Ginsenoside Rb1 affects mitochondrial Ca2+ transport and inhibits fat deposition and fibrosis by regulating the wnt signaling pathway to treat rotator cuff tears via docking with SFRP1

Abstract

Background: Rotator cuff tears (RCTs) are among the most common musculoskeletal disorders that affect quality of life. This study aimed to investigate the efficacy of ginsenoside Rb1 in RCTs and the mechanisms involved.

Methods: First, a fibrotic model of FAPs was induced, and FAPs were cultured in media supplemented with different concentrations of ginsenoside Rb1. Next, a rat model of RCTs was constructed and treated with ginsenoside Rb1. Molecular docking was subsequently utilized to detect the binding of ginsenoside Rb1 and SFRP1. Finally, SFRP1 was knocked down and overexpressed in vivo and in vitro to investigate the mechanism of ginsenoside Rb1 and SFRP1 in RCTs.

Results: Compared with the Normal group, FAP viability was decreased, but Collagen II, FN and α-SMA levels were increased in the Control group. After treatment with different concentrations of ginsenoside Rb1, FAP viability increased, but Collagen II, FN and α-SMA levels decreased. Among them, 60 µM ginsenoside Rb1 had the best effect. In vivo experiments revealed that ginsenoside Rb1 improved RCTs in rats. Molecular docking revealed the binding of ginsenoside Rb1 to SFRP1. Additionally, SFRP1 levels were lower in the Control group than in the Normal group. After treatment with ginsenoside Rb1, SFRP1 levels increased. In vivo, overexpressing SFRP1 along with ginsenoside Rb1 treatment further alleviated tendon tissue fibroblast infiltration and fat accumulation and further reduced the expression of Collagen II, FN, and α-SMA. In vitro, overexpressing SFRP1 along with ginsenoside Rb1 treatment further decreased the expression of CaMKII, PLC, PKC, Wnt, and β-catenin, further decreased the Ca²⁺ fluorescence intensity and mitochondrial length, increased the red/green intensity, and decreased the MitoSOX fluorescence intensity. Additionally, overexpressing SFRP1 along with ginsenoside Rb1 treatment further increased cell proliferation, decreased apoptosis, reduced the protein expression of Collagen II, FN, and α-SMA in muscle tissue, and further reduced the levels of TNF-α, IL-1β, and IL-6 in the cell supernatant.

Conclusions: Ginsenoside Rb1 inhibited the activation of the Wnt signaling pathway by promoting SFRP1 expression, thereby inhibiting mitochondrial function and Ca²⁺ absorption to treat fat infiltration and muscle fibrosis caused by RCTs.

Keywords: Ca2+; Ginsenoside Rb1; Rotator cuff tears; SFRP1; Wnt.

Fig. 1

Screening of the optimal ginsenoside Rb1 concentration. A. IF staining for α-SMA. B. Cell proliferation was assessed using the CCK-8 assay. C and D. The levels of the Collagen II, FN and α-SMA mRNAs and proteins were detected via qRT‒PCR and Western blot. *P < 0.05 compared with the Normal group and #P < 0.05 compared with the Control group

Fig. 2

Ginsenoside Rb1 improved RCTs in rats. A. HE staining of the injured tissue from each group. B. Masson’s trichrome staining of infiltrated tissue fibers. C. Oil red O staining of fat accumulation in the tissue. D. qRT‒PCR and Western blot were performed to detect the expression of lipid-related molecules (Pparγ, C/ebpα, Fas, Fabp4, and Apn). E. Collagen II, FN and α-SMA mRNA levels. F. Protein expression of Collagen II, FN, α-SMA and SFRP1. G. IF staining showing SFRP1 levels. *P < 0.05 compared with the Sham group and #P < 0.05 compared with the RCTs group

Fig. 3

Ginsenoside Rb1 targeted SFRP1. A. Structural analysis of the main active components of ginsenoside Rb1. B. Molecular docking analysis of ginsenoside Rb1 binding to SFRP1. C. The DARTS assay was used to investigate the interaction between ginsenoside Rb1 and SFRP1. D and E. SFRP1 mRNA and protein expression. *P < 0.05 compared with the Normal group and #P < 0.05 compared with the Control group

Fig. 4

Ginsenoside Rb1 improved RCTs by influencing SFRP1 to inhibit fat deposition and fibrosis. A. HE staining of the injured tissue from each group. B. Masson staining of infiltrated tissue fibers. C. Oil red O staining of fat accumulation in the tissue. D. qRT‒PCR and Western blot were utilized to measure the expression of lipid-related molecules (Pparγ, C/ebpα, Fas, Fabp4, and Apn). E. Collagen II, FN, α-SMA and SFRP1 mRNA and protein expression. F. IF staining showing Collagen II, FN, and α-SMA levels. *P < 0.05 compared with the Sham group, #P < 0.05 compared with the RCTs group, @P < 0.05 compared with the Rb1 + oe-NC group, and &P < 0.05 compared with the Rb1 + si-NC group

Fig. 5

Ginsenoside Rb1 affected mitochondrial Ca²⁺ transport by regulating the Wnt signaling pathway through SFRP1. A and B. CaMKII mRNA and protein expression. C and D. ICC analysis of the distribution of β-catenin. E and F. Changes in Ca²⁺ absorption were detected via fluorescence calcium imaging. G. MitoTracker Red CMXRos staining of mitochondrial morphology. H and I. The mitochondrial membrane potential was evaluated with a JC-1 kit. J and K. MitoSOX staining was utilized to measure mitochondrial ROS. *P < 0.05 compared with the Control group, #P < 0.05 compared with the Rb1 + oe-NC group, and &P < 0.05 compared with the Rb1 + si-NC group

Fig. 6

Ginsenoside Rb1 regulated the Wnt signaling pathway through SFRP1 to affect mitochondrial Ca²⁺ transport and inhibit cell damage and fibrosis. A. The CCK-8 assay was performed to detect cell proliferation in each group. B. Apoptosis was determined by flow cytometry. C. Western blot analysis of Caspase-3, Bax, and Bcl-2 levels. D. Western blot analysis of Collagen II, FN and α-SMA levels in cells. E. The contents of TNF-α, IL-1β and IL-6. *P < 0.05 compared with the Control group, #P < 0.05 compared with the Rb1 + oe-NC group, and &P < 0.05 compared with the Rb1 + si-NC group

Fig. 7

Graphical abstract. The mechanism by which ginsenoside Rb1 treats fat infiltration and muscle fibrosis caused by RCTs