Salvianolic acid-B improves fat graft survival by promoting proliferation and adipogenesis

Our previous study proved that Salvia miltiorrhiza could enhance fat graft survival by promoting adipogenesis. However, the effect of salvianolic acid B (Sal-B), the most abundant and bioactive water-soluble compound in Salvia miltiorrhiza, on fat graft survival has not yet been investigated. This study aims to investigate whether salvianolic acid B could improve fat graft survival and promote preadipocyte differentiation. The underlying mechanism has also been studied. In vivo, 0.2 ml of Coleman fat was transplanted into nude mice with salvianolic acid B. The grafts were evaluated by HE and IF at 2 and 4 weeks posttransplantation and by micro-CT at 4 weeks posttransplantation. In vitro, the adipogenesis and proliferative activities of salvianolic acid B were analyzed in cultured human adipose-derived stem cells (h-ADSCs) and 3T3-L1 cells to detect the mechanism by which salvianolic acid B affects graft survival. In vivo, the weights and volumes of the fat grafts in the Sal-B-treated groups were significantly higher than those of the fat grafts in the control group. In addition, higher fat integrity and more viable adipocytes were observed in the Sal-B-treated groups. In vitro, salvianolic acid B showed the ability to promote 3T3-L1 and h-ADSC proliferation and adipogenesis. Our in vitro experiments demonstrated that salvianolic acid B can promote the proliferation of adipose stem cells and enhance the differentiation of adipose stem cells. Simultaneously, in vivo experiments showed that salvianolic acid B can improve the survival rate of fat transplantation. Therefore, our research shed light on the potential therapeutic usage of salvianolic acid B in improving the survival rate of fat transplantation.

treatment [7]. In 2019, The American Aesthetic Society reported that 102,155 autologous fat grafting procedures were performed for buttock augmentation (34,086), breast reconstruction (24,892) and face filling (43,177), demonstrating a steady upward trend from previous years [8]. However, autologous fat transplantation also presents inherent limitations, which means that the fat grafting has some disadvantages, such as high and unpredictable tissue absorption rates, which lead to uncertain efficacy. Meanwhile, autologous fat transplantation may involve a variety of complications, such as fibrous tissue formation, cyst formation, fat liquefaction necrosis and calcification, which may manifest after autologous fat transplantation [9]. Therefore, these disadvantages will influence the effect of autologous fat grafting.
With the development of fat grafting research, two key factors are involved in improving the survival rate of transplanted fat: accelerating the revascularization of transplanted adipose tissue (revascularization theory [10]) and promoting the differentiation of preadipocytes into adipocytes (preadipocyte theory [11,12]). Revascularization theory holds that transplanted adipose tissue can only maintain its nutrient supply by infiltration of surrounding tissue fluid, which is only 150-200 microns away, until sufficient blood supply is established between the graft and the host tissue. However, the host's neovasculature usually grows into the graft 5 days after transplantation and can only invade the periphery of the graft. Therefore, the survival rate of transplanted fat can be improved to some extent by accelerating the reconstruction of blood supply in the transplanted body, increasing the blood supply of the transplanted body, shortening the ischemic period of transplanted adipocytes, and increasing the reconstruction of blood supply in the transplanted body. For example, adipose tissue-derived stromal cells (ADSCs) [13], stromal vascular fraction (SVF) [14,15], VEGF [16], etc. are used to increase the blood supply of transplantation. However, there are some restraints, such as the need for in vitro culture, cumbersome operation, easy pollution, consumption of a large amount of fillable adipose tissue, and poor patient compliance. Nonetheless, the survival rate of large volume fat transplantation is not satisfactory even if these auxiliary methods are used.
From another point of view, preadipocyte theory suggests that compared with mature adipocytes, preadipocytes are smaller in size and more tolerant to ischemia and hypoxia. In the early stage of fat transplantation, many mature adipocytes are necrotic and apoptotic due to ischemia, hypoxia and malnutrition. Some mature adipocytes are transformed into preadipocytes. When the environment of the fat transplantation recipient area is improved and blood supply and nutrition are sufficient, preadipocytes differentiate into mature adipocytes again [12]. According to the theory of preadipocytes, we should promote the proliferation of poorly differentiated preadipocytes, transform them into mature adipocytes, and maintain the volume and weight of adipose tissue as much as possible to improve the survival rate of fat transplantation.
Salvia miltiorrhiza is an important traditional Chinese medicine that plays an important role in the treatment of cardiovascular diseases [17]. Our previous study found that Salvia miltiorrhiza can effectively improve the survival rate of free fat transplantation in a fat graft rabbit model [18], and the fat retention rate of patients with autologous fat transplantation was significantly improved (SM group vs. non-SM group, 60.06 ± 16.12% vs. 34.04 ± 11.15%) [19]. Second, Salvia miltiorrhiza can promote the differentiation of preadipocytes. Salvianolic acid B (Sal-B) is the most abundant and bioactive watersoluble compound in Salvia miltiorrhiza. Studies [20] have shown that salvianolic acid B increases the mRNA expression of adipogenic transcription factors, including PPARγ, C/EBPα and PPARα, in 3T3-L1 preadipocytes to increase glucose uptake and mitochondrial respiration, reduce glycerol release and promote adipocyte differentiation. However, the effect of Sal-B on graft fat survival and its specific mechanism have not been investigated. In this study, we attempted to investigate whether Sal-B improves the survival rate of fat transplantation by promoting preadipocyte differentiation.

