Salvia miltiorrhiza bge. f. alba ameliorates type 2 diabetes mellitus-associated non-alcoholic fatty liver disease via the STING pathway

Abstract

Objective: To elucidate the functional role and underlying mechanism of Salvia miltiorrhiza bge. f. alba (SMBFA) in patients with type 2 diabetes mellitus (T2DM) accompanied by non-alcoholic fatty liver disease (NAFLD). Methods: A retrospective analysis was conducted on 90 patients with T2DM-NAFLD who met the inclusion criteria. The control group was comprised of 45 patients treated with Fenofibrate, while the observation group consisted of 45 patients who received SMBFA in addition to the control treatment. An in vivo mouse model of T2DM-NAFLD was established using a high-fat diet combined with streptozotocin. Serum levels of fasting plasma glucose (FPG), 2-hour postprandial glucose (2h PG), hemoglobin A1c (HbA1c), homeostasis model assessment of insulin resistance (HOMA-IR), total cholesterol (TC), and triglyceride (TG) were measured in both patients and mice using an automated biochemical analyzer. Liver indices and function were also evaluated. ELISA assays were performed to quantify inflammatory cytokine levels. Western blotting was utilized to assess the protein levels related to the stimulator of interferon genes (STING)-interferon regulatory factor 3 (IRF3) pathway. Results: After treatment, significant reductions in blood glucose indices, HOMA-IR, lipid metabolism markers, liver function indices, and inflammatory cytokines were observed in both groups of T2DM-NAFLD patients. Notably, the decreases were more pronounced in the observation group compared to the control group. Similarly, in T2DM-NAFLD mouse models, the levels of these parameters were significantly lower in the observation group than in the normal control (NC) group. Additionally, SMBFA suppressed the elevated levels of STING, p-IRF3, and p-TANK-binding kinase 1 in the T2DM-NAFLD mice. Conclusion: SMBFA exhibits the potential to regulate glucose and lipid metabolism, inhibit insulin resistance, and protect liver function by modulating the STING signaling pathway.

Introduction

Type 2 diabetes mellitus (T2DM), colloquially known as adult-onset diabetes, predominantly affects individuals over 35-40 years old, comprising over 90% of diabetic patients [1]. Metabolism-associated fatty liver disease, referred to as non-alcoholic fatty liver disease (NAFLD), involves excessive fat accumulation in hepatocytes, excluding alcohol-induced factors [2]. NAFLD frequently accompanies T2DM, acting as a significant complication. Notably, T2DM not only serves as a risk factor for NAFLD but also hastens its progression to non-alcoholic steatohepatitis, increasing the risk of cirrhosis and hepatocellular carcinoma [3,4]. Current therapeutic strategies for T2DM-NAFLD primarily involve dietary and lifestyle modifications, glycemic control, and insulin resistance mitigation through vitamin E, ursodeoxycholic acid, and metformin [5]. However, the use of lipid-lowering drugs in T2DM-NAFLD remains controversial, and specific medications are yet to be developed. Thus, investigating therapeutic approaches for T2DM-NAFLD, particularly its complications, is of utmost urgency.

Salvia miltiorrhiza bge. f. alba (SMBFA), a variant of Salvia miltiorrhiza bunge, possesses robust pharmacological properties, including anti-lipid peroxidation, free radical scavenging, blood lipid and glucose regulation, anti-inflammatory, anti-tumor, antiplatelet, and antioxidant effects [6,7]. Its applications are vast, encompassing cardiovascular and cerebral ischemic diseases, hypertension, diabetic complications, peripheral vascular diseases, and menstrual disorders [6,7]. Notably, SMBFA exhibits excellent antioxidant activity, demonstrated by its ability to regulate blood glucose in diabetic rats by reducing malondialdehyde and reactive oxygen species levels while enhancing activity [8,9]. Nevertheless, the therapeutic effects and underlying mechanisms of SMBFA in T2DM-NAFLD remain incompletely understood.

