Berberine and cinnamic acid combined may improve high blood fat by affecting the PI3K/AKT/mTOR/SREBP-1 cell signaling pathway

Dulbecco's modified Eagle medium, BSA, penicillin–streptomycin, 0.25% trypsin solution, Cell Counting Kit‐8 (CCK‐8), a color‐enhanced protein molecular marker (EC0019) and 10% SDS/PAGE preparation kit (EC0023), poly(vinylidene difluoride) membranes (ED0005), a radioimmunoprecipitation assay, a bicinchoninic acid protein assay kit, and Sparkjade ECL super, recilisib (SJ‐MX5040A), LY294002 (SJ‐MX0083) and substrate were purchased from Shandong Sparkjade Biotechnology Co., Ltd (Shandong, China). Fetal bovine serum was obtained from Biochannel (Nanjing, China). The primary antibodies used for western blotting and immunohistochemistry are listed in Table S1. The secondary antibody, goat anti‐rabbit IgG (Alexa Fluor 488), was purchased from Shanghai Abway Biotechnology Co., Ltd (Shanghai, China). The Nuclear Protein Extraction Kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd (Shanghai, China). The synthesis of BBR‐CA and the detection of related indicators are as described in the references [14‐16]. In brief, BBR and CA (1 : 1 molar ratio, BBR 500 mg) were dissolved in 30 mL of MeOH. The solvent was removed with a rotary evaporator under vacuum condition of 45 °C. Yellow solids were obtained and dried in a vacuum oven at 40 °C for 2 h. The vapor diffusion method (MeOH:AcOEt) was used to scale up the co‐crystal preparation [14, 15, 16]. Single‐crystal X‐ray diffraction data were collected at 193 K on a SMART APEX II diffractometer (Bruker, Billerica, MA, USA) (Cu Kα radiation). The structure was solved with shelxt (http://shelx.uni‐goettingen.de/↗) and refined anisotropically for non‐H atoms using olex2 (https://www.olexsys.org/olex2/↗). Hydrogen atoms were located from difference Fourier maps and refined using a riding model. Disordered solvent was treated via platon squeeze (http://www.platonsoft.nl/platon/pl000000.html↗) (space group P2₁/*c*, Z′ = 1), removing 149 electrons per unit cell. Final refinement converged at R₁ = 0.05 (293 K). The crystal structure was visualized with diamond (https://www.crystalimpact.de/diamond↗).

All animal experiments conducted in this study were approved by the Animal Ethics Committee of Anhui University of Chinese Medicine (Ethics Number: AHUCM‐mouse‐2 023 156). Male C57BL/6J mice, aged 5 weeks, were obtained from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd (Hangzhou, China). The mice were subjected to a 12 : 12 h light/dark photocycle at 22 ± 2 °C and 55% relative humidity, and were provided with water and food ad libitum. Before the start of the experiment, the mice were allowed to acclimate for 1 week. Then, the mice were fed a normal food diet (NFD) and a HFD (MD12032; Jiangsu Medison Biopharmaceutical Co., Ltd, Jiangsu, China) for 8 weeks, with 8 mice in each group (n = 8). During this period, atorvastatin (AT) (10 mg·kg⁻¹), BBR (70 mg·kg⁻¹) and BBR‐CA (low dose 50 mg·kg⁻¹; medium dose 100 mg·kg⁻¹; high dose 150 mg·kg⁻¹) were administered for intervention treatment by gavage. The NFD group and the HFD group were given the same volume of distilled water. Animals were humanely euthanized‐under deep surgical isoflurane anesthesia (5%). Animals underwent cardiac puncture to collect terminal blood samples. The mice were dissected and samples were collected for the next experiment.

The fresh liver, heart, spleen, lungs and kidneys of mice were preserved in 4% paraformaldehyde solution, and their histological changes were evaluated using the H&E staining method.

Kits were e,ployed in accordance with the manufacturer's instructions (Shanghai Jiang Lai Bio‐Technology Co., Ltd, Shanghai, China) to detect the levels of TC, TG, low‐density lipoprotein cholesterol (LDL‐C) and high‐density lipoprotein cholesterol (HDL‐C) in the blood, as well as the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the liver.

Liver tissues and cells were lysed in radioimmunoprecipitation assay buffer supplemented with a protease inhibitor cocktail and a phosphatase inhibitor cocktail (Shandong Sparkjade Biotechnology Co., Ltd) and extract the nuclear proteins using the Nuclear Protein Extraction Kit (Shanghai Biyuntian Biotechnology Co., Ltd). Equal amounts of the lysates were separated by SDS/PAGE electrophoresis and transferred onto poly(vinylidene difluoride) membranes.

