Study On The Protective Effect And Mechanism Of Rhoifolin On Alcoholic Liver Injury

Objective:In order to provide a certain experimental basis for the clinical application of the authentic medicinal material Citrus grandis ’Tomentosa’ and further promote the comprehensive development and utilization of the medicinal resources of it,the active component rhoifolin from Citrus grandis ’Tomentosa’ was extracted and purificated,and the protective effect and mechanism of it on alcoholic liver injury by animal and cell experiments in vivo and in vitro were analyzed.Method:1 Extraction,isolation,purification and identification of rhoifolinRhoifolin was extracted and purified by percolation and recrystallization,and its purity was determined by thin layer chromatography(TLC),nuclear magnetic resonance(NMR)and high performance liquid chromatography(HPLC).2 Protective effect of rhoifolin on mice with chronic alcoholic liver injuryA total of 70 balb/c mice were randomly divided into blank group,model group,total flavonoids group,high dose of rhoifolin group,medium dose of rhoifolin group,low dose of rhoifolin group and biphenyldicarboxylate group.The experiment was conducted for 7 weeks,in which the blank group was fed with normal saline at 10 mL·kg-1,and the mouse models of alcoholic liver injury in the remaining groups were replicated by gradient ethanol gavage(53 degrees liquor was used for 7 weeks.The liquor dose is 6mL·kg-1 for the first week,and then the liquor dosage increases by 1 mL every week until it increases to 10 mL·kg-1).Simultaneous administration during model building,in which the blank group was given normal saline at 10 mL·kg-1,the model group was given orally 0.2%carboxymethyl-cellulose sodium at 10 mL·kg-1,the biphenyldicarboxylate group was given 150 mg·kg-1 bifendate drop solution,the total flavonoids group dose was 100 mg·kg-1,and the high,medium and low doses of rhoifolin were 40,20 and 10 mg·kg-1,respectively.At the last day of modeling,all mice fasted for 12h and weighed.After blood was taken from the eyeball,the serum was centrifuged at 4℃,3500 r·min-1 for 10min,and the serum levels of ALT and AST and the expressions of inflammatory factors(TNF-α,IL-6,IL-8)in the serum were detected.The liver was rapidly dissected,washed with cold saline and weighed to calculate the liver index.Liver was saved at-20℃ and used for histological analysis and detection of the expression levels of TG,MDA,SOD and GSH.3 Study on the protective effect and mechanism of rhoifolin on ethanol-induced LO2 cells3.1 MTT method was used to screen the concentration of rhoifolinThe effect of rhoifolin on the growth of LO2 cells was determined by MTT.The experiment was divided into blank group and different dose groups of rhoifolin(the doses were 6.25,12.5,25,50,100,200,400 and 500 μmol·L-1).3.2 The concentration of ethanol was screened by MTT methodThe effect of ethanol on the growth of LO2 cells was measured by the MTT.The experiment was divided into blank group and the concentration of ethanol was respectively set as 50,100,200,300,400,500 and 600 mmol·L-1.3.3 Effects of rhoifolin on the activity of alcohol-induced LO2 cellsThe experiment was divided into the blank group,the alcohol model group,the high-dose group(100 μmol·L-1),the medium-dose group(50 μmol·L-1),and the low-dose group(25μmol·L-1).Except for the blank group,all the other groups were stimulated with 200 mmol·L-1 alcohol for 24 hours,and the rhoifolin was added for 24 hours.The effect of rhoifolin on alcohol-induced LO2 cell growth was determined by MTT method,while the effect of rhoifolin on alcohol-induced LO2 cell apoptosis was detected by flow cytometer.3.4 Protective effect of rhoifolin on ethanol-induced LO2 cells and its mechanismLO2 cells with excellent logarithmic growth stage were selected and counted with the Count Star cell counter.The cell concentration was 1×105 cell/mL.For each well,2mL cell suspension was added to the 6-well plate and incubated for 24 h at 37℃ in a 5%CO2 incubator.The supernatant was then added to each well with different concentrations of rhoifolin(25,50,100μmol·L-1)and ethanol(final