LIU Lulu,CHEN Yanxu,LIU Lulu,LI Man,XU Rui,QU Wensheng,YIN Jiye,ZHANG Xin,ZHOU Zhenhua,GAO Yating,NIE Dan,SHAN Junjie,GAO Yueqiu

LIU Lulu,Institute of Chinese Materia Medica,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China

CHEN Yanxu,LI Man,ZHANG Xin,ZHOU Zhenhua,GAO Yating,GAO Yueqiu,Shanghai Key Laboratory of Traditional Chinese Clinical Medicine,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China

XU Rui,QU Wensheng,YIN Jiye,NIE Dan,SHAN Junjie,Institute of Pharmacology and Toxicology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China

Abstract OBJECTIVE:To investigate the chemical characters of water-extract of Baqi Lingmao formula (巴芪灵猫方,BQLM formula)and its effects on anti-liver injury in model mice and live cells.METHODS:BQLM formula was composed of ten herbal medicines.We determined the contents of alkaloids,saponins,phenolic acids and flavonoid in BQLM formula by UV spectrophotometry.The active components of alkaloids and phenolic acids in BQLM formula were identified by HPLC chromatography.The anti-hepatic injury effects of BQLM formula were investigated with concanavalin A (ConA)-induced hepatitis model of mice,human liver LO2 and HepG2.2.15 cells.RESULTS:BQLM formula(2 and 10 g/kg,orally)significantly improved the damages of liver tissues and functions caused by ConA in mice,reduced the infiltration of inflammatory cells into liver and inhibited the inflammatory cytokine secretion of interferon-γ,tumor necrosis factor-α and interleukin-6.BQLM formula simultaneously decreased the levels of alanine aminotransferase and aspartate aminotransferase of liver and serum,and recovered the superoxide dismutase and catalase activities of liver to normal levels in ConA-induced hepatic-injury mice.The serum of BQLM formula group stimulated the human liver LO2 cell proliferation in vitro.Further,BQLM formula obviously promoted the proliferation of normal hepatocytes (LO2 cells) and inhibited the hepatocytes death induced by ConA.It also significantly inhibited the proliferation of HepG2.2.15 cells and decreased the secretion of HBsAg and HBeAg in vitro.CONCLUSIONS:BQLM formula has anti-inflammation and anti-hepatitis virus Beffects,and is capable of improving liver injury in vivo and in vitro.

Keywords:hepatitis B,chronic;concanavalin A;injury;hepatocytes;Baqi Lingmao formula

INTRODUCTION

Chronic hepatitis B (CHB) virus infection is a global public health problem,affecting more than 400 million people worldwide.In China,the number of people living with hepatitis B virus (HBV) infection is greatest.1CHB patients contain a wide clinical spectrum ranging from a subclinical inactive carrier state to progressive chronic hepatitis,cirrhosis and liver carcinoma.2

Traditional Chinese Medicine(TCM)was used to treat chronic liver diseases for a long history and it is still very important and major medicines today in China.In recent decades,a number of empirical formulations have been treated HBV patients in clinical to assess their efficacy and safety.For examples,"Fufang Biejia Ruangan Tablet" (复方鳖甲软肝片) was used for treatment of hepatic fibrosis in patients with CHB.3"Fu-Zheng-Jie-Du-Tang" (扶正解毒汤) markedly decreased the concentration of serum HBsAg,increased interferon-γ (IFN-γ) levels,and modulated host immune function of HBV patients.4Clinical studies showed that "Yin-chen-hao-decoction" (茵陈蒿汤)plays crucial roles in CHB treatment,significantly improved the clinical symptoms and viral replication of patients,and restoring liver function.5

Among these clinical formulations,"Lingmao formula"(灵猫方) and "Bushen recipe"(补肾方) were both established by Prof.Gao Yueqiu (Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine),and the two prescriptions were used for HBV patients in their hospital for ten years.6,7"Bushen recipe" combined with entecavir significantly decreased the serum HBV DNA,increased HBeAg loss for HBeAg-positive HBV patients and mildly elevated alanine aminotransferase (ALT) without any serious adverse events.8The expressions of granulysin and IFN-γ or CD4+T cells and the expressions of granzyme,tumor necrosis factor-α (TNF-α) and IFN-γ or CD8+T cells were declined in the treatment group.9HBeAg-positive CHB patients receive Lingmao formula combined with entecavir significantly decreased serum HBV DNA levels and reduced HBeAg titer of.8 On the base of the two formulas,Prof.Gao created a new formula which was called "Baqi Lingmao formula" (巴芪灵猫方,BQLM formula) for treatment of CHB.BQLM formula is composed of ten herbal drugs.BQLM formula improved stronger effects on invigorating kidney,strengthening spleen and enhancing blood circulation than "Lingmao formula" or "Bushen formula"in clinical treatments.10