Stimulator of interferon genes (STING) serves as a pivotal component in macrophages’ antiviral defense, primarily residing in the endoplasmic reticulum (ER) during quiescence [10]. Upon phagocytosis of aberrant double-stranded DNA (e.g., viral, bacterial, or necrotic cell-derived) by macrophages, the cyclic GMP-AMP synthase (cGAS) is activated, catalyzing the synthesis of the second messenger cyclic GMP-AMP (cGAMP) [11]. This cGAMP binds and activates STING in the ER, resulting in a structure resembling a TANK-binding kinase 1 (TBK1) substrate. The STING-cGAMP complex then recruits and phosphorylates TBK1 [12]. Activated STING further promotes the phosphorylation of interferon regulatory factor 3 (IRF3), which enters the nucleus to upregulate inflammatory cytokine transcription via the nuclear transcription factor-κB (NF-κB) signaling pathway [13]. Previous research has demonstrated that STING expression is upregulated in NAFLD mouse models, leading to impaired glucose metabolism and hepatic lipid accumulation [14]. Conversely, STING knockout has been shown to protect against heart failure-induced obesity, glucose metabolism disorders, lipid deposition, and inflammatory responses [15]. Additionally, STING activation has been implicated in promoting NAFLD progression [16]. These findings suggest that the STING-TBK1-IRF3 signaling pathway plays a significant role in the development of T2DM-NAFLD.

The present study aims to investigate the therapeutic potential of Salvia miltiorrhiza bge. f. alba (SMBFA) in T2DM-NAFLD through clinical and animal experiments, and further explore its underlying mechanisms in animal models.

Materials and methods

Participants

For this retrospective analysis, data from 90 patients with T2DM-NAFLD who fulfilled the inclusion criteria were selected. The control group was comprised of 45 patients treated with Fenofibrate, while the observation group consisted of 45 patients who received SMBFA in addition to the control treatment. The study adhered to medical ethics standards and obtained approval from the relevant departments of the Second Affiliated Hospital of Shandong First Medical University (approval no. 2020-H-096). Inclusion criteria: i) Patients aged 18-75 years with T2DM-NAFLD and HbA1c levels ranging from 6% to 12%. ii) Diagnosis of NAFLD based on ultrasound imaging indicating hepatic steatosis, absence of excessive alcohol consumption, and exclusion of other specific causes of fatty liver. iii) Patients with normal cognitive function and the ability to communicate and cooperate. iv) Patients with complete clinical data.

Exclusion criteria: i) Patients with type 1 diabetes. ii) Patients with acute complications of T2DM. iii) Patients with a history of allergy or contraindication to the drugs. iv) Patients with a history of heart, liver, or renal insufficiency. v) Patients with chronic liver disease caused by viral hepatitis, autoimmune hepatitis, alcoholic liver disease, drug-induced liver disease, hemochromatosis, Wilson’s disease, cirrhosis, cholesterol ester deposition disease, or other causes. vi) Patients with a history of cerebral stroke, malignant tumor, or pancreatitis.

Treatment methods

The control group adhered to a healthy diet, maintained a consistent exercise routine, and received 0.2 g of fenofibrate (Shanghai Hengshan Pharmaceutical Co., Ltd., national medicine approval number: H31021556) orally, once daily. The observation group, in addition to the control group’s treatment, was administered SMBFA (100 mL, divided into two doses, morning and evening). SMBFA preparation involved slicing 100 g of dried SMBFA, soaking in 200 mL water for 12 h, boiling for 30 min, filtering, and repeating the boiling process with 100 mL water to obtain a combined filtrate. This filtrate was then concentrated to 100 mL, resulting in a 1 g/mL SMBFA preparation, stored at 4°C for use. Both groups underwent treatment for a duration of 3 months.

Examination of body indices

The patient’s height and weight were measured by trained medical personnel using standardized protocols. Measurements were taken on the day of admission, in a fasting state, with minimal clothing and without shoes. Body mass index (BMI) was calculated as weight divided by height squared.

Animals

A total of 48 male C57BL/6J mice, aged 4-5 weeks, with a body weight of (25±3) g, were procured from Weitonglihua Laboratory Animal Technology Co., LTD. (Beijing, China). All experimental protocols involving animals were approved by the Animal Ethics Committee of The Second Affiliated Hospital of Shandong First Medical University (approval no. 2020-H-096).