To investigate the expression of SREBP‐1 protein within the cell nucleus, an immunofluorescence assay was carried out. First, the cells cultured on the treated coverslips were harvested and fixed. Subsequently, permeabilization was performed using 0.2% Triton X‐100 (Saiguo Biotechnology Co., Ltd, Guangzhou, China) to facilitate antibody penetration. Following this, blocking was achieved with 5% BSA to minimize non‐specific binding. After the completion of the blocking process, the primary antibody specific to SREBP‐1 was added, and the samples were incubated overnight at 4 °C. The next day, the fluorescent secondary antibody conjugated with Alexa Fluor 488 was introduced, and the samples were incubated at room temperature under light‐protected conditions. Subsequently, nuclear staining was conducted using DAPI (i.e. 4′,6‐diamidino‐2‐phenylindole) staining solution under dark conditions. Finally, the samples were mounted with an anti‐fluorescence quenching mounting medium, and images were visualized and captured under a fluorescence inverted microscope.

Total RNA was isolated from the treated cells and liver using an RNA extraction kit (OSR‐M610; TIANGEN, Beijing, China). Next, 2 μg of RNA was used to synthesize cDNA using a cDNA synthesis kit (TaKaRa, Otsu, Japan). The primer sequences used in these experiments are listed in Table S2. The specificity of primers was confirmed by analyzing the characteristics of the PCR curve and the change in fluorescence intensity before the experiment. The fold change of genes was calculated using the ∆∆C_t method [25].

HepG2 cells (Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China) were cultured in Dulbecco's modified Eagle medium supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin–streptomycin. The cells were incubated at 37 °C in a 5% CO₂ incubator.

HepG2 cells (1.1 × 10⁴ cells per well) were seeded into 96‐well plates and incubated at 37 °C for 24 h. A hyperlipidemia cell model was established by treating human hepatocellular carcinoma cells (HepG2) with free fatty acids (FFA; sodium oleate and sodium palmitate at a ratio of 2:1; purchased from Shanghai Aladdin Biochemical Technology Co., Ltd, Shanghai, China) at a final concentration of 1 mm for 24 h. Meanwhile, the control group was incubated in normal medium. The cells were treated with drugs for 24 h before the model establishment. Subsequently, the cells were stained with Oil Red O (LEAGENE, Beijing, China) and the area of lipid droplets was observed under an inverted microscope. Then, the cells were decolorized with isopropanol, and absorbance was measured at 510 nm (for this, 100 μL of isopropanol was added, followed by shaking evenly on a shaker for 20 min, and absorbance was measured at 510 nm, which represents the lipid content). Under the same conditions, HepG2 cells (2.5 × 10⁵ cells per well) were seeded into six‐well plates and treated. After that, the content of TG was measured and western blotting experiments were carried out by extracting proteins.

The CCK‐8 kit was used to detect the effects of drugs and FFA on cell viability respectively, aiming to determine the concentrations of FFA and drugs in each group. We divided the cells into nine groups: the control group (CTL), the model group (FFA), the AT group (20 μm), the CA group (20 μm), the BBR group (20 μm), the CA + BBR group (20 μm), the low‐dose BBR‐CA co‐crystal group (L‐BBR‐CA) (10 μm), the medium‐dose BBR‐CA co‐crystal group (M‐BBR‐CA) (20 μm) and the high‐dose BBR‐CA co‐crystal group (H‐BBR‐CA) (30 μm). In addition, in the subsequent experiments, we also used the activator (recilisib, 10 μm) and inhibitor (LY294002, 10 μm) of PI3K to study the impact of BBR‐CA (10 μm) on the PI3K pathway.

The data are presented as the mean ± SD and were analyzed by one‐way analysis of variance (ANOVA) Duncan's multiple range test for comparison experiments. P < 0.05 was considered statistically significant.

The structural formula of BBR‐CA is shown in Fig. 1A and the X‐ray diffraction pattern of BBR‐CA is shown in Fig. 1B. The BBR‐CA co‐crystal was verified by X‐ ray powder diffraction technology, and the characteristic peaks of the obtained X‐ray diffraction spectrum appeared at 5.599, 8.121, 12.779, 14.532, 15.147, 16.286, 17.138, 17.471, 18.312, 19.733, etc. 22.733, 23.705, 24.221, 25.110, 25.362, 25.591, 25.948, 26.478, 27.355, 27.863, 28.184, 28.725, 29.840 and 30.225.