MATERIALS AND METHODS

Medicine and major reagents

BQLM formula is a clinical prescription for treatment of CHB patients in Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine.It is composed of ten herbal medicines:Bajitian (Radix Morindae Officinalis) 30 g,Yinyanghuo (Herba Epimedii Brevicornus)15 g,Dihuang(Radix Rehmanniae)10 g,Huangqi(Radix Astragali Mongolici)30 g,Baizhu(Rhizoma Atractylodis Macrocephalae) 15 g,Kushen (Radix Sophorae Flavescentis)15 g,Maozhuacao(Radix Ranunculi Ternati) 15 g,Dangshen (Radix Codonopsis) 15 g,Lingzhi (Ganoderma Lucidum) 15 g,Qingpi (Fructus Citri Reticulatae Immaturus) 10 g.Glycyrrhizin capsules were purchased from Beijing Kawin Technology Share-Holding Co.,Ltd.,(Beijing,China.) Entecavir tablet was produced by Suzhou Dawnrays Pharmaceutical Co.,Ltd.,(Suzhou,China).The standards substances(salvianic acid A sodium,protocatechuic acid,protocatechuic aldehyde,caffeic acid,rosmarinci acid,salvianolic acid B,oxymatrine,oxysophocarpine,sophoridine,matrine and sophocarping) were purchased from National Inatitute for Food and Drug Control in China.Commercial kits of mouse superoxide dismutase(SOD),catalase (CAT),malondialdehyde (MDA),ALT and aspartate aminotransferase (AST) were purchased from Nanjing Jiancheng Biotechnology co.Ltd.Enzyme-linked immunosorbent assay (ELISA) kits of mouse IFN-γ,TNF-α and interleukin-6 (IL-6) were purchased from Neobioscience Technology Co.,Ltd.The ELISA kits of mouse HBsAg and HBeAg were produced by Shanghai Kehua Bioengineering Co.,Ltd.Fetal bovine serum was purchased from Lonsera (Uruguay),1640 cell culture medium was purchased from Hyclone (Logan,UT,USA).MTT was from Amresco(Washington,USA).Varioskau Flash version 2.4.3 ELISA is produced by Thermo Scientific(MA,USA).

Experimental animals and cells

20-22 g specific psthogen-free (SPF) female Balb/C mice [Animal license No.SCKK (Jing) 2016-0011]were purchased from Beijing Vital River Laboratory Animal Technology Co.,Ltd.(Beijing,China).These mice were kept under standard environment condition(23-25 ℃,humidity 50%-70 %,12 h light:12 h dark cycle) with free access to standard diet and water ad libitum.All animal procedures were performed according to the Guide for the Care and Use of Laboratory Animal of the National Institute of Health and Guide of the Animal Welfare Act.

Human LO2 liver cell line and HepG 2.2.15line were kept in our laboratory.The two cell lines were cultured with 1640 DMEM medium containing 10% fetal bovine serum (FBS) at 37 ℃,5% CO2and saturated humidity conditions.

Preparing water-extract of BQLM formula

Six folds weight of BQLM formula(170 g×6=1020 g)were mixed,crushed and passed through 20 mesh sieve.The powder was added 15 L water and soaked for 30 min,then decocted for 1h.The decocted material was passed through 100 mesh sieve.The residue was added 15 L water again and continued decocting 1 h and filtered.The two filtrates were combined and centrifuged (3000 r/min × 20 min),the supernatant was concentrated to thin paste under reduced pressure at 50-55 ℃.The thin paste was loaded on plate and heated at 70-80 ℃to concentrate into thick paste.The thick paste was dehydrated in vacuum at 55 ℃to obtain dry power and named BQLM formula.

Qualitative analyzes of main components in BQLM formula

In previous qualitative tests,we found that there were alkaloids,saponins,phenolic acids,terpenes,flavonoid glycosides and sugars as main components in BQLM formula.

(a)Total flavonoid content

Total flavonoid content was determined based on a method of Weiet al.1140 mg dried BQLM formula was added 10 mL deionized water to dissolve.2 mL BQLM formula solution was added 1 mL 5% NaNO2and 1 mL 10% Al(NO3)3.After 6 min,5 mL 5%NaOH was added to the solution and vortexed for 15 s.The absorbance values were measured at 510 nm by using spectrophotometer.The flavonoid content of BQLM formula was calculated as rutin equivalent by using the equation obtained from the rutin calibration curve (0.03-0.30 mg/mL).All samples were analyzed in triplicates.

(b)Total phenolic acids content

Total phenolic acids content was determined based on a method of Wanget al.1210 mg dried BQLM formula was added 25 mL deionized water to dissolve.2 mL BQLM formula solution was added ethanol to 5 mL,then 2 mL 0.3% SDS was added into the solution and vortexed.Further,1 mL mixed solution of 0.6% ferric chloride-0.9% potassium ferricyanide (1∶0.9,v/v) was added to the reactive system and against for light for 5 min.Finally,the reactive system was added 0.1 mol/L HCl solution to 25 mL volume.After 20 min in dark,the system solution was measured the absorbance at 760 nm using a spectrophotometer.A standard calibration curve of protocatechuic acid (2.06-10.29 μg/mL)was plotted.All samples were analyzed in triplicates.

(c)Total saponins content

Total saponins content was determined based on a method of Wanget al.1310 mg dried BQLM formula was added 10 mL deionized water to dissolve.0.5 mL BQLM formula solution was taken to a tube,and heated to remove solvents.0.2 mL 5% vanillin (prepared with glacial acetic acid) and 0.8 mL perchloric acid were added the tube,then put on a bath (70 ℃) for 20 min.The tube was removed to an ice-water bath for 10 min and added glacial acetic acid to 10 mL,and stirred.The system solution was measured the absorbance at 550 nm using a spectrophotometer.A standard calibration curve of diosgenin (40-400 μg) was plotted.All samples were analyzed in triplicates.

(d)Total alkaloids content

Total alkaloids content was determined based on a method of Shaoet al.1430 mg dried BQLM formula was added 10 mL deionized water to dissolve.2.0 mL BQLM formula solution was taken to an erlenmeyer flask,and added 6 mL 0.02% bromothymol blue buffer and standed for 5 min,then added 6 mL chloroform into the system.After shaking for 2 min,the system solution was transfered into a tap funnel and standing for 30 min.The layer of chloroform was removed and measured the absorbance at 415 nm using a spectrophotometer.A standard calibration curve of matrine (9.91-59.46 μg/mL) was plotted.All samples were analyzed in triplicates.