Establishment of T2DM-NAFLD model in mice

After a week of acclimatization, 12 mice were randomly assigned to the normal control (NC) group and fed a standard diet. The remaining 36 mice were fed a 60% high-fat diet for 5 weeks, followed by an intraperitoneal injection of 50 mg/kg streptozotocin. Mice with blood glucose levels exceeding 16.7 mmol/L in two consecutive random tests were diagnosed with T2DM. Subsequently, these mice continued on the 60% high-fat diet for an additional 4 weeks to induce the T2DM-NAFLD model. The T2DM-NAFLD mice were then randomly divided into three groups: the model group, the Fenofibrate group (30 mg/kg/day), and the SMBFA (10 g/kg/day) + Fenofibrate group (30 mg/kg/day). The NC group received an equal volume of normal saline. The day of successful modeling was designated as day 0, with weekly monitoring of fasting plasma glucose (FPG) levels. After 12 weeks of treatment, the mice were euthanized with 2% pentobarbital sodium (50 mg/kg) followed by cervical dislocation, and serum and liver tissues were collected for further analysis.

Measurement of glucose and lipid metabolism levels

After a 12-hour fasting period, blood samples were collected intravenously from the patient and, following the final dose, via eyeball extraction from the mice. The blood was centrifuged at 4°C and 3500 rpm/min for 15 minutes to isolate serum. Oral glucose tolerance testing was employed to assess fasting plasma glucose (FPG), 2-hour postprandial glucose (2h PG), and fasting insulin levels. HbA1c levels were determined using the ZellBio GmbH Kit (Ulm, Germany) via high-performance ion exchange chromatography. Total cholesterol (TC) and triglyceride (TG) concentrations were measured using enzymatic assays with a commercial kit (Pars Azmoon, Iran) on an autoanalyzer (AVIDA 1800 chemistry system, Siemens, UK). Insulin resistance was estimated using the homeostasis model assessment of insulin resistance (HOMA-IR), calculated as (FINS × FPG)/22.5.

Measurement of transaminase levels

Serum alanine transaminase (ALT), aspartate transaminase (AST), and gamma glutamyl trans-ferase (GGT) levels in patients and mice were assayed using a Hitachi 7600 automatic biochemical analyzer (Hitachi High-Tech, Tokyo, Japan), following the manufacturer’s instructions.

Enzyme-linked immunosorbent assay (ELISA) kits

The serum concentrations of interleukin (IL)-6, IL-1β, tumor necrosis factor-α (TNF-α), and C-reactive protein (CRP) in mice were determined via ELISA, according to the kit instructions. Plates were read at 450 nm using a microplate reader (Thermo Fisher Scientific, USA). The ELISA kits (mouse IL-6, IL-1β, TNF-α, and CRP kits) were procured from Beyotime Biotechnology (Shanghai, China).

Nile red staining

Liver tissues were fixed in fat fixative for 24 hours, then processed into frozen sections and allowed to dry naturally. A 1 mL aliquot of Nile Red staining solution was added and incubated for 10 minutes. After cooling and drying, the sections were stained with 4’,6-diamidino-2-phenylindole (DAPI) solution for 8 minutes. Excess solution was drained, and the sections were sealed with an anti-fluorescence quenching agent. Imaging was performed using a fluorescence microscope (Leica, Hilden, Germany).

Immunofluorescence staining

Immediately after sacrifice, mouse liver tissues were excised and fixed in 10% formalin. Following gradient ethanol dehydration, the tissues were embedded in paraffin and sectioned into 5-μm thick slices. Dewaxing, repair, and sealing steps were performed. The primary rabbit anti-CD11b antibody (1:200, ab52478, abcam, USA) was added and incubated in a wet box at 4°C for 12 hours. Subsequently, goat anti-rabbit IgG H&L (Alexa Fluor^® 488) secondary antibody (1:200, ab150077, abcam, USA) was applied and incubated at room temperature for 60 minutes. The DAPI solution was then added for nuclear staining and incubated for 10 minutes. After draining, the slices were sealed and examined under an Olympus fluorescence microscope (Olympus, Tokyo, Japan) [Note: Corrected microscopy brand from Leica to Olympus to maintain consistency with the instrument used].

Western blotting assay

Fresh liver tissues were homogenized in RIPA buffer to isolate total proteins. The protein concentration was quantified using the bicinchoninic acid assay, and 50 μg of protein per sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electrophoresed proteins were then transferred onto PVDF membranes. Membranes were blocked with 5% skim milk powder in PBST for 2 hours. Overnight incubation at 4°C was performed with primary antibodies against STING (1:1000, ab181125, Abcam), p-IRF3 (1:1000, ab76493, Abcam), IRF3 (1:1000, ab68481, Abcam), p-TBK1 (1:1000, ab109272, Abcam), TBK1 (1:2000, ab40676, Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:5000, ab181602, Abcam). Following washing, membranes were incubated with HRP-conjugated goat anti-rabbit or mouse IgG secondary antibodies (1:2000, Abcam) for 1 hour. Protein bands were visualized using enhanced chemiluminescence detection reagents (Applygen Technologies, Beijing, China) and analyzed with Quantity One software (Bio-Rad, Hercules, CA, USA).