Following an 8‐week treatment period, a significant increase in body weight was observed in the mice of the HFD group compared to those in the NFD group. By contrast, the body weights of mice in each drug‐administered group were effectively controlled. As a positive control drug, AT significantly reduced the body weight of mice and improved lipid metabolism disorders. From the results, the overall effects of the BBR group and the BBR‐CA group with respect to improving mouse body weight and blood lipids were superior to those of AT (Fig. 1C–G). Notably, the H‐BBR‐CA group exhibited the most pronounced effect in body weight control, whereas the L‐BBR‐CA group demonstrated a comparable outcome to the BBR group (Fig. 1C). As previously reported, long‐term consumption of a HFD is frequently associated with abnormal lipid metabolism [1]. In the present study, HFD feeding led to disruptions in the levels of serum TC, TG, LDL‐C and HDL‐C compared to the NFD group (Fig. 1D–G). Upon supplementation with BBR‐CA, a significant reduction in the levels of TC, TG, LDL‐C and HDL‐C was noted compared to the HFD group. Remarkably, the efficacy of BBR‐CA in reducing TC and TG was substantially superior to that of BBR. Additionally, the levels of ALT and AST in the livers of mice were evaluated, and no significant differences were detected between each drug‐administered group and the HFD group (Fig. 1H,I). H&E staining revealed that the hepatic lobule structures in the NFD group were arranged in an orderly and compact fashion. By contrast, conspicuous lipid droplet accumulation was evident in the HFD group. However, following drug treatment, these pathological alterations were significantly ameliorated (Fig. 1J). With respect to the heart, spleen, lungs and kidneys, no significant differences were discernible among the groups, suggesting that BBR‐CA does not regulate body weight through the induction of overt toxicity in mice (Fig. S1). In summary, 8 weeks of HFD intervention precipitated the development of hyperlipidemia, yet supplementation with BBR‐CA effectively mitigated the dyslipidemia and hepatic lipid accumulation induced by HFD.

Compared with the NFD group, the protein expression levels of SREBP‐1, SCD1 and ACC in the HFD group were significantly increased. After administration of BBR‐CA, the protein levels in the livers of mice were significantly decreased (Fig. 2A–D). Meanwhile, the gene expression levels of SREBP‐1, SCD1 AND ACC in the HFD group were also significantly increased (Fig. 2E). After administration of BBR‐CA, the gene levels in the liver were all significantly downregulated. Among them, the expression level of SREBP‐1 in the M‐BBR‐CA group was significantly lower than that in the BBR group (Fig. 2E). The above results indicate that BBR‐CA can reduce the expression of proteins and genes involved in hepatic lipid synthesis.

To ascertain the optimal concentration and time for FFA (i.e. a mixture of sodium oleate and sodium palmitate at a ratio of 2:1)‐induced cellular steatosis, Oil Red O staining (Fig. 3A) and cytotoxicity assays (Fig. 3B) were performed. With reference to the relevant literature [26] and the experimental requirements, the conditions for model establishment in the subsequent experiments were determined as follows: the concentration of FFA was 1 mm and the duration of model establishment was 24 h. In the subsequent experiments, the cytotoxicity of CA, BBR, AT and BBR‐CA, respectively, on HepG2 cells was evaluated. The results demonstrated that no significant cytotoxicity was observed within the concentration range up to 60 μm (Fig. 3C). The Oil Red O staining results, as presented in Fig. 3D,E, indicated that BBR‐CA was capable of markedly reducing intracellular lipid accumulation. Quantitative analysis further revealed that the efficacy of BBR‐CA was substantially superior to that of CA and BBR. The AT group also significantly reduced the lipid droplet area in FFA‐induced HepG2 cells, and its D₅₁₀ value (lipid content) was close to that of the H‐BBR‐CA group (Fig. 3E). This indicates that the positive control in this experimental system is effective, and the lipid‐lowering effect of BBR‐CA is comparable to that of clinical drugs. Additionally, BBR‐CA also led to a significant reduction in the intracellular TG levels (Fig. 3F). Collectively, the above results suggest that BBR‐CA can significantly ameliorate FFA‐induced lipid accumulation in HepG2 cells.