HPLC chromatographic conditions

(a)Phenolic acids in BQLM formula

The HPLC analyses were performed on Agilent HPLC system (PA,CA,USA) equipped with YMC-Pack ODS-A column (4.6×250 mm,5μm) and a photodiode array detector coupled with chemstation B .04.03 software.The mobile phase consisted of methanol (A)and 0.4% aqueous formic acid (v/v,B) using a linear gradient program of 10%-40%A in 0-30 min,40%A in 30-55 min,40%-70%A in 55-70 min,70%-100%A in 70-80min and 100%A in 80-90 min.The flow rate was 1.0 ml/min and column temperature 30 ℃.UV detector was set at 280 nm.

(b)Alkaloids in BQLM formula

The HPLC system was Thermo Syncronis C18 column (4.6×250 mm,5μm) and a photodiode array detector coupled with chemstation B .04.03 software.The mobile phase consisted of 10 mmol/L ammonium acetate (pH=8.0 adjusted with ammonia solution) as A solution,and acetonitrile-10 mmol/L ammonium acetate (v/v=9∶1) as B solution using a linear gradient program of 0-25 min,10%-13%B;25-30 min,13%B;30-80 min,13%-50%B;80-90 min,50%-100%B and 90-100 min,100% B.The flow rate was 0.8 mL/min and column temperature 35 ℃.UV detector was set at 220 nm.

Determination of protective activity in ConA-induced liver injury mice15,16

Fifty Balb/C female mice (20-22 g) were randomly divided into 5 groups (n=10 each group):(a) normal control group (received water,i.g);(b) ConA-induced liver-injury model group (received 10 mg/kg ConA alone dissolved in saline,i.v);(c) ConA (10 mg/kg,i.v.) +high dose of BQLM formula (10.0 g/kg,dissolved in water,i.g);(d) ConA (10 mg/kg,i.v.)+low dose of BQLM formula (2.0 g/kg,dissolved in water,i.g);(e)ConA (10 mg/kg,i.v)+glycyrrhizin capsules (50mg/kg,dissolved in water,i.g) group.Mice were injected ConA one time each week and received BQLM formula one time every day from treated ConA until end of the test.After the last injection of ConA (total three times),BQLM formula was continued same treatment for 3 d.

After the test ended,these mice were blooded from eyes and sacrificed by dislocation.The liver,kidney and thymus of mice were enucleated,weighed and accounted their weight indexes.The liver samples of these mice were sliced at same sites and fixed in 10%formalin,then dehydrated,embedded and stained with hematoxylin and eosin.The retain liver of each mouse was weighted and homogenated with saline.The liver homogenate was centrifugated (4000 r/min×10 min),and the supernatant was analyzed some parameters of liver functions (such as AST and ALT),anti-oxidative stress(such as SOD and CAT)with commercial kits.

The spleen of these mice was selected under aseptic conditions,then grinded in DMEM medium(containing 10% fetal bovine serum,100 U/mL penicillin and 100 μg/mL streptomycin),and passed through 200 mesh to obtain a homogeneous cell suspension.The erythrocytes in the cell suspension were split with Tris-NH4Cl buffer,and these spleen cells were washed three times with the DMEM medium and resuspended in the essential medium.The splenocytes density was counted with a haemocytometer (cell viability >95%).Splenocyte proliferation was assayed by MTT methods.The splenocytes were seeded into 96-well plate at 5 × 106cell/mL in triplicates,then added 100 μL ConA (final concentration 4 μg/mL),or LPS (final concentration 15 μg/mL)or essential medium and incubated at 37 ℃in a humid atmosphere with 5% CO2.After 48 h incubation,20 μL of 2.5 mg/mL MTT solution was added into each well and continued to incubate for 4 h.The untransformed MTT was removed from these wells and added 10%SDS(100 μL),and incubated overnight at 37 ℃.The absorbance was evaluated in an ELISA reader at 570 nm.

The splenocytes (5 × 106cells/well) of ConA-induced hepatitis mice were incubated with ConA(4 μg/mL)in 24-well culture plates at 37 ℃in 5% CO2.After 72 h,the plate was centrifuged (1000 r/min×10 min) and the supernatants were collected for detecting IFN-γ and IL-6 levels using commercial ELISA kits.

Determination proliferation and survivorship of liver LO2 cells in vitro17

LO2 cell suspensions(1.0×104cells/well)were seeded into 96-well plate in triplicates.After 12 h,four final concentrations of BQLM formula (10,50,100 and 250 μg/mL)were added into the LO2 cells respectively and incubated for 72 h.Cell proliferation rates were determined by MTT methods.After 72 h incubation,the culture medium was aspirated and added 100 μL MTT (1.0 mg/mL),and continued to incubat for 4 h.The medium was aspirated and replaced with 100 μL DMSO to dissolve the formazan crystals.The culture plates were shaken for 5 min,read OD value at 540 nm and counted the proliferative rate of LO2 cells.Proliferative rate (%)=(OD experimental group－ OD control group)/OD control group ×100%.

LO2 cell suspensions(1.0×104 cells/well)were seeded into 96-well culture plate in triplicates.After 12 h,these cells were exposed to ConA(0 or 500 μg/mL)for 1 h,then 0,10,50 or 250 μg/mL of BQLM formula(final concentration) were added respectively and incubated at 37 ℃for 72 h.Cell survival rate were determined by MTT methods.After 72 h incubation,the medium in each well was aspirated and replaced with 100 μL MTT (1.0 mg/mL) and incubated at 37 ℃for 4 h.The medium was aspirated and replaced with 100 μL DMSO to dissolve the formazan crystals.The culture plates were shaken for 5 min,and the absorbance value of each well was read at 540 nm.The inhibitive rate of LO2 cells was counted.

Inhibitive ratio (%)=(OD experimental group－ OD solvent group)/OD solvent group×100%.