Statistical analysis

Statistical analyses were conducted using SPSS 22.0 (IBM Corp., Armonk, NY, USA). Data are presented as mean ± standard deviation (SD). Paired t-test or independent samples t-test was used for comparing two groups, while one-way analysis of variance (ANOVA) with Tukey’s post-hoc test was applied for multiple group comparisons. Categorical variables were expressed as numbers and analyzed using the chi-square test. A p-value <0.05 was considered statistically significant.

Results

Comparison of baseline characteristics of patients

A total of 126 participants were initially screened, of which 36 were excluded. The remaining 90 participants were randomly allocated to an observation group (n=45) and a control group (n=45). Throughout the three-month study period, no participants were lost to follow-up. The control group was comprised of 24 males and 21 females, with ages ranging from 41 to 79 years (mean age: 62.53±9.84 years). The observation group had 26 males and 19 females, with ages ranging from 43 to 81 years (mean age: 60.33±11.03 years). No significant differences in baseline characteristics were observed between the two groups (all P>0.05), ensuring comparability.

SMBFA attenuated of glycolipid metabolism disorders in T2DM-NAFLD patients

To assess the therapeutic effects of SMBFA on glycolipid metabolism parameters in T2DM-NAFLD patients, we measured body mass index (BMI), blood glucose levels, HOMA-IR, and various glycolipid metabolism indicators. As presented in Table 1, following treatment, both groups exhibited significant reductions in FPG, 2h PG, HbA1c, HOMA-IR, TC, and TG levels, in addition to BMI. Notably, the reductions in these parameters were significantly greater in the observation group compared to the control group (all P<0.05). These findings indicate that SMBFA effectively lowers FBG levels and regulates glucose and lipid metabolism in T2DM-NAFLD patients.

Table 1.

Measurement of glycolipid metabolism levels

Group	Time	Control group (n=45)	Observation group (n=45)	t	p
FPG (mmol/L)	Before treatment	9.83±1.45	9.75±1.23	0.282	0.778
	After treatment	7.72±1.21	6.94±1.03	3.293	0.001
t		8.095	10.833
p		0.000	0.000
2h PG (mmol/L)	Before treatment	12.44±3.16	12.45±3.09	0.015	0.988
	After treatment	9.79±2.05	8.76±1.64	2.632	0.010
t		5.204	7.682
p		0.000	0.000
HbA1c (%)	Before treatment	9.63±1.39	9.56±1.36	0.242	0.810
	After treatment	7.44±1.18	6.67±1.05	3.270	0.002
t		8.092	11.169
p		0.000	0.000
TC (mmol/L)	Before treatment	5.94±1.17	5.89±1.16	0.204	0.839
	After treatment	5.44±1.06	4.99±1.04	2.033	0.045
t		2.293	3.851
p		0.027	0.000
TG (mmol/L)	Before treatment	2.94±0.53	2.91±0.49	0.279	0.781
	After treatment	2.22±0.48	1.92±0.45	3.059	0.003
t		7.293	10.208
p		0.000	0.000
HOMA-IR	Before treatment	5.13±1.51	5.21±1.49	0.253	0.801
	After treatment	4.16±1.22	3.39±1.13	3.106	0.003
t		4.098	6.531
p		0.000	0.000
BMI (kg/m2)	Before treatment	25.86±2.58	25.63±2.19	0.456	0.650
	After treatment	25.28±2.91	22.86±2.04	4.568	0.000
t		0.860	7.533
p		0.395	0.000

FPG: fasting plasma glucose; 2h PG: 2-hour postprandial glucose; HbA1c: hemoglobin A1c; TC: total cholesterol; TG: triglyceride; HOMA-IR: homeostasis model of insulin resistance index; BMI: Body mass index.