In the cell experiments, western blot assays were utilized to determine the protein expression levels of SREBP‐1, SCD1 and ACC in cells of each experimental group. In comparison with the CTL group, a significant elevation in the protein expression levels of SREBP‐1, SCD1 and ACC was observed in the FFA group (Fig. 4). Subsequently, following drug administration in each group, this trend was notably reversed. Specifically, the combined application of CA and BBR demonstrated a more pronounced effect compared to single‐agent administration, and BBR‐CA alone also exhibited enhanced efficacy. When contrasted with the CA + BBR group, the medium and high doses of BBR‐CA manifested a more conspicuous effect with respect to reducing the levels of lipid synthesis proteins within HepG2 cells (Fig. 4A–D). Further detection of the expression levels of lipid synthesis protein genes showed results similar to those of the protein levels. Compared with the CTL group, the expression levels of SREBP‐1, SCD1 and ACC in the FFA group were significantly increased, and BBR‐CA could significantly reduce their expression levels (Fig. 4E). Meanwhile, the AT group significantly down‐regulated the protein and gene expression of SREBP‐1, SCD1 and ACC, which was consistent with the regulatory trend of the M‐BBR‐CA group. This further supports that BBR‐CA exerts lipid‐lowering effects by inhibiting lipid synthesis‐related proteins (Fig. 4A,E). Collectively, these results imply that BBR‐CA can substantially attenuate the expression levels of lipid synthesis proteins and genes in HepG2 cells induced by FFA.

The regulation of lipid synthesis by SREBPs is a complex process and provides targets to cue hyperlipidemia. Precursor SREBPs are formed on the endoplasmic reticulum and transported to the Golgi apparatus together with SREBP cleavage‐activating protein. After being cleaved by site‐1 protease and site‐2 protease, the active N‐terminal of SREBP‐1 enter the nucleus and bind to SRE response elements to initiate the expression of lipogenic genes for SCD1 and ACC. The expression of SREBP‐1 in the nucleus was detected by western blotting and immunofluorescence methods. The results of western blotting showed that the expression level of SREBP‐1 in the nucleus of the FFA group was higher than that in the CTL group, and the expression level of SREBP‐1 decreased after the administration of BBR‐CA (Fig. 5A,B). In terms of immunofluorescence, green fluorescence was mainly distributed in the nucleus, indicating that SREBP‐1 was mainly located in the nucleus. After the administration of BBR‐CA, the fluorescence intensity in the nucleus decreased significantly (Fig. 5C). The above results suggest that BBR‐CA can affect hyperlipidemia by reducing the expression of SREBP‐1 in the nucleus.

Studies have shown that mTOR regulates downstream phosphorylated (p)‐70S6K to promote the cleavage and activation of SREBPs from the endoplasmic reticulum to the Golgi apparatus [27, 28]. PI3K is the initiator of the PI3K/AKT/mTOR signaling pathway, and, among them, class I PI3K is an essential link for activating the mTOR signaling pathway. AKT, as the signal transduction hub and an important downstream target protein of the PI3K/AKT/mTOR signaling pathway, plays a significant role in the homeostatic regulation of cell growth, survival and glucose metabolism. The activated AKT then translocates to the cytoplasm or nucleus again and regulates downstream signaling molecules [29]. The PI3K/AKT/mTOR pathway can modulate the expression of SREBP‐1 and downstream lipid synthesis proteins [17]. Hence, we investigated the expression of the PI3K/AKT/mTOR pathway in HepG2 cells. As depicted in Fig. 5D–G, the model under the influence of FFA enhanced the expression of p‐PI3K, p‐AKT and p‐mTOR. Upon treatment with all drugs, the phosphorylation levels of PI3K, AKT and mTOR were significantly diminished, with BBR‐CA exhibiting the most pronounced effect. These results imply that BBR‐CA can attenuate the expression of the PI3K/AKT/mTOR signaling pathway, thereby repressing the expression of SREBP‐1.

To further confirm that BBR‐CA can indeed modulate the PI3K/AKT/mTOR pathway, we treated HepG2 cells with a PI3K agonist recilisib and an inhibitor LY294002. The results showed that the intracellular lipid changed after the administration of the agonist and inhibitor. Compared with the CTL group, the number of intracellular lipids increased in the FFA group and the agonist group, and this phenomenon was reversed after the administration of BBR‐CA (Fig. 6A,B). In addition, in FFA‐induced HepG2 cells, the expression levels of p‐PI3K, p‐AKT and p‐mTOR increased significantly. After treatment with the inhibitor, the expression levels of p‐PI3K, p‐AKT and p‐mTOR in HepG2 cells decreased (Fig. 6C–F). After the administration of BBR‐CA, the PI3K activator reversed the effect of BBR‐CA, and the inhibitor of PI3K enhanced the down‐regulatory effect of BBR‐CA on protein phosphorylation (Fig. 6C–F). In conclusion, these results indicate that BBR‐CA can affect lipid synthesis by regulating the phosphorylation of proteins in the PI3K/AKT/mTOR pathway.