Determination of liver LO2 cells proliferation with blood serum from ConA-induced hepatic-injury mice

LO2 cell suspensions(1.0×104 cells/well)were seeded into 96-well plate in triplicates.After 12 h,three final concentrations of serum (1/4,1/8 and 1/16 of serum)which treated with BQLM formula were added into the LO2 cells respectively and incubated for 72 h.Cell proliferation rates were determined by MTT methods.After 72 h incubation,the culture medium was aspirated and added 100 μL MTT (1.0 mg/mL),and continued to incubate for 4 h.The medium was aspirated and replaced with 100 μL DMSO to dissolve the formazan crystals.The culture plates were shaken for 5 min,read OD value at 540 nm and counted the proliferative rate of LO2 cells.

Proliferative rate(%)=(OD experimental group–OD control group)/OD control group×100%.

Determination of HepG2.2.15 cells proliferation in vitro

HepG2.2.15 cells in DEME containing 10%heat-inactivated FBS at 37 ℃in a humid 5% CO2air atmosphere.Briefly,the cells were plated in 96-well culture plates at a density of 0.5×104cells/well in 100 μL culture medium for 5 h,then different concentrations of BQLM formula (0,10,50,250 μg/mL) in 100 μL were added the culture wells.At the time points of 72 h,20 μL 0.5% MTT was added to each well and the cells were incubated for 4 h.Then,the culture medium in each well was token way,and 200 μL 10%SDS was added into each well and continued to incubate 37 ℃in a humid 5%CO2air atmosphere.Finally,ODs were monitored at 570 nm and counted the proliferative rate of HepG2.2.15 cells.

Analysis of HBsAg and HBeAg secretion from HepG2.2.15 cells17

HepG2.2.15 cells(5.0×103/well)were seeded plates in triplicates.After incubation for 24 h,the HepG2.2.15 cells were treated with 0,10,100,250 and 500 μg/mL of BQLM formula (final concentrations) respectively for 48 h.After the culture medium was centrifugated,the supernatant was collected and analyzed the concentration of HBsAg and HBeAg in the supernatant with ELISA kits.

Statistical analysis

The data were expressed as mean ± standard deviation(±s) and examined for their statistical significance of difference with SPSS 13.0 and one-way analysis of variance.P-values of less than 0.05 were considered to be statistically significant.

RESULTS

Total alkaloid,saponin and triterpene,flavonoid and phenolic acid contents in BQLM formula

Totally 1020 g initiative material of BQLM formula was decocted two times with water to obtain 411.4 g dried water-extract (BQLM formula),its yield was 40.34%.The extract contains alkaloids (0.60%),saponin and triterpene (32.95%),flavonoid (1.61%),phenolic acid(2.04%)and some carbohydrate.

Identification of phenolic acids and alkaloids components in BQLM formula by HPLC chromatography

Because phenolic acid ingredients were mainly come from water-extract of Salvia miltiorrhiza and alkaloids mainly was source of Sophora flavescens in BQLM formula (Figure 1A,1B),two HPLC fingerprint profiles of BQLM formula were established to identify these phenolic acids and alkaloids(Figure 2A,2B).

Form Figure 1A,there were five marker compounds in water-extract of Salvia miltiorrhiza was performed under our separation conditions and UV detection(280 nm),and the five markers were salvianic acid A sodium,protocatechuic aldehyde,caffeic acid,rosmarinci acid and salvianolic acid B respectively.The five markers compounds were also found in BQLM formula (Figure 2A).However,another marker component protocatechuic acid of Danshen(Salvia miltiorrhiza) was extracted in BQLM formula.The result suggested that the decoction of mixed ten medicinal herbs were in favour of protocatechuic acid extraction than single Salvia miltiorrhiza.

Form Figure 1B,there were four marker compounds in water-extract of Kushen (Sophora flavescens),and the four markers were oxymatrine,oxysophocarpine,matrine and sophocarpine respectively.In BQLM formula,we found only two alkaloid markers (matrine and sophocarpine),oxymatrine and oxysophocarpine were lost (Figure 2B).The result suggested that oxymatrine and oxysophocarpine might be changed into matrine and sophocarpine or other compounds by mixed-extract of ten medicinal herbs.

Figure 1 HPLC chromatograms of five phenolic acids and four alkaloids in water-extract of Sophora flavescens

Figure 2 HPLC chromatograms of six phenolic acids and three alkaloids in BQLM formula

Protective effect of BQLM formula on ConA-induced liver injury in mice

(a) Survival rate and organ weight of liver,kidney and thymus

After the first injection of ConA at dose of 10 mg/kg,these mice were administrated orally 2.0 g/kg (low dose) and 10.0 g/kg (high dose) of BQLM formula for 18 d.When the test ended,there were four mice died in ConA-treated alone group,two died in high dose group,two died in low dose group of BQLM formula,and three died in glycyrrhizin group.The organs of liver,kidney and thymus of every mouse were enucleated and weighed,and calculated their weight indexes (Table 1).These data indicated that the liver weight index of ConA-treated model group significantly increased(P <0.01),and the thymus index remarkably decreased (P <0.05),but kidney index had no obvious difference (P >0.05).Treatment of 2 and 10 g/kg BQLM formula both ameliorated the swelling of liver(P <0.05).Glycyrrhizin did not show good changes compared to ConA-treated alone.

Liver hematoxylin-eosin (HE) staining of mice showed that injection of ConA alone caused severe liver damage.There was abundant infiltration of inflammatory cells (mostly lymphocytes) around the central veins and large areas of centrilobular necrosis and hepatocyte apoptosis (Figure 2B).Administration of low and high dose of BQLM formula both clearly attenuated the extent of the liver pathological damage induced by ConA.The necrotic areas in the livers of ConA+(BQLM formula)treated mice were significantly smaller compared with the ConA alone group,and BQLM formula significantly decreased inflammatory infiltration,inhibited apoptosis or necrosis of hepatocytes (Figure 3C,3D).Glycyrrhizin also improved the liver changes,but its effect was weaker than BQLM formula (Figure 3E).