Human adipose-derived stem cell (h-ADSC) isolation
The use of human tissue was approved by the local ethics committee of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. Adipose tissue particles were harvested during thigh liposuction from adult female human patients (n = 5; mean age, 28 years; mean BMI, 24.5) at the Department of Plastic and Reconstruction Surgery, Shanghai Ninth People's Hospital. All tissues were waste materials collected as a byproduct of surgery, and the samples were immediately transported to the tissue culture laboratory for processing.
The collected fat particles were washed with phosphate-buffered saline (PBS) and allowed to stand for 5 min at room temperature, and then the lower layer of tumescent fluid and blood components was discarded. Then, the fat particles were mixed with an equal volume of prepared type IV collagenase solution (Gibco, USA). The solution's final concentration was 0.2%, which was dissolved in low-sugar Dulbecco's modified Eagle's medium (low-sugar DMEM, HyClone, USA), and the solution was filtered twice with a 0.22 μm filter (Falcon, USA). Then, the samples were placed on a 37 °C shaker and shaken at a speed of 175 rpm for 1 h. After shaking, the solution was centrifuged at 1500-2000 rpm for 5 min and the supernatant and fat suspension were discarded. Ten milliliters of low-sugar DMEM with 10% fetal bovine serum (FBS, Gibco, USA) and 1% antibiotic-antimycotic (Gibco, USA) was added to the centrifuge tube (Falcon, USA), pipetted evenly and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and cell pellets were observed at the bottom of the tube. Three milliliters of FBS-containing medium was added and pipetted evenly, and then 8 ml of serum-containing medium was added, mixed well and inoculated in a 10 cm petri dish. The cells were then placed in an incubator containing 20% oxygen and 5% carbon dioxide at 37 °C.

3T3-L1 and h-ADSCs expansion
The culture medium was changed every 48 h, and when the cells reached approximately 90% confluence, the 3T3-L1 cells (ATCC, USA) and h-ADSCs were washed three times using PBS. These adherent cells were then passaged using a solution of 0.25% trypsin plus 0.02% ethylenediaminetetraacetic acid (EDTA) (Gibco, USA) for 1 min. The cell suspension was further divided into three culture flasks along with growth medium. Cells at passage three were used for the experiment.

Adipogenic differentiation
The h-ADSCs from the 5 liposuction donors were mixed, and used for follow-up experimental research. 3T3-L1 cells and h-ADSCs at passage three were used for adipogenic differentiation, and the cell density was adjusted to 2 × 10 4 cells/L. Once the cells reached confluency, the growth medium was switched to induction medium A. After three days, the medium was replaced by induction medium B (all from Cyagen Biosciences, USA). Induction A and B were mixed with various concentrations of salvianolic acid B (0, 10, 50, 100 μmol/L) (Sal-B, Selleck, USA) before use. The cells were used on days 4 and 8 for follow-up experimental research.