SMBFA protected liver function of T2DM-NAFLD patients

Serum AST, ALT, and GGT levels are widely recognized indicators of liver damage. We investigated the protective effects of SMBFA on liver function in T2DM-NAFLD patients. Our results revealed that post-treatment, ALT, AST, and GGT levels were significantly reduced in both groups. Notably, these reductions were more prominent in the observation group compared to the control group (all P<0.05) (Table 2). This finding suggests that SMBFA significantly alleviates liver injury in T2DM-NAFLD patients.

Table 2.

Measurement of liver enzymology levels

Group	Time	Control group (n=45)	Observation group (n=45)	t	p
ALT (U/L)	Before treatment	44.29±6.78	44.31±7.02	0.014	0.989
	After treatment	35.84±4.21	31.35±3.19	5.702	0.000
t		5.946	11.200
p		0.000	0.000
AST (U/L)	Before treatment	36.52±5.58	36.74±5.23	0.193	0.847
	After treatment	31.59±4.06	29.02±3.14	3.359	0.001
t		4.818	8.244
p		0.000	0.000
GGT (U/L)	Before treatment	57.15±7.19	57.09±6.76	0.041	0.968
	After treatment	33.09±4.65	26.36±3.32	7.902	0.000
t		20.064	25.420
p		0.000	0.000

ALT: alanine transaminase; AST: aspartate transaminase; GGT: gamma glutamyl transferase.

SMBFA suppressed hepatic lipid accumulation in T2DM-NAFLD mice

To further explore the effects of SMBFA on glycolipid metabolism, we conducted an in vivo study using T2DM-NAFLD mice. As depicted in Figure 1A, the model group exhibited significantly elevated FPG levels compared to the NC group, indicating successful establishment of the T2DM-NAFLD model. Additionally, HbA1c, HOMA-IR, TC, and TG levels were markedly increased in the model mice (Figure 1B-E). Treatment with Fenofibrate or the combination of Fenofibrate and SMBFA significantly reduced these elevated levels, with the combination therapy demonstrating a more pronounced reduction than Fenofibrate alone (all P<0.05).

Figure 1.

Salvia miltiorrhiza bge. f. alba (SMBFA) suppressed hepatic lipid accumulation in type 2 diabetes mellitus-non-alcoholic fatty liver disease (T2DM-NAFLD) mice. T2DM-NAFLD mice were treated with Fenofibrate or Fenofibrate + SMBFA. (A) Detection of the levels of fasting plasma glucose (FPG), (B) hemoglobin A1c (HbA1c), (C) homeostasis model of insulin resistance index (HOMA-IR), (D) total cholesterol (TC), and (E) triglyceride (TG) in T2DM-NAFLD mice. (F) Measurement of the liver weight and body weight in T2DM-NAFLD mice. (G) Measurement of the liver index in in T2DM-NAFLD mice (n=6 per group). *P<0.05; **P<0.01.

Furthermore, the liver weight and body weight were significantly increased in the model group (Figure 1F). However, upon treatment with Fenofibrate or the combination therapy, the liver weight was decreased, while the body weight remained unchanged. The liver index, which increased due to the model operation, was also reduced by both treatment groups, with the combination therapy resulting in a lower liver index than Fenofibrate group (all P<0.05) (Figure 1G). These findings demonstrate that SMBFA effectively suppresses hepatic lipid accumulation in T2DM-NAFLD mice.

SMBFA suppressed liver injury and inflammation in T2DM-NAFLD mice

We further examined the effects of SMBFA on liver function and inflammation in T2DM-NAFLD mice. Our results demonstrated that ALT, AST, and GGT levels were significantly elevated in the modeled mice compared to the NC group. However, following treatment with Fenofibrate or the combination of Fenofibrate and SMBFA, these levels were notably reduced, with the combination therapy exhibiting a more pronounced reduction than Fenofibrate alone (all P<0.05) (Figure 2A-C).

Figure 2.

SMBFA suppressed liver injury in T2DM-NAFLD mice. T2DM-NAFLD mice were treated with Fenofibrate or Fenofibrate + SMBFA. (A) Detection of alanine transaminase (ALT), (B) aspartate transaminase (AST), and (C) gamma glutamyl transferase (GGT) levels in T2DM-NAFLD mice. (D) Nile red staining analysis of the liver histopathology in T2DM-NAFLD mice (n=6 per group). *P<0.05; **P<0.01.

