Reagents
The four crude herbs, Poria cocos (PC, FuLing, Cat. No.20200102-1), Ramulus Cinnamomi (RC, GuiZhi, Cat. No.20200201-1), Rhizoma Atractylodis Macrocephalae (RAM, BaiZhu, Cat. No.2011010) and Radix Glycyrrhizae (RG, GanCao, Cat. No.20191001) were purchased from the Affiliated Hospital of Integrated Traditional Chinese and Western Medicine and authenticated by Professor Fangshi Zhu. All the crude drugs were morphologically authenticated according to Chinese Pharmacopoeia (2020 Edition). Reference substances including cinnamaldehyde, Atractylenolide II and glycyrrhizic acid were purchased from PUSH Bio-Technology Co. Ltd (Chengdu, China). Lipopolysaccharide(LPS), STING specific inhibitor C176 and STING specific agonist DMXAA were purchased from MedChemExpress (MCE, NJ, USA). High fat diet(HFD) was purchased from New Brunswick(Cat. No. D12492, NJ, USA). Antibodies against STING, F4/80, TBK1, Phosphorylated TBK1(p-TBK1), TNFα, 8-OHdG, 4-hydroxy-2,3-E-nonenal (4-HNE) and 3-Nitrotyrosine (3-NT) were purchased from CST(CST, GER). Enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D (R&D Systems, US). DNA extraction kit, First Strand cDNA Synthesis Kit and 2× SYBR Green/ROX qPCR Mix kit were purchased from Vazyme Biotech (Nanjing, Jiangsu, China). TRIzol reagent was purchased from Invitrogen (Carlsbad, CA, USA). Microplate assay kits for Blood gamma glutamyl transferase (GGT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG) and total cholesterol (TC) measurement were purchased from Beyotime Biotechnology(Beyotime, Beijing, China).
Preparation of LGZG
LGZG, consisting of PC, RC, RAM and RG in the ratio of 4:3:3:2, were boiled twice with 10 times of deionized water (ddH2O, w/v) for one hour per time. The raw drug extracts was concentrated and lyophilized. The lyophilized powder was weighed and dissolved in ddH2O for further use. The HPLC analysis was performed to identify the main compounds in LGZG (5 mg/mL) as previously reported [20]. Briefly, HPLC analysis was performed to identify the main compounds in LGZG. Chromatographic
separation was performed using an Waters XBridge C18 column (4.6 mm × 250 mm, 5 μm; Waters,USA) at 30 °C. The mobile phase consisted of water containing 0.1% Formic acid (A) and Methanol (B). The gradient program was set as follows: 0–5 min, 5–12% B; 5–20 min, 12–14% B; 20–40 min, 14-30% B; 40–50 min, 30-35% B; 50–60 min, 35–43% B; 60–70 min, 43-50% B; 70–75 min, 50-75% B; 75–90 min, 75-85% B; 90–95 min, 85-95% B; 95–95.1 min, 95-5% B. The mobile phase flow rate was 1 mL/min, and the sample injection volume was 100 μL.
Animals, feeding, and treatment
6 to 8-week-old male C57BL/6J mice(18~20 g)were feed in Experimental Animal Center, Affiliated Hospital of Integrated Traditional Chinese and Western Medicine (Nanjing, China). Mice were group-housed in a pathogen-free animal facility under a 24 h light/dark cycle with free access to water and food. The experiments program were reviewed and approved by the Animal Ethics Committee of Affiliated Hospital of Integrated Traditional Chinese and Western Medicine with reference to European Community guidelines for the use of experimental animals. The mice were fed with standard chow diet (10% kcal from fat, set as the normal control group, n=9) or a high-fat diet (HFD) composed of 60% fat and 20% carbohydrate(n =36). After 8 weeks feeding, the HFD fed mice were randomly divided into four groups (n=9) including Model (Mod), LGZG-L (low dose of LGZG, at a dose of 11g/kg/day ), LGZG-H (high dose of LGZG, at a dose of 22g/kg/day ),and C176 (STING specific inhibitor, C-176, 6.7mg/kg/day). Mice were fed with HFD and orally administered LGZG for an additional 9 weeks and then sacrificed for sampling. The mice body weight and fasting blood glucose (FBG) levels were measured weekly before sacrifice. Intra peritoneal glucose tolerance test (IPGTT) and insulin tolerance tests (ITTs) were performed at 3 day before sacrifice. For IPGTT, mice were fasted for 8 h and then intraperitoneally injected with glucose (2mg/g body weight). The blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 min after glucose challenge respectively. For the insulin tolerance test (ITT), mice were fasted for 6 h and then intraperitoneal injected with human insulin (0.75 U/kg body weight). The blood glucose level were measured at 0, 30, 45, 60, 90 and 120 min after insulin injection respectively. At the end of experiment, blood samples for further assays were collected through abdominal heart puncture. The freshly separated liver tissues was quickly divided into several aliquots and stored in a -80 oC refrigerator and 4% paraformaldehyde respectively for further use. Blood GGT, ALT, AST, TG and TC were measured by commercially available assay kits according to the standard manual.