Oil red O staining
To measure the degree of adipogenesis and differentiation, the cells were washed with PBS, fixed in 4% paraformaldehyde for 30 min at room temperature, and stained with fresh Oil Red O solution (Solarbio, China) for 30 min. Then, the stained cells were washed with PBS two times. Adipogenic differentiation was visualized by the presence of numerous intracellular lipid droplets using an inverted fluorescence microscope (Nikon, Japan). The image analysis was performed using ImageJ according to the method of Deutsch [21] and Lin [22]. After the scale of the image was established, the area of the lipid droplets was measured and was displayed by ImageJ as the surface area in square micrometers (μm 2 ). Three fields were measured for each of the 3 experiments, and three technical replicates were performed in each of the 3 experiments. The significance of differences between the control and treated groups was set at P < 0.05 and assessed by ANOVA with GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA).

Triglyceride assay
Lipid accumulation was used as a marker of adipogenic differentiation and was assessed through quantitation of triglycerides in the cell. Salvianolic acid B was added to the samples during differentiation. On days 4 and 8 of differentiation, the triglyceride level in the culture medium was measured by a triglyceride assay kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions. Briefly, the assay was initiated with the enzymatic hydrolysis of triglycerides by lipase to produce glycerol and free fatty acids. The glycerol released was subsequently measured by a microplate reader at a wavelength of 510 nm. The protein concentration was measured using the BCA concentration (Boster, China). The triglyceride content (mmol/gprot) = Triglyceride concentration (mmol/L)/Protein concentration (gprot/L).

5-Ethynyl-2′-deoxyuridine (EdU) proliferation assay
Cells were seeded in 24-well plates and incubated under standard conditions with various concentrations of salvianolic acid B (0, 10, 50, 100 μmol/L). Twenty-four hours after incubation, cell proliferation was detected using the EdU Cell Proliferation Assay Kit (Invitrogen, USA) according to the manufacturer's protocol. Briefly, cells were incubated with 50 μM EdU for 2 h before fixation, permeabilization, and EdU staining. Then, cell nuclei were stained with Hoechst 33,342 (Invitrogen, USA) for 30 min. The proportion of cells that incorporated EdU was determined by inverted fluorescence microscopy (Nikon, Japan). The cells were counted manually in each field, three fields were counted for each of the 3 experiments, and three technical replicates were performed in each of the 3 experiments. The proportion of Edu + cells (%) = 100*The number of Edu + (Green) cells/The number of total cells(Hoechst33342 + cells, Blue). The significance of differences between the control and treated groups was set at P < 0.05 and assessed by ANOVA with GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA).

Cell viability assays
The effects of salvianolic acid B on the viability of 3T3-L1 cells and h-ADSCs were tested by a Cell Counting Kit-8 (Beyotime, China) according to the manufacturer's instructions. Briefly, 5000 cells/well were seeded in a 96-well plate. The cells were cultured in growth medium or adipogenic differentiation medium with various concentrations of salvianolic acid B (0, 0.1, 0.5, 1, 5, 10, 25, 50, 75, 100, 125 μmol/L). At 72 h of culture, 10% CCK-8 reagent was mixed with medium and added to each well. The 96-well plate was incubated at 37 °C for 2 h. The relative number of cells was measured by absorbance at 450 nm using a microplate reader (Thermo, USA).

Flow cytometry
After 3 days in culture with various concentrations of salvianolic acid B pretreatment, h-ADSCs were resuspended in PBS buffer according to the number of cells (5000/ml). For Annexin V and propidium iodide staining, 195 μL of cell suspension was mixed well with 5 μL Annexin V-FITC and incubated at room temperature for 10 min. The cells were washed with PBS and resuspended in 190 μL of deliquated binding buffer, and then 10 μL of 20 µg/ ml propidium iodide was added. The samples were analyzed by flow cytometry using CytoFLEX LX (Beckman Coulter, USA). The data were analyzed by CytExpert (Beckman Coulter, USA).