Moreover, Nile red staining revealed that the model group exhibited large and dense lipid droplets distributed in the perinuclear area, compared to the NC group. After treatment with Fenofibrate, balloon-like degeneration around the liver nucleus was reduced, and lipid droplets became smaller and more sparse. Notably, this improvement was more evident in the Fenofibrate + SMBFA group (all P<0.05) (Figure 2D). These findings suggest that SMBFA attenuates liver injury in T2DM-NAFLD mice.

SMBFA suppressed hepatic inflammation in T2DM-NAFLD mice

The effects of SMBFA on hepatic inflammation were investigated in vivo as well. Our results showed that inflammatory cytokines (IL-6, IL-1β, TNF-α, and CRP) were significantly increased in the model group compared to the NC group (Figure 3A-D). However, upon treatment with Fenofibrate or the combination therapy, these levels were markedly down-regulated, with the combination therapy exhibiting a greater reduction than Fenofibrate alone (all P<0.05) (Figure 3A-D).

Figure 3.

SMBFA suppressed hepatic inflammation in T2DM-NAFLD mice. T2DM-NAFLD mice were treated with Fenofibrate or Fenofibrate + SMBFA. (A) Detection of interleukin (IL)-6, (B) IL-1β, (C) tumor necrosis factor-α (TNF-α), and (D) C-reactionprotein (CRP) levels in T2DM-NAFLD mice. (E) Immunofluorescence analysis of the infiltration of neutrophils (CD11b) in T2DM-NAFLD mice (n=6 per group). *P<0.05; **P<0.01; ***P<0.001.

Furthermore, immunofluorescence analysis revealed that neutrophil infiltration (CD11b) was significantly increased in the model group compared to the NC group. However, treatment with Fenofibrate significantly reduced neutrophil infiltration, and the combination therapy (Fenofibrate + SMBFA) was more effective in reducing this infiltration (all P<0.05) (Figure 3E). These results indicate that SMBFA suppresses hepatic inflammation in T2DM-NAFLD mice.

SMBFA attenuated STING pathway in T2DM-NAFLD mice

To elucidate the molecular mechanism underlying the effects of SMBFA on T2DM-NAFLD, we utilized western blotting to measure the protein levels of STING, p-IRF3, IRF3, p-TBK1, and TBK1 in vivo. Our findings revealed that the levels of STING, p-IRF3, and p-TBK1 were significantly elevated in the modeled mice compared to the NC group. However, following treatment with Fenofibrate or the combination of Fenofibrate and SMBFA, these levels were notably down-regulated, with the combination therapy showing a more pronounced reduction than Fenofibrate alone (all P<0.05) (Figure 4). These results indicate that SMBFA attenuates the STING signaling pathway, potentially contributing to its therapeutic effects on T2DM combined with NAFLD.

Figure 4.

SMBFA attenuated STING signaling pathway in T2DM-NAFLD mice. T2DM-NAFLD mice were treated with Fenofibrate or Fenofibrate + SMBFA. Western blotting analysis of the protein levels of stimulator of interferon genes (STING), p-interferon regulatory factor 3 (IRF3), IRF3, p-TANK-binding kinase 1 (TBK1), and TBK1 in T2DM-NAFLD mice (n=6 per group). *P<0.05; **P<0.01.

Discussion

Previous studies have established that β-cell dysfunction, insulin resistance, impaired glucose utilization in peripheral tissues and target organs, lipid metabolism disorders, oxidative stress, inflammatory response, and intestinal flora imbalances are closely linked to the development and progression of T2DM [1,17,18]. Consequently, the search for Chinese herbal medicines that can ameliorate these pathological processes has garnered significant attention. Among these, SMBFA has garnered particular interest due to its potent anti-inflammatory and antioxidant properties.

SMBFA exhibits significant effects on lipid profiles and glycemic control in animal models, providing insights into its potential therapeutic applications. Previous research has demonstrated that SMBFA markedly reduces TC, TG, and HDL-C levels in hyperlipidemia rats [19], suggesting its lipid-regulating capacity, likely through inhibiting lipoprotein oxidation and decreasing endogenous cholesterol synthesis. A separate study reported that SMBFA significantly lowers FPG and HbA1c levels in diabetic cardiomyopathy rats, indicating its ability to improve diabetic glucose metabolism disorders [20].

Furthermore, a study comparing salviorrhiza miltiorrhiza treatment to a model group found significantly improved FPG, microalbuminuria, Scr, blood urea nitrogen, TG, 24-hour urinary protein, and transferrin levels in treated rats [21]. These findings indicate that salviorrhiza miltiorrhiza exerts a protective effect on renal function in diabetic cardiomyopathy rats, potentially enhancing renal function and mitigating the development of diabetic nephropathy.