Histology
Liver specimens were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4μm thick sections. Sections were stained with hematoxylin and eosin(H&E) according to a standard procedure for assessment histopathological changes. For Oil Red O staining, The fixed liver specimens were embedded in optimum cutting temperature compound (OCT), and cut into 10 μm thick sections. Sections were stained with 0.5% Oil Red O solution according to a standard procedure for assessment lipid accumulation. The degree of hepatic lipid deposition and oil red O positive area were quantified with Image J software.
Immunofluorescence and Immunohistochemistry
Paraffin-embedded mouse liver blocks from different groups were sliced into 4 μm-thick sections for immunofluorescence and immunohistochemistry staining. The antibodies against STING(1:200, CST, 16029T), F4/80 (1:200, CST,70076S),3-Nitrotyrosine (1: 100, Abcam, ab61392) , 4 Hydroxynonenal (1: 100, Abcam, ab48506) were used according to manufacturer's protocol. The fluorescence intensity was analyzed by using Image J software and normalized by nuclei fluorescence intensity (6-diamidino-2-phenylindole, DAPI, blue).
Detection of mtDNA Content in Cytosolic Extracts by real time PCR(qPCR)
Cytosolic mtDNA content was measured according to the previous work [21]. Briefly, freshly separated mice livers from different groups were divided into two aliquots. One aliquot was used to extract total DNA as the control for total mtDNA by using the DNA extraction kit according to the standard manual(Axygen US). The other one was used to extract cytosolic DNA. For the extraction of cytosolic DNA, the liver tissue was homogenized in 500 μL buffer (150 mM NaCl, 50 mM Hepes, 25 μg/mL digitonin, pH 7.4) and then incubated for 10 min to permeabilize the plasma membrane. The homogenate was centrifuged (980×g, 5min) for 3 times to pellet intact cells. The cytosolic supernatants was further centrifuged at 17,000 × g for 25 min to pellet any remaining cellular debris, yielding cytosolic preparations free of nuclear, mitochondrial, and endoplasmic reticulum contamination. DNA was then isolated from these pure cytosolic fractions by using the DNA extraction kit according to the standard manual.
Total RNA of liver tissues from sacrificed mice were prepared using TRIzol reagent. RNA (1 μg) was reverse-transcribed into cDNA using First Strand cDNA Synthesis Kit according to the standard protocol. qPCR was performed on ABI 7500 qPCR systems according to the standard protocol. Data was analyzed using the ΔΔCT relative quantification method. Primers sequences and corresponding genes are shown in Table 1.
Cell separation, culture and drug treatment
Bone marrow cells were isolated from free-fed C57 mice and differentiated into bone-marrow-derived macrophages (BMDMs) as previously described [22] . Briefly, bone marrow cells were isolated from the tibias and femurs of the mice and cultured in DMEM containing 10% fetal bovine serum and 20% L929 culture supernatant for 6–8 days to obtain the BMDMs. To detect the effect of LGZG on the activation of STING mediate signal pathway, BMDMs were pretreated with (7.5% NaHCO3) or LGZG (10-4or 10- 3 g/mL) for 2 h respectively as previously reported [19]. Then the cells were incubated with DMXAA (75 μg/mL) for 24 h in the absence or presence of LPS (100 ng/mL, dissolved in 1× phosphate buffered saline). After incubation, the cell lysates from different groups were harvest respectively for the detection of expression of STING and other related proteins. Furthermore, the cell culture supernatant from each group were collected for the detection of IFNβ and TNF by ELISA.
For HepG2 cells and macrophage co-culture study, the BMDMs treated with different doses LGZG (10-4or 10-3 g/mL)were respectively added to PA-stimulated HepG2 cells at a ratio of 1:10 and then incubated for another 48 hours and assayed for hepatocyte fat deposition and inflammatory responses as previously reported[13]. The lipid deposition was detected by Oil Red O staining. For the STING activation, cells were treated with DMXAA (75 μg/mL) at the last 24 hours. Details are refer to Luo et al.’s work[13].
Immunoblotting
For the immunoblotting, mice liver homogenates from and cultured cells were lysed with RIPA buffer. The denatured samples were loaded for SDS-PAGE, transferred, incubated with antibodies and visualized with enhanced chemiluminescence. The antibodies against F4/80, STING, TBK1, pTBK1, TNFα and tubulin were used. Ratios of p-TBK1 to TBK1 were normalized to Tubulin.
Statistical analysis
For cell studies, data are representative of three independent experiments. The Western blot images were semi quantified with the Image J program. Statistical analysis of the data was performed using GraphPad Prism 7. Significance was assessed by performing an unpaired two-tailed Student’s t test as indicated in individual figures. Quantitative data are presented as Mean ± SEM. Statistical significance was set at *P< 0.05, **P<0.01, ***P< 0.001, ****P< 0.0001.