mechanism of action. The results show that Guinea pig's meniscus width, as well as the area of meniscus calcification and meniscus and articular cartilage injury score, were significantly reduced in the HBP-A intervention group compared to the ACLT group. The expression levels of mtrix metalloproteinase 13 (MMP13), runt-related transcription factor 2 (Runx2), Indian hedgehog (Ihh), alkaline phosphatase (ALP), and ankylosis homologue (ANKH) at the protein and gene level significantly decreased in the HBP-A intervention group compared to the ACLT group. In vitro study, apoptosis, hypertrophy, and calcification of rat PMFs after 10% stretch force were significantly improved with HBP-A intervention. Western blot and RT-qPCR showed that hypertrophy, calcification, and p38 MAPK signalling pathway-related markers of PMFs were incredibly depressed in the HBP-A intervention group compared to the 10% stretch force group. In conclusion, HBP-A can slow down meniscus hypertrophy and mineralisation induced by abnormal mechanical loading, and its mechanism of action may be through the p38-MAPK signalling pathway.

Keywords: HBP‐A; KOA; abnormal mechanical damage; hypertrophy and mineralisation; meniscus; p38‐MAPK signalling pathway.

FIGURE 1

HBP‐A slows down meniscal hypertrophy and mineralisation. Right knee joints: ACLT group. The left joints: Self‐controlled study. Con: Normal guinea pig. (A, B) Width measurement and quantification analysis of hypertrophy of medial and lateral meniscus after intervention with different concentrations of HBP‐A. Quantitative comparison of the left and right knee joints in the medial or lateral menisci. # vs left knee, p > 0.05; * vs left knee, p < 0.05. L‐M‐M, Left Medial Meniscus; R‐M‐M, Right Medial Meniscus; L‐L‐M, Left Lateral Meniscus; R‐L‐M, Right Lateral Meniscus. (C, D) Alizarin red staining and quantification analysis showed meniscal mineralisation and its area of the right medial meniscus after intervention with different concentrations of HBP‐A. Data are shown as mean ± SD, n = 4, # vs Con, p < 0.05; *, **, *** vs ACLT, p < 0.05.

FIGURE 2

HBP‐A reduced the degeneration of the right medial meniscus and tibial articular cartilage. (A, C) Safranin O/Fast Green staining and OA menisci damage grade exhibited the damage of medial meniscus of the right knee joints and the effect of different concentrations of HBP‐A intervention. (B, D) The overall shape and Safranin O/Fast Green of the tibial articular cartilage and OA cartilage damage scores depicted the damage of the tibial articular cartilage of the right knee joints and the effect of different concentrations of HBP‐A intervention. Data are shown as mean ± SD, n = 4, # vs Con, p < 0.05; *, **, *** vs ACTL, p < 0.05.

FIGURE 3

HBP‐A reduced overexpression of relevant markers related to pathological hypertrophy and mineralisation. (A, B) Immunohistochemical staining and its quantitative analysis showed expression situations of hypertrophy‐related protein markers MMP13, Ihh, and Mineralisation‐related markers Runx2, ALP, ANKH in the medial meniscus. Positive signals: Claybank particles (show by black arrow). (C) RT‐qPCR showed the mRNA expression levels of meniscus hypertrophy marker IL‐1β, MMP13, Ihh, and mineralisation marker Runx2. * vs Con, p < 0.05; ** vs ACTL, p < 0.05.

FIGURE 4

0.3 mg/mL HBP‐A improved inhibition of PMF proliferation in vitro. (A) Immunofluorescence showed high expression of collagen type II and V in all cultured PMFs (red fluorescent: collagen II or X in the cytoplasm; blue fluorescent: nucleus). (B, C) Western blot and quantitative analysis showed expression of MMP13 in PMFs at different time points after 10% stretch intervention. ** vs Con, p < 0.05; * VS Con, p > 0.05. (D) CCK‐8 assays showed the effect of HBP‐A on the proliferation of PMFs. ** vs Con, p < 0.05. (E) CCK‐8 assay showed 0.3 mg/mL HBP‐A and SB203580 improved the inhibition of 10% stretch‐induced proliferation of PMFs. ** vs Con, p < 0.05; *** vs Con, p < 0.05; ## vs 10% stretch, p < 0.05.

FIGURE 5

0.3 mg/mL HBP‐A improved apoptosis and morphological changes of PMFs in vitro. (A, B) Flow cytometry and analysis showed a significant increase in PMF apoptosis in the 10% stretch group and that 0.3 mg/mL HBP‐A and SB differentially reduced PMF apoptosis induced by 10% stretch. (C, E) Staining by rhodamine‐labelled ghost pen cyclic peptide showed the morphology of PMFs in different groups. (D, F, G) Alizarin red and toluidine blue staining demonstrated the mineralisation and hypertrophy of the PMFs in different groups. * vs Con, p < 0.05, # vs 10% stretch.

FIGURE 6

HBP‐A also altered the expression of genes associated with hypertrophy, mineralisation, and the P38‐MAPK signalling pathway in PMFs. (A–I) RT‐qPCR showed that the overexpression of the genes associated with excessive hypertrophy, degeneration, and mineralisation of PMFs resulted from 10% stretch force in vitro and that HBP‐A reduces hypertrophy and mineralisation of PMFs by inhibiting the p38‐MAPK signalling pathway. * vs Con, p < 0.05, # vs 10% stretch, p < 0.05.

FIGURE 7

HBP‐A altered the protein expression related to hypertrophy, mineralisation, and the P38‐MAPK signalling pathway in PMFs. (A–H) Western and analysis showed that the overexpression of the protein markers associated with excessive hypertrophy, degeneration, and mineralisation of PMFs resulted from 10% stretch force in vitro and that HBP‐A and inhibitors reduced hypertrophy and mineralisation of PMFs by inhibiting the p38‐MAPK signalling pathway. * vs Con, p < 0.05, # vs 10% stretch, p < 0.05.