Figure 3 Representative images showing HE staining of mouse liver sections(×100)

(c) Liver functional markers of plasma and liver homogenate

Compared to normal control group,the activities of AST and ALT (markers of liver function) of liver homogenate increased (P >0.05) and plasma levels of AST and ALT significantly elevated (P <0.05 andP <0.01) in ConA-treated alone group.However,compared to ConA-treated alone mice,the AST and ALT of liver homogenate in ConA+BQLM formula groups were completely reversed to normal levels,and plasma AST levels were similar to normal group (P <0.05).

There was no significant dose-effect relation between 2 and 10 g/kg(Table 2).

(d)Release of inflammatory cytokines in liver homogenate

ConA-induced hepatitis is associated with the production of various inflammatory cytokines,such as TNF-α,IFN-γ and IL-6.As shown in Table 3,compared with normal control group,ConA-treatment alone leaded to elevation of TNF-α,IFN-γ and IL-6 levels in liver homogenates of mice (P >0.05).Treatment of 10 g/kg of BQLM formula to the ConA-injected mice resulted in significant suppression of TNF-α and IFN-γ levels (P <0.05),and also decreased IL-6 level(P>0.05).The activity of 2 g/kg BQLM formula was weaker than 10 g/kg dose.Glycyrrhizin also showed a depressed activity for three inflammatory cytokines.

(e)Antioxidant activity of liver

Effects of BQLM formula on the levels of SOD,CAT and MDA of liver homogenates of mice were shown in Table 4.These data showed that injection of ConA decreased the activities of SOD and CAT,but did not reach significant difference (P >0.05).Compared with the ConA alone group,administration with two doses of BQLM formula resulted in an increase SOD and CAT to normal level (P >0.05),and high dose was slight stronger than low dose.There was no markedly change of MDA level among these groups.

Effect of BQLM formula on proliferation of human LO2 hepatocytes and anti-damage induced by ConA in vitro

BQLM formula exhibited significant effect of anti-liver damage induced by ConA in mice.Further,we were investigated BQLM formula on the proliferation and anti-ConA damage with human hepatocytes LO2 cellsin vitro.Three final concentrations of BQLM formula(10,50 and 250 μg/mL) were co-cultured with human LO2 hepatocytes or simaltaneously added ConA(500 μg/mL)for 72 h.The results were shown that 10,50 and 250 μg/mL of BQLM formula significantly improved the proliferation of LO2 cells,and their proliferative rates were 9.8%,20.8% and 76.0% (P <0.01,<0.001) respectively.ConA (500 μg/mL) was added to the LO2 cells and induced the death of 55.8% cells.However,10,50 and 250 μg/mL of BQLM formula significantly inhibited LO2 cells to death,and the death rates of three concentrations were 49.3%,39.3%and-3.0% respectively (Table 5).There was obviously concentrationdependent manner for BQLM formula for improving the proliferation of LO2 cells and inhibiting cell death induced by ConA.

Table 1 Effect of BQLM formula on indexes of organ weight of ConA-induced liver injury mice(±s)

Table 1 Effect of BQLM formula on indexes of organ weight of ConA-induced liver injury mice(±s)

Notes:the mice of normal control group received water (i.g);the mice of ConA-induced liver-injury model group received ConA alone(10 mg/kg dissolved in saline,i.v);the mice of BQLM formula(high dose)group received ConA(10 mg/kg,i.v.)+BQLM formula(10.0 g/kg,dissolved in water,i.g);the mice of BQLM formula(low dose)group received ConA(10 mg/kg,i.v.)+BQLM formula(2.0 g/kg,dissolved in water,i.g);the mice of glycyrrhizin goroup received ConA(10 mg/kg,i.v.)+glycyrrhizin capsules(50 mg/kg,dissolved in water,i.g).BQLM formula:Baqi Lingmao formula;ConA:concanavalin.Compared with normal control group,aP <0.01,cP <0.05;compared with ConA model group,bP<0.05.

Table 2 Effect of BQLM formula on AST and ALT from the liver and serum of ConA-induced liver injury mice(±s)

Table 2 Effect of BQLM formula on AST and ALT from the liver and serum of ConA-induced liver injury mice(±s)

Notes:the mice of normal control group received water (i.g);the mice of ConA-induced liver-injury model group received ConA alone(10 mg/kg dissolved in saline,i.v);the mice of BQLM formula(high dose)group received ConA(10 mg/kg,i.v.)+BQLM formula(10.0 g/kg,dissolved in water,i.g);the mice of BQLM formula(low dose)group received ConA(10 mg/kg,i.v.)+BQLM formula(2.0 g/kg,dissolved in water,i.g);the mice of glycyrrhizin goroup received ConA(10 mg/kg,i.v.)+glycyrrhizin capsules(50 mg/kg,dissolved in water,i.g).BQLM formula:Baqi Lingmao formula;ConA:concanavalin;H:high dose;L:low dose.Compared with normal control group,aP <0.05,bP<0.01;compared with ConA model group,cP<0.05.

Table 3 Effect of BQLM formula on inflammatory cytokines in liver homogenates of ConA-induced liver injury mice(pg/g,±s)

Table 3 Effect of BQLM formula on inflammatory cytokines in liver homogenates of ConA-induced liver injury mice(pg/g,±s)

Notes:the mice of normal control group received water (i.g);the mice of ConA-induced liver-injury model group received ConA alone(10 mg/kg dissolved in saline,i.v);the mice of BQLM formula(high dose)group received ConA(10 mg/kg,i.v.)+BQLM formula(10.0 g/kg,dissolved in water,i.g);the mice of BQLM formula(low dose)group received ConA(10 mg/kg,i.v.)+BQLM formula(2.0 g/kg,dissolved in water,i.g);the mice of glycyrrhizin group received ConA(10 mg/kg,i.v.)+glycyrrhizin capsules(50 mg/kg,dissolved in water,i.g).BQLM formula:Baqi Lingmao formula;ConA:concanavalin.Compared with ConA model group,aP<0.05.