Nude mouse coleman fat graft model
The process of the animal experiment is shown in Additional file 2: Figure S2. All animal experiments were approved by Shanghai Ninth People's Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, China. Female nude mice (aged 6 to 8 weeks) were housed in individual cages with a 12-h light/dark cycle and provided with standard food and water ad libitum. The mice were randomly divided into three groups (6 mice per group): saline, 10 μmol/L, and 50 μmol/L. Each mouse was injected subcutaneously on the left and right flank of the back with 0.2 ml of Coleman fat using a 1 ml syringe with a blunt infiltration cannula. The grafts were injected into a spherical shape. The mice were locally injected with 0.2 ml of saline or salvianolic acid B (10 μmol/L, 50 μmol/L) once every 2 days. At 14 days, the grafts on the left back were harvested and carefully separated from surrounding tissue, and their volumes and weights were measured. The wounds were closed with size 6-0 nylon sutures, and antibiotic ointment was applied to the affected area for 1 week to prevent local infection. Each harvested sample was assessed histologically and immunohistochemically. At 28 days, the fat grafts were scanned via micro-CT, the grafts were harvested and carefully separated from surrounding tissue, and their volumes and weights were measured. Each harvested sample was assessed histologically and immunohistochemically.

Histological analysis and immunofluorescence staining
Tissues were fixed in paraformaldehyde overnight, embedded in paraffin, and cut at a thickness of 5 μm and then stained with hematoxylin and eosin. We used the methods of Shoshani [23] and Yu [24] to evaluate the histologic parameters, such as cell integrity, tissue inflammation, presence of cysts/vacuoles, and the extent of fibrosis. Each parameter was scored as 0 = absence, 1 = minimal presence, 2 = minimal to moderate presence, 3 = moderate presence, 4 = moderate to extensive presence, and 5 = extensive presence. The scoring was performed independently by 3 authors who were unaware of the grouping.

RNA extraction and real-time RT-PCR
To investigate the adipogenic differentiation potential of 3T3-L1 cells and h-ADSCs, we assessed the transcriptional levels of PPARγ, C/EBPα and FABP4 in 3T3-L1 cells and h-ADSCs by real-time PCR assays. Initially, total RNA of 3T3-L1 cells and ADSCs was extracted using a total RNA miniprep kit (Axygen, USA), and RT-qPCR was performed with an ABI 7900HT system using SYBR Premix (Takara, Japan) according to the manufacturer's instructions. mRNA quantification was performed using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for normalization. The SYBR green primers for qRT-PCR are listed in Additional file 4: Table S1.

Micro-CT analysis
The fat grafts were scanned via micro-CT (PerkinElmer, USA), and the fat grafts were analyzed by ProPlan CMF 3.0.

RNA-Seq analysis
RNA sequencing samples were acquired after the addition of Sal-B (50 μmol/L) or solvent to h-ADSCs which were from two patients for 4 days and 8 days in differentiation medium. RNA quantity and quality were measured using a NanoDrop ND-1000.

Statistical analysis
In this study, all in vitro experiments were conducted 3 times. Single blinding was used for the statistical analysis. Two blinded data analysts independently analyzed the data. The final data were consistent between the two analysts. Data are expressed as the mean ± SD. The continuous variables between the two groups were compared by the independent samples t-test. One way ANOVA with the Tukeys post-hoc test was employed for pairwise comparisons among multiple groups. The significance of differences between the control and treated groups was set at P < 0.05 and assessed by GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA).

Salvianolic acid B facilitates the survival rate of transplanted fat.
Our previous study found that Salvia miltiorrhiza could improve the survival rate of fat transplantation in fat graft rabbit models [18] and in patients with autologous fat grafting to the breast [19]. To explore whether salvianolic acid B, one of the main active ingredients of Salvia miltiorrhiza, plays an important role in improving the survival rate of fat transplantation, a nude mouse fat transplantation model was established. The nude mice were randomly divided into three groups: a control group (physiological saline) and two experimental groups (salvianolic acid B: 10 and 50 μmol/L). The results showed that in the second week, there was no significant difference between the control group and the experimental group. We attributed it to inflammation-led tissue swelling that made the difference not obvious. However, in the fourth week, we found that the volume retention rate of the 50 μmol/L salvianolic acid B treatment group was significantly higher than that of the control group (50 μmol/L vs control: 0.142 ± 0.026 vs 0.058 ± 0.031 ml, **P < 0.01) (Fig. 3a). In addition, to further explore the structural changes of the fat grafts, we conducted H&E staining and immunofluorescence detection. We found that the inflammatory cell infiltration level of the salvianolic acid B treatment groups was less than that of the control group in the second or fourth week, and the integrity of fat cells was better than that of the control group (Fig. 3b, d). Further, through immunofluorescence, the results showed that the number of Perilipin + living adipocytes in the salvianolic acid B treatment groups was significantly higher than that in the control group (2 weeks, 50 μmol/L vs 10 μmol/L vs control: 50.98 ± 9.87 vs 46.57 ± 7.12 vs 31.78 ± 5.99, *P < 0.05, **P < 0.01; 4 weeks, 50 μmol/L vs 10 μmol/L vs control: 59.59 ± 6.80 vs 49.08 ± 9.42 vs 28.10 ± 7.00, **P < 0.01) (Fig. 3c, e). By using micro-CT to analyze subcutaneous fat grafts, we found that salvianolic acid B increased the survival rate of fat grafts (50 μmol/L vs 10 μmol/L vs control: 59.36 ± 23.20% vs 41.36 ± 24.96% vs 20.45 ± 14.51%, * P < 0.05) (Fig. 4a, b). In general, salvianolic acid B can promote the survival rate of fat grafts.

