Patients
The study protocols were approved by the ethics committee of Shanghai Punan Hospital (approval number: 2013–12-18) and all enrolled subjects provided signed informed consent forms. Two hundred sixty patients with NAFLD were enrolled in Shanghai Punan Hospital from January 2013 to May 2016. The Chinese guidelines for the management of NAFLD were used for patient diagnosis [16]. Exclusion criteria were: patients with alcoholic liver, viral, autoimmune or hereditary liver disease; organ failure; other serious diseases such as heart, liver, kidney, hematopoietic system, mental illness and cancer; pregnant and lactating women; had used drugs affecting lipid metabolism in the 4 weeks prior to the study; long-term use of other drugs and combination therapies that may affect efficacy and safety; incomplete information and inability to cooperate in completing the study.
Traditional Chinese medicine (TCM) criteria were applied according to the NASH diagnosis and treatment project of the key specialty collaboration group of The Eleventh Five-Year Plan of State Administration of TCM of the People’s Republic of China (PRC). Syndrome differentiation was as follows: (1) Liver-QI stagnation accompanied by spleen deficiency and phlegm stagnation; (2) Stagnation of phlegm, stasis of the blood and heat conversion of dampness stagnancy; (3) Damp depression, blood stasis and liver-YIN deficiency. TCM symptoms and sign scores were: (1) Hypochondriac pain; Implicit swelling pain, no impact on work, 2 points; relatively heavy swelling pain that was continuous, 4 points; swelling pain was severe and affects work, 6 points. (2) Fatigue: Limb slightly tired, does not affect work, 2 points; limb weakness, barely impinges on work, 4 points; the whole body is weak and patient unwilling to move at all, 6 points. (3) Abdominal distension: Abdominal distension after eating, relieved later, 2 points; postprandial abdominal distension with slow relief, 4 points; abdominal distension all day, 6 points. (4) Stools: Defecate rarefied or uncomfortable, 1–2 times a day, 2 points; a loose stool more than 3 times a day, 4 points; diarrhea immediately after eating, 6 points.
Treatment procedures
All NAFLD patients were given guidelines on requirements for diet control and appropriate exercise. The patients were randomly assigned to 4 groups: rosiglitazone, 30 patients (Ros); Silibinin, 50 patients (Sil); DSSG, 130 patients as well as 50 patients DSSG combined with Sil (DSSG + Sil). In total 90 patients in the DSSG group were screened and subdivided according to TCM classifications into a D1 group (liver-QI stagnation with spleen deficiency plus phlegm stagnation), a D2 group (phlegm stagnation, blood stasis, heat conversion of the dampness stagnancy) and a D3 group (damp depression, blood stasis, deficiency of liver-YIN), with 30 patients in each group (Fig. 1). The remaining 40 patients of the DSSG group did not belong to one of these classifications, but belonged to other TCM categories (such as liver depression with QI stagnation, etc.).
Medications
Group Ros
Rosiglitazone hydrochloride (Yilixi, Zhejiang Wan Ma Pharmaceutical Co., Ltd. Manufacturing Approval Number: H20041408), 4 mg/time, once a day.
Group Sil
Silibinin capsules (Manufacturer: Tianjin TASLY Pharmaceutical Co, LTD, manufacturing approval number: 450707024); orally, 3 × 35 mg containing capsules, 3 times a day.
Group DSSG
Patients received DSSG (Composition: Salvia miltiorrhiza Bunge Lamiaceae 6 g, Paeonia veitchii Lynch Paeoniaceae 6 g and Bupleurum komarovianum Lincz. Apiaceae 5 g, Curcuma aromatica Sallsb. Zingiberaceae 6 g, Cyperus rotundus L. Cyperaceae 6 g, Angelica sinensis (Oliv.) Diels Apiaceae 5 g, Fallopia japonica Houtt. Polygonaceae 6 g, Radix Astragalus membranaceus (Fisch. ex Link) Bunge Fabaceae 8 g, Artemisia capillaris L. Asteraceae 9 g, Isatis tinctoria L. Brassicaceae 12 g, Patrinia scabiosaefolia Fisch Caprifoliaceae 12 g, Artemisia argyi H.Lév. and Vaniot Asteraceae 5 g, Crataegus wilsonii Sarg. Rosaceae 6 g, Raphanus sativus (L.) Domin Brassicaceae 6 g, Cynanchum bungei Decne. Apocynaceae 12 g, and Schisandra chinensis (Turcz.) Baill. Schisandraceae 5 g). The hospital entrusted Shanghai Yuan Pharmaceutical Co., Ltd to produce (Manufacturing Approval No: Z09130001), 12 g/packages, each containing the above dose proportions; 2 packages/time, were administered 3 times a day.
Group DSSG + Sil
DSSG (orally, 2 packages, 3 times a day) + Silibinin capsules (orally, 3 tablets, 3 times a day).
