Shenfu Injection: A Famous Chinese Prescription That Promotes HCN4 Activity in Bone Marrow Mesenchymal Stem Cells

Abstract

We investigated the effects of Shenfu Injection (SFI) on HCN4 activity in bone marrow mesenchymal stem cells (BMSCs). The sample of BMSCs was divided into six groups: a control group, a high-dose SFI group (0.25 ml/ml), a middle-dose SFI group (0.1 ml/ml), a low-dose SFI group (0.05 ml/ml), an adenovirus-encoded control vector group, and an adenovirus-encoded HCN4 group. Cell ultrastructure was observed using a transmission electron microscope. Quantitative reverse transcription PCR (RT-qPCR) was performed to detect HCN4 expression, and HCN4 activity was detected using the whole-cell patch clamp technique. An enzyme-linked immunosorbent assay was performed to detect cAMP content. Application of flow cytometry confirmed that the isolated cells showed BMSC-like phenotypes. Differentiation of BMSCs in both the SFI and the adenovirus-encoding HCN4 groups occurred according to the cellular ultrastructure. Application of the whole-cell patch clamp technique revealed that SFI could activate the inward pacing current of BMSCs in a concentration-dependent manner. The RT-qPCR results showed that HCN4 expression was significantly higher in the high-dose SFI group than in the medium- and low-dose groups, whereas the cAMP content in the overexpressed HCN4 group decreased significantly; this content in the high-dose SFI group increased significantly. In conclusion, SFI promotes HCN4 activity in BMSCs, which could explain its treatment effect when administered to patients with cardiovascular diseases.

Figure 1

Flow cytometry used to detect the surface markers of bone marrow mesenchymal stem cells (BMSCs). The possession of a BMSC-like phenotype by the isolated cells was confirmed by the positive rates of CD45, CD29, and CD44 (1.19%, 98.9%, and 98.19%, respectively).

Figure 2

Transfection efficiency of HCN4. A representative image of transfection. Green fluorescent protein (GFP) expression was observed under the microscope in both empty and overexpressed groups 48 h after transfection (a). Scale bar: 100 μm. The reverse transcription polymerase chain reaction (RT-PCR) results revealed that HCN4 expression in the overexpressed group was higher than that in the control group and in the NC group (b), indicating that the transfection was successful. ^∗P < 0.05, compared with the control group; ^#P < 0.05, compared with scrambled control (NC) (N = 6 in each group).

Figure 3

The ultrastructure of bone marrow mesenchymal stem cells (BMSCs) observed with a transmission electron microscope. In the normal saline group, the nuclei of the BMSCs, which had clear nucleoli and nuclear membranes, were positioned on one side. In the Shenfu Injection-treated group, differentiation of BMSCs was conspicuous, evidenced by rough endoplasmic reticulum, increased mitochondria, cell expansion, and increased stromal vesicles. In the overexpressed HCN4 group, the nuclei were larger, the membrane structure was intact, and there was a clear increase in the rough endoplasmic reticulum, mitochondria, and stromal vesicles compared with those of the control group. Scale bar: 2 μm.

Figure 4

Effect of Shenfu Injection on I-V curve in bone marrow mesenchymal stem cells (BMSCs). (a) Representative traces; (b) quantified I-V curve; (c) max HCN current. BMSCs in the overexpressed HCN4 group evidenced hyperpolarization activated inward pacing current, while BMSCs in Shenfu Injection treatment group also showed increased inward pacing current in a concentration-dependent manner (^∗P < 0.05; N = 10 in each group).

Figure 5

Effect of Shenfu Injection on HCN4 and cAMP expression in bone marrow mesenchymal stem cells (BMSCs). The expression of HCN4 at the mRNA level in BMSCs after treatment with Shenfu Injection increased in a concentration-dependent manner (a). The cAMP content was detected by performing an enzyme-linked immunosorbent assay (ELISA) (b). The cAMP content in the high-dose Shenfu Injection group increased, whereas the cAMP content in the overexpressed HCN4 group decreased significantly. Compared with the control group, ^∗P < 0.05 (N = 6 in each group).