RNA-Seq analysis suggests that salvianolic acid B can promote adipocyte differentiation
To further clarify the effect of salvianolic acid B on adipose stem cells, we collected cells on the 4th and 8th days and then performed RNA sequencing in both the case and control groups. The results indicated that large amounts of genes were differentially expressed between the case and control groups on the 4th and 8th days (Fig. 5a). On the one hand, adipogenesis genes, such as Cebpα, Fabp4, CD36, and LPL, were significantly upregulated (Fig. 5b). In addition, upregulated genes were enriched in the PPAR signaling pathway through KEGG analysis (Fig. 5c, Additional file 3: Figure S3A). On the other hand, studies have shown that extracellular matrix remodeling is necessary for adipose cell differentiation [26]. Notably, genes related to the extracellular matrix, such as FN1, ITGA4, ITGA5, ITGB3, SDC4, THBS1, and TNXB (Fig. 5b), were downregulated. Meanwhile, they were enriched in the ECM receptor response pathway through KEGG analysis (Fig. 5c, Additional file 3: Figure S3B). Furthermore, the focal adhesion pathway was also downregulated (Fig. 5c). It has been reported that reassembly of focal adhesion (FA) is essential for fat differentiation and that decreasing the expression of focal adhesion can improve adipogenesis [27]. Our experiments indicated that the expression of focal adhesion-related genes in the drug group was downregulated. Moreover, our DE heat map further confirmed that salvianolic acid B could change the expression of genes of preadipocytes in adipogenesis (Fig. 5d). At the same time, the GSEA results also verified the upregulation of the PPAR pathway and the downregulation of ECM-and FA-related pathways (Fig. 5e). In general, our sequencing results imply that salvianolic acid B is likely to promote adipose stem cell differentiation by enhancing the expression of adipogenic-related genes while restraining the gene expression of extracellular matrix-related pathways. The promoter effect of salvianolic acid B on the adipogenic-related genes described above was also demonstrated by quantitative RT-PCR and western blot (Fig. 6a, b). Meanwhile, at a concentration of 50 μmol/L, salvianolic acid B showed the best effect, which is consistent with our previous conclusion.