Observation indices
Biochemical values
(1) Fasting serum lipids: total cholesterol (TC), triacylglycerol (TG). (2) Liver functions: alanine aminotransferase (ALT), aspartate transaminase (AST) and γ-glutamyl transpeptidase (GGT) concentrations; (3) Fasting plasma glucose (FPG). Biochemical indexes were carried out by the hospital laboratory and the results recorded before and after 16 weeks of treatment.
Imaging indexes
B-ultrasound examinations were performed before and after treatment for 16 weeks (the same technician used light, medium and heavy scans to evaluate the degree of fatty liver damage). The efficacy evaluation was based on clinical research guidelines for new TCMs to formulate the efficacy evaluation criteria for the severity of fatty liver using B-ultrasound. Efficacy was graded according to the following observations: Clinical cure: B-ultrasound reexamination found that the characteristics of NAFLD disappeared; Obvious effect: Grade of fatty liver B-ultrasound reduced by 2 levels; Effective: Grade of fatty liver B-ultrasound reduced by 1 level; None-effective: Failed to meet effective standards.
In vivo rat experimental methods
Procedures involving laboratory rats were conducted following the ethical standards of Shanghai Punan Hospital and Chinese humane guidelines (Policy No. 2006 398). Specific-pathogen-free Sprague–Dawley rats (33 males, 8 weeks-old, weight range 220 ± 20 g) were supplied by Shanghai SLRC Laboratory Animal Co., Ltd. with Certificate No. SCXK (Shanghai) 2008–0016), Rats were housed and fed ad libitum for 5 days in a clean quiet environment (23 – 25 °C, relative humidity 50 – 70%).
For the in vivo experiments, 33 rats were assigned to a control group with a normal diet (n = 10) and a model group fed a 45% high-fat diet (n = 23). The normal group was fed with purified feed with 10% fat content, while the NAFLD group was fed with purified feed with a 45% fat content, and all of the animals had access to drink water ad libitum. After successful model establishing, the remaining rats were subdivided into normal control (NC, n = 9), model control (MC, n = 10) and model DSSG (MDSSG, n = 10) groups, respectively. The NC and MC rats were given saline, whereas the DSSG group received DSSG. The dosage of DSSG in 200 g rats was 0.018 times than that for a normal weight person leading to DSSG doses of 0.108 g/100 g body weight, which was applied intragastrically (Fig. 2). Only male rats were chosen, since previous findings proposed male rats as more suitable for high-fat diet NAFLD models [17]. After 8 weeks, 1 rat in the control and 3 rats in the high-fat diet groups were sacrificed and liver tissue slices were examined and pathological sections of liver tissues stained with H&E. The presence of significant macrovesicular steatosis in the pathological sections represented the successful establishment of the model (Fig. 3A and B).
Specimen collection and index determinations
The rats were humanely killed and weighed after 8 weeks of interventions. Rats were deeply anesthetized with chloral hydrate (0.3 mL/100 g body weight) and blood samples taken from the abdominal aorta to measure blood glucose and lipid concentrations, and to assess liver functions. After the rats were killed, the liver was immediately removed and washed in sterilized saline at 4 °C in an ice bath before being dried on filter paper. A small quantity of liver tissue was fixed in formalin for subsequent sectioning after paraffin-embedding. Liver homogenate was produced after immersing liver tissue (1 g) in normal saline (10 mL). The concentration of superoxide dismutase and malondialdehyde in liver tissues were measured, and expression of NF-κB by western blotting.
Western blotting
Liver tissue (50 mg) was taken from the animals and l mL RIPA protein lysate was added. Next, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40 and a complete protease inhibitor cocktail were added. The tissue was ground and homogenized in a ball mill. The resulting homogenate was centrifuged (4 °C, 16,000 rpm, 30 min) and the supernatant removed for analysis. A standard curve was constructed using Xylene brilliant cyanin G to determine the protein concentration of the liver tissue in each group. The samples were uniformly concentrated after determination of their protein concentrations. Subsequently, denaturation of 25 µg/mL of protein in loading buffer was carried out for 5 min at 100 °C.
After gel electrophoresis and transfer to PVDF membranes, blots were incubated with a primary antibody against NF-κB (rabbit, Cell Signaling Technology, Inc. Danvers, MA, US) and GAPDH (mouse, Shanghai KangChen Bio-teck Co., Ltd.) at 4 °C overnight. After the application of appropriate secondary antibodies, electrochemiluminescence was employed to visualize the signals emitted by protein bands.
Data analysis
SPSS Statistics for Windows (ver. 21.0) was employed for analysis of data. If the measured data were normally distributed, the mean ± SD was used to express the results. Single factor variance analysis was employed to make pairwise comparisons across multiple data groups. A t-test was used for normal distributions and variance homogeneity and a rank sum test for abnormal distribution. For enumeration data, a chi-squared, Wilcoxon rank sum or Mann Whitney U tests were used. P < 0.05 was deemed to be a significant finding.