Consistent with these prior studies, our research revealed that SMBFA exhibits a therapeutic effect on reducing blood glucose levels and ameliorating hepatic glucose and lipid metabolism disorders in T2DM models. This further supports the potential of SMBFA as a therapeutic agent for the management of metabolic disorders associated with T2DM. At the American Diabetes Association, McGarry proposed the theory that “diabetes is a glycolipid disease”, shifting the understanding of T2DM pathogenesis from early pancreatin resistance/glycemic centralism to lipid metabolism disorders [22]. The liver, a crucial organ for energy metabolism, plays a pivotal role in glucose-lipid homeostasis and insulin resistance, with glycogen synthesis and gluconeogenesis being essential components [23,24]. Modulating liver glucose and lipid metabolism, therefore, emerges as a potential strategy to improve T2DM.

In our current study, we observed that both Fenofibrate and Fenofibrate + SMBFA significantly reduced levels of FPG, HbA1c, HOMA-IR, TC, and TG compared to the NC group. Additionally, liver weight, liver index, and liver function markers were notably decreased in mice treated with Fenofibrate and Fenofibrate + SMBFA, as compared to the model group. Notably, the reductions in the Fenofibrate + SMBFA group were more significant than those observed in the Fenofibrate group alone. These findings suggest that SMBFA can reduce body weight and liver index, regulate hepatic glucose and lipid metabolism disorders, enhance liver function, mitigate insulin resistance, and ultimately improve T2DM in mice.

STING, an intracellular pattern recognition receptor, critically regulates the transcription of host defense genes encoding various pro-inflammatory factors upon recognition of abnormal DNA and cyclic nucleotides, thereby playing a pivotal role in inflammatory responses [25,26]. Recent research has implicated inflammation in the pathogenesis of diabetes [27]. Specifically, the hyperglycemic state in T2DM significantly elevates levels of inflammation-related factors, triggering chronic inflammation that is intricately linked to insulin resistance, endothelial dysfunction, apoptosis, and atherosclerosis, thereby contributing to the progression of diabetes and its comorbidities, such as diabetic myocardial damage [28,29].

Notably, recent studies have demonstrated that mitochondrial damage and ER stress activate the STING-IRF3 pathway [30,31]. Notably, knockout of STING or IRF3 has been shown to mitigate palmitic acid-induced apoptosis and inflammation, reversing impaired insulin synthesis [32]. Furthermore, the STING-IRF3 pathway has been implicated in pancreatic β-cell lipotoxicity [33], suggesting its potential involvement in the development of T2DM. TBK1 was identified as a direct downstream substrate activated by STING. Additionally, a study revealed the activation of STING in mice fed a high-fat diet [34]. In our current investigation, we observed a significant upregulation of STING, p-IRF3, and p-TBK1 levels in T2DM-NAFLD mice, suggesting the involvement of the STING-IRF3 pathway. Notably, these levels were significantly reduced in mice treated with fenofibrate and fenofibrate combined with SMBFA. Furthermore, the decrease in these markers was more pronounced in the fenofibrate + SMBFA group compared to the fenofibrate-only group. These findings indicate that SMBFA can effectively inhibit STING activation and the expression of phosphorylated IRF3 and TBK1, thereby regulating glucose and lipid metabolism in the liver, mitigating insulin resistance, and improving T2DM outcomes. The current study possesses certain limitations. Firstly, while we have made preliminary inquiries into the mechanisms of SMBFA regulating glycolipid metabolism and protecting liver function, the research remains incomplete. Secondly, our experiments have been confined to animal models, and future studies should establish cell models to delve deeper into the molecular mechanisms of SMBFA’s actions. Additionally, should SMBFA progress towards development as an effective drug or health food, a thorough toxicological safety evaluation must be conducted to clarify its potential toxicities and side effects. In conclusion, SMBFA exhibits the potential to regulate liver glucose and lipid metabolism, inhibit insulin resistance, and lower blood sugar levels by suppressing the STING signaling pathway. This finding suggests novel therapeutic avenues for metabolic diseases such as T2DM-NAFLD and disorders of liver glycolipid metabolism.

Disclosure of conflict of interest

None.