Effect of blood serum of ConA-induced liver-damage mice on LO2 cell proliferation

The serum of ConA-induce liver-damage mice were incubated with LO2 cells for 72 h.The effect on cell proliferation was shown in Table 6.These data exhibited that the serum from all groups could significantly stimulate LO2 cell proliferation at 1/4,1/8 and 1/16 dilution compared to saline (P <0.001).The serum of BQLM formula (L) and BQLM formula (H) groups were shown more strongly improved the proliferation of LO2 cells at 1/4 dilution compared to normal mice group(P<0.05).

Effect of BQLM formula on proliferation and secretion of HBsAg and HBeAg of HepG2.2.15 hepatocytes

In order to investigate whether BQLM formula might have anti-proliferation effect on HepG2.2.15 cells,a series concentration of BQLM formula (10,50 and 100 μg/mL) were treated with HepG2.2.15 cells for 48 h.Compared to cells treated with saline,some HepG2.2.15 cells were induced to death after treated with BQLM formula and in a concentration-dependent manner.Especially,100 μg/mL of BQLM formula exhibited significant anti-proliferation effect on HepG2.2.15 cells(P<0.001)(Table 7).

The effect of BQLM formula on HBsAg and HBeAg secretion of HepG2.2.15 cells was investigated,and the result was shown in Table 7.After 48 h co-culture of HepG2.2.15 cells with 100,250 and 500 μg/mL BQLM formula,BQLM formula could significantly reduce HBsAg secretion by 16.6%,34.0% and 43.4%(P <0.05,<0.01) and the secretion of HBeAg by 15.8%,22.5% and 44.7% (P <0.05,<0.01) respectively.BQLM formula exhibited a good concentration-effect relationship on inhibiting the secretion of HBsAg and HBeAg in HepG2.2.15 cells.

Table 4 Effect of water decocted extract and two sites on oxidative stress from the liver of ConA-induced liver injury mice(±s)

Table 4 Effect of water decocted extract and two sites on oxidative stress from the liver of ConA-induced liver injury mice(±s)

Notes:the mice of normal control group received water (i.g);the mice of ConA-induced liver-injury model group received ConA alone(10 mg/kg dissolved in saline,i.v);the mice of BQLM formula(high dose)group received ConA(10 mg/kg,i.v.)+BQLM formula(10.0 g/kg,dissolved in water,i.g);the mice of BQLM formula(low dose)group received ConA(10 mg/kg,i.v.)+BQLM formula(2.0 g/kg,dissolved in water,i.g);the mice of glycyrrhizin group received ConA(10 mg/kg,i.v.)+glycyrrhizin capsules(50 mg/kg,dissolved in water,i.g).BQLM formula:Baqi Lingmao formula;ConA:concanavalin;SOD:superoxide Dismutase;CAT:catalase;MDA:malondialdehyde.

Table 5 Effect of BQLM formula on proliferation and anti-death of human liver LO2 cells(±s)

Table 5 Effect of BQLM formula on proliferation and anti-death of human liver LO2 cells(±s)

Notes:there were three groups (saline control,ConA control and (ConA+BQLM formula)) in the test.The concentration of ConA was 500 μg/mL,and the three concentrations of BQLM formula were 10,50 and 250 μg/mL respectively.LO2 cells were treated with saline,ConA and BQLM formula for 72 h to determine the proliferation rate and death rate induced by ConA(500 μg/mL)in vitro;ConA:concanavalin A;BQLM formula:Baqi Lingmao formula.aP<0.01,bP<0.001,compared to saline group;cP<0.01,dP<0.001,compared to ConA group,n=3.

Table 6 Effect of serum from mice which were treated BQLM formula on proliferation of human liver LO2 cells(±s)

Table 6 Effect of serum from mice which were treated BQLM formula on proliferation of human liver LO2 cells(±s)

Notes:the mice of normal control group received water (i.g);the mice of ConA-induced liver-injury model group received ConA alone(10 mg/kg dissolved in saline,i.v);the mice of BQLM formula(high dose)group received ConA(10 mg/kg,i.v.)+BQLM formula(10.0 g/kg,dissolved in water,i.g);the mice of BQLM formula(low dose)group received ConA(10 mg/kg,i.v.)+BQLM formula(2.0 g/kg,dissolved in water,i.g);the mice of glycyrrhizin group received ConA(10 mg/kg,i.v.)+glycyrrhizin capsules(50 mg/kg,dissolved in water,i.g).The serum LO2 cells were treated with the serum of mice from the five groups respectively using three diluted concentrations (16,8 and 4 folds) to determine the proliferation rate;BQLM formula:Baqi Lingmao formula.aP <0.01 andbP <0.001,compared to saline group;cP<0.05,compared to normal mice group,n=3.

Table 7 Effect of BQLM formula on proliferation of Hep2.2.15 cells(±s)

Table 7 Effect of BQLM formula on proliferation of Hep2.2.15 cells(±s)

Notes:there were three groups (saline control,BQLM formula and glycyrrhizin) in the test.The concentrations of BQLM formula and glycyrrhizin both were 10,50 and 100 μg/mL respectively.Hep2.2.15 cells were treated with saline,BQLM formula and glycyrrhizin for 48 h to determine the proliferation rate in HepG2.2.15 in vitro.BQLM formula:Baqi Lingmao formula.Compared to saline group,aP<0.001,n=3.