Discussion
Autologous fat grafting has become a common technique for repairing volume and contour deficiencies in plastic and reconstructive surgery [28]. The biggest problem related to autologous fat transplantation is the unpredictable absorption rate [29]. In previous experimental studies, the absorption rate of fat transplantation was as high as 80% [30], which is consistent with the results of the control group in our experiment (survival rate in the control group: 20.45 ± 14.51%). Hence, it is still needed to further explore how to improve the survival rate of autologous fat transplantation. In other studies, researchers have tried to improve the blood supply by using ADSCs [13], SVF [14,15], and VEGF [16] to improve the survival rate of transplanted fat. However, there are some restraints, such as the need for in vitro culture, cumbersome operation, easy pollution, consumption of a large amount of fillable adipose tissue, and poor patient compliance. Nonetheless, the survival rate of large volume fat transplantation is not satisfactory, even if these auxiliary methods are used. In our previous research, we observed that a mixture of traditional Chinese medicine ingredients including Salvia miltiorrhiza, which contains Sal-B, can improve the survival rate of fat transplantation in a rabbit model and in patients with autologous fat grafting to the breast [18,19]. In the present study, we identified Sal-B as the main active molecule in Salvia miltiorrhiza and showed that it can improve the survival rate of fat transplantation in a nude mouse fat graft model (50 μmol/L vs 10 μmol/L vs control: 59.36 ± 23.20% vs 41.36 ± 24.96% vs 20.45 ± 14.51%, *P < 0.05).
Researchers believe that later in fat transplantation, a large number of adipocytes, especially those near the center, become necrotic. Instead, preadipocytes proliferate and differentiate, producing new adipocytes to fill the necrotic area. Active cell proliferation appeared approximately 1 week after transplantation and lasted until 12 weeks after transplantation [12]. Therefore, researchers have suggested that enhancing the amount of preadipocytes in the graft or promoting the proliferation and adipogenic differentiation of more preadipocytes can retain more fat grafts, thereby improving the efficiency of transplantation. Our in vitro results demonstrated that Sal-B can promote the proliferation capability of 3T3-L1 cells and h-ADSCs and down-regulate the pathway of cell cycle/senescence and death pathways according to RNA-Sequencing. But high concentration such as 100 umol/L was actually preventing cell proliferation and showing more modest effects on adipose differentiation, since there was higher apoptosis in 3T3-L1 and lower TG production in both 3T3-L1 and h-ADSCs at D8. PPARγ and C/EBPα are key regulatory factors in adipogenesis [31]. PPARγ is necessary for inducing the differentiation of preadipocytes into adipocytes [32] and can promote adipogenesis in C/EBPα-deficient cells. In contrast, C/EBPα is unable to promote adipogenesis in PPARγ-deficient cells, which indicates that PPARγ is the main regulator of adipogenesis [33]. Otherwise, cross-regulation between C/EBPα and PPARγ is important for maintaining the differentiated state of cells [34]. In addition to PPARγ and C/EBPα, other transcription factors are also involved in adipocyte differentiation. These transcription factors function at different stages of adipogenesis to produce mature adipocytes. Among them, fatty acid binding protein 4 (FABP4) is one of the representatives, and it is responsible for the formation of mature adipocytes. In this study, through RNA-Seq analysis and subsequent PCR/Western blot verification, we revealed that Sal-B can promote the expression of adipogenesis-related genes, such as PPARγ, C/EBPa and FABP4, which indicated that Sal-B can promote ADSC adipogenic differentiation. Consistent with our findings, a recent study has shown that Sal-B can improve the mitochondrial activity of 3T3-L1 cells and increase the expression of adipogenesis-related genes [20]. This finding also revealed the potential of Sal-B in promoting adipogenesis. Finally we think the Sal-B could be injected into the local transplantation area or mix with fat graft in future clinical usages, such as breast reconstruction, face filling and other clinical usages.
In addition, we observed some interesting results in our experiment, and the degree of inflammatory cell infiltration in the salvianolic acid B-treated group was lower than that of the control group. Meanwhile, in the GSEA analyze of RNA-seq, we found the pathways of IL-17 and NF-kB were down-regulation in Sal-B treated group (Additional file 3: Figure S3C). Researchers have found that inflammation could inhibit adipogenesis [35]. Moreover, overactivation of inflammation reduces the survival rate of fat transplantation [12,36,37]. Therefore, salvianolic acid B may have an anti-inflammatory effect in fat transplantation, although this supposition requires more in-depth research. Otherwise, according to the results of RNA-Sequencing, we found that the salvianolic acid B could up-regulate the pathways of thermogenesis and oxidative phosphorylation in day 4 and metabolism genes in day 8, which was consistent with other researches in the field of the obesity studies [38,39]. Therefore, we will conduct in-depth research on the anti-obesity mechanism of salvianolic acid B in subsequent studies.

Conclusions
Salvianolic acid B can promote the proliferation of adipose-derived stem cells and enhance the differentiation of adipose stem cells by increasing the expression of adipogenesis-related genes. Our in vivo experiments indicated that salvianolic acid B can improve the survival rate of fat transplantation. Therefore, salvianolic acid B is a promising agent to improve the survival rate of fat transplantation.