Table 8 Effect of BQLM formula for HBsAg and HBeAg secretions of HepG2.2.15 cells(±s)

Table 8 Effect of BQLM formula for HBsAg and HBeAg secretions of HepG2.2.15 cells(±s)

Notes:there were three groups (saline control,BQLM formula and glycyrrhizin) in the test.The concentrations of BQLM formula and glycyrrhizin both were 10,50 and 100 μg/mL respectively.Hep2.2.15 cells were treated with saline,BQLM formula and glycyrrhizin for 48 h to determine the secretion of HBsAg and HBeAg in HepG2.2.15 in vitro.BQLM formula:Baqi Lingmao formula.aP <0.05,bP <0.01,cP<0.001,compared to saline group,n=3.

DISCUSSION

The HBV is one of the most common infectious agents worldwide.In China,where about 120 000 000 persons are chronically infected.Chronic infection with HBV can significantly impair the quality of life and life expectancy of patients because of the potential for disease progression,which can lead to fibrosis,cirrhosis,liver failure,and hepatocellular carcinoma.18Currently,the available antiviral drugs for HBV include immunomodulatory drugs (IFN-α and pegylated IFN-α) and HBV polymerase inhibitors (nucleosideanalogs:lamivudine,telbivudine,and entecavir and nucleotide analogs:adefovir and tenofovir).19However,these therapeutic drugs are not satisfactory with obvious drug resistance and dose-dependent side effects.20

In China,Traditional Chinese medicine (TCM) has a long history of treating liver diseases and serves as an established segment of the public health system.At present,it has been estimated that 30%-50%of total medicine consumption of hepatitis B treatment in China is attributed to Chinese medicine with low cost and low toxicity,and 80% of the patients with CHB in China rely on TCM.21The strategy of TCM treatment of CHB is:(a) ameliorating uncomfortable symptoms and improve the quality of life of patients;(b) alleviating liver inflammation;(c)inhibiting or delaying hepatic fibrosis;(d) regulating immune function;(e) improve lipid metabolism.21Unlike western medicines,TCM strictly relies on the two therapeutic pillars of holism and syndrome differentiation.In modern TCM,the definition of chronic liver diseases in TCM was gradually refined to encompass:(a) weakening of theQi(biological substances/activities that preserve life);(b) blockage of meridians (circulation channels ofQi)by blood stasis;(c) generation of dampness and heat(inflammatory pathogens).22

BQLM formula was composed of ten herbal medicines.Among these medicinal herbs,Bajitian (Morinda Officinalis How)and Yinyanghuo(Epimedium Brevicornu) warm "Yang Qi" and invigorate kidney;Dihuang(Rehmannia Glutinosa) enrich the kidney and replenish"Yin";Hunagqi (Astragalus Membranaceus),Baizhu(Atractylodis macrocephalae) and Lingzhi (Ganoderma lucidum)fortify"spleen function"(nursing and regulating digestive functions) and replenish "Qi";Maozhacao (Ranunculi Ternati) and Kushen (Sophorae Flavescentis) clear "heat",drive away dampness and eliminate residual pathogen;Danshen (Salvia Miltiorrhiza) and Qingpi (Citri Reticulatae) activate blood circulation and promote "Qi".The whole formula enriches the kidney,fortifies the spleen,clear heat,detoxify and eliminate dampness.

Among the ten medicinal herbs of BQLM formula,some single herbs and their some components have been confirmed possess stronger anti-HBV activities in clinical and pharmacological applications.For example,matrine and oxymatrine preparation from Sophorae flavescentis had been used to treat HBV patients.The two alkaloids exhibited significant effect on the clearance of hepatitis B virus DNA,HBV surface antigen and HBV e antigen,and were beneficial to the normalization of serum ALT and AST levels.23The crude polysaccharides from Sophorae flavescentis also exhibited good activity of anti-hepatic injury in ConA-induced damage model of mice.17A clinical evaluation ofRadix Astragalusmembranaceus was performed in 208 patients with CHB.The treatment group (n=116)was treated with compound from the Radix and the control group (n=92) was treated with regular drugs used for viral hepatitis.The results indicated that negative conversion rates of HBeAg and HBV DNA were significantly higher in the treatment group than in the control group.24In a clinical evaluation,30 HBV patients were treated with Salvia miltiorrhiza for 3 months of treatment.The results exhibited that the negative conversion rate of HBeAg was 16.7%.A follow up of 3 and 9 months after the end of treatment,the negative conversion rates of HBeAg were 22.7%and 25.0%,respectively.25The major active constituents of water-solubility in root of Salvia miltiorrhiza were phenolic acids,such as salvianic acid,protocatechuic aldehyde,rosemarinic acid andsalvianolic acid B.26 Sodium tanshinone ⅡA sulfonate (STS) is a water-soluble derivative of tanshinone ⅡA (an active component of Salvia miltiorrhiza).C57BL/6 mice pretreated with STS released less ALT into plasma in response to ConA challenge and had reduced inflammatory infiltration and hepatocyte apoptosis,abrogated TNF-α and IFN-γ production in the liver.27Ranunculus ternatus polysaccharide exerts significant hepato-protective effectin vivoandin vitro.The polysaccharides increased human liver cell proliferation,reduced cell damage by ConA,and inhibited the secretion of HBsAg and HBeAg by HepG2.2.15 cells.28Ganoderic acid from Ganoderma lucidum inhibited replication of HBV in HepG2.2.15 cells,and protected the mice from liver injury induced by bacille Calmette-Guerin(BCG) plus lipopolysaccharide.29Administration of Ganoderma lucidum polysaccharide (GLP) reversed above hepatic injury stimulated by BCGin vivo.Moreover,GLP dose-dependently inhibited activities of CYP2E1,CYP1A2 and CYP3A in hepatic microsomesin vitro,it suggested that inhibition of GLP on P450 oxidative metabolism might participate in the hepato-protective mechanism.30Atractylodes macrocephala polysaccharide also significantly reduced the elevated expression of markers of liver dysfunction and the hepatic morphologic changes induced by hepatic ischemia-reperfusion injury (IRI) in rats.It also markedly inhibited lipid peroxidation,increased the activities of SOD and suppressed the expression of interleukin-1β and NF-kB in IRI-treated rats.31Water-extract of Citri reticulatae significantly decreased the activity of ALT and AST of serum,degrade the content of MDA of hepatic tissues,increased SOD activity of chemical-induced liver injury.32These studies suggested that Baqi Lingmao formula is effective for treating chronic hepatitis B seems to relate with these active components.

ConA is a powerful immunostimulant and inflammogenin vivo.Kimuraet al33developed a chronic liver injury mouse model that was induced by repeated ConA injections.ConA model has unique features with respect to its pathogenesis and important similarities to immune-mediated hepatitis in humans,such as autoimmune hepatitis,acute viral hepatitis or distinct entities of drug toxicity leading to immune activation.34This chronic liver injury model was similar to autoimmune liver disease and HBV hepatitis with these features of T cell activation and chronic inflammation.These inflammatory mediators including TNF-α,IFN-γ and IL-6 are secreted by hepatic macrophages(Kupffer cells)and activated T cells,and act as major mediators to initiate,amplify,and promote the inflammatory response in inflammation-induced hepatocyte apoptosis,result in liver-specific inflammation and apoptosis.35,36Oxidative stress is thought to be a sub-cardinal contributor to ConA-induced hepatic failure.37SOD,CAT and MDA has been frequently used as a marker of cellular oxidation status.38Liver damage is routinely assessed by histology and by quantification of serum AST and ALT activities.ConA injection into the tail vein caused a great increase in the serum AST and ALT levels.39

About the mechanism of BQLM formula on anti-CHB and liver-protection,we find some information from "Bushen recipe" and "Lingmao formula"."Bushen recipe"is composed of Bajitian(Morinda Officinalis),Dihuang (Rehmannia Glutinosa),Huangqi(Atractylodis Macrocephalae),Baizhu (Asarum Sagittarioides),Yinyanghuo (Epimedium Koreanum),Cuqipi(Citri reticulatae),Nvzhenzi (Ligustri Lucidui);Jigucao(Abrus Cantoniensis);and Fuling (Poria Cocos)."Bushen recipe"can decline the expressions of immune effect molecules (granzyme B,TNF-α and IFN-γ) of peripheral T cells in CHB patients with liver-kidneyYindeficiency.9The recipe plus ETV promotes the reduction of HBsAg level and the clearance of HBeAg,and regulates B-cell differentiation,and increases B cell activation factor(BAFF)levels in CHB patients.40Nieet al41found that "Bushen recipe" could improve the proportion of CD3,CD4,CD4/CD8 T lymphocytes,and reduce the percentage of CD8 cells and increase the proportion of Th1/Th2.The recipe also reduced the proportion of Th17,increased Treg ratio,and improved the Treg/Th17 ratio,decreased the expression of IL-17 and IL-4,and increased IL-6 and IFN-α in CHB patients.The ConA-induced hepatic injury mice were treated with "Bushen recipe",and the levels of IL-6,IL-1,TNF-α and TNF-β in peripheral blood were decreased,and the expression levels of TBK1,IRF3,TLR9,TLR4,TLR3,NF-kB,and MyD88 were also decreased.42These results suggest that "Bushen Recipe"can inhibit the inflammatory mediators through TLRs-mediated inflammatory signaling pathway and immunoregulation to exhibit its anti-liver injury effects."Lingmao formula" is composed of Huangqi (Astragalus Membranaceus),Maozhuacao(Ranunculi Ternati),Huhuanglian (Picrorrhiza Scrophulariflora),Yinyanghuo(Epimedium Koreanum),Cuqingpi(Citri Reticulatae) and Nvzhenzi (Ligustri Lucidui).The prepared drug serum of rats treated "Lingmao formula" was incubated with HepG2.2.15 cells,which could inhibit starvation-induced autophagy.43 HepG2.2.15 were intervened by the aqueous-extract of "Lingmao formula"at concentration of 500 and 1000 mg/L,and Lingmao Formula could promote the degradation of pgRNA,sRNA and cccDNA by inhibiting La protein expression in HepG2.2.15 and induced the inhibition of HBV replication.44

In our present studies,BQLM formula significantly repaired the damage of liver tissue caused by ConA,reduced the infiltration of inflammatory cells,and inhibited the secretion of inflammatory cytokines IFN-γ,TNF-α and IL-6.BQLM formula also improved the activities of ALT and AST (functional markers of liver)of liver and serum in ConA-induced hepatic-injury mice.We found that ConA also induced some oxidative damages in liver of mice,but BQLM formula could recover SOD and CAT activities of liver to normal levels.Further,BQLM formula obviously promoted the proliferation of human LO2 hepatocytes and inhibited hepatocytes death induced by ConA in vitro.It also inhibited the proliferation and the secretion of HBsAg and HBeAg of HepG2.2.15 cells.

In conclusion,these results of the present study revealed that BQLM formula treatment showed significantly hepato-protective and anti-HBV effectsin vivoandin vitro.The underlying mechanism might be by inhibiting inflammatory and oxidative damages of liver,improved hepatocytes proliferation,and reduced the secretion of HBsAg and HBeAg.These researches suggested that BQLM formula was a potential natural medicine to protect the liver from the viral and autoimmune hepatitis in human.