concentration was 200 mmol·L-1)and incubated at 37℃ in 5%CO2 incubator for 24 hours.Further,the cell supernatant was used to detect the expression of ALT,AST and LDH in LO2 cells.In addition,ELISA was used to determine the content of inflammatory factors TNF-α,IL-6,IL-8.GAPDH was used as the internal reference gene,and real-time PCR was used to detect expression of apoptotic factors(TNF-α,IL-6,IL-10,Bax,Bcl-2,Bax,Caspase-3,CYP2E1).Results:1 Extraction,separation,purification and identification of rhoifolin.Rhoifolin was identified by TLC and the results showed that,compared with the standard of rhoifolin,it had the same flaxen spots and specific shift value(0.385).The structure was identified by NMR technique,which was consistent with the structure of the standard.Through comprehensive analysis,the samples were identified as rhoifolin.The results of high performance liquid chromatography(HPLC)showed that the samples showed a single main peak,and the purity of rhoifolin was calculated to be over 98%by area normalization method.Meet the requirements of laboratory animal and cell research.2 Protective effect of rhoifolin on chronic alcoholic liver injury mice.Liver index results showed that the liver coefficient of the blank group was 4.03±0.24%,and the model was 4.82±0.34%,and there was a significant difference between the two(P<0.05).Compared with the model group,the liver index of mice in the high-dose rhoifolin group,the total flavonoid group and the positive drug group was significantly reduced,with a significant difference(P<0.05).Pathological examination results showed that:in the model group,the liver cells around the central vein and interlobular meridians were disordered,the hepatocytes were disordered,the boundaries were unclear,the liver sinus became narrower,the liver cells were cytosolic,and they showed watery degeneration and inflammatory cells in some cases,spotty and focal necrosis can be seen;after treatment with the total flavonoids and rhoifolin with different concentrations,hepatocytes tended to be normal,the infiltration phenomenon weakened,and local necrosis was reduced.The measurement results of ALT and AST in serum showed that the content of ALT and AST in the blank group were 47.92±4.35,56.02±3.66,and the ALT and AST in the model group were 70.60±5.56,90.96±12.90,there is a significant difference between the two(P<0.05).Compared with the model group,ALT and AST in the low-dose group of rhoifolin,middle-dose group of rhoifolin,high-dose group of rhoifolin,the total flavonoid group,and the positive drug group were significantly decreased(P<0.05).The results of TG and MDA in mouse liver showed that the blank group was 0.12±0.01 mmol/gprot,0.21±0.02 mmol/gprot,and the model group was 0.16±0.02 mmol/gprot,0.26±0.03 mmol/gprot,respectively.There was a significant difference between the two(P<0.05).Compared with the model group,TG and MDA decreased significantly in the low-dose group,middle-dose group,the high-dose rhoifolin group,the total flavonoid group and the bifendate group(P<0.05).The results of GSH and SOD in mouse liver showed that the blank group was 11.48±1.73 mmol/gprot,35.40±2.13 U/gprot,the model group was 5.64±1.23 mmol/gprot,19.05±1.48 U/gprot,and there was a significant difference between the two(P<0.05).The ratios of GSH and SOD in the liver of mice in the low-dose group,the middle-dose group,the high-dose group of rhoifolin,the total flavonoids group,and the bifendate group were significantly lower than those in the model group(P<0.05).3 Study on the protective effect and mechanism of rhoifolin on ethanol-induced LO2 cells.3.1 MTT method was used to screen the concentration of rhoifolin.The MTT results showed that after 24 hours of intervention of LO2 cells by rhoifolin,the cell activity was 100.73±2.64%,98.99±3.27%,98.66±3.63%,98.15±4.38%,97.25±3.68%,93.9112.98%when the concentration of rhoifolin was 12.5,25,50,100,200,300μmol·L-1.Compared with the blank group,there was no significant difference in the activity of LO2 cells(P>0.05).When the concentration of rhoifolin was 400 and 500 μmol·L-1,the cell activities were 80.54±3.93%and 68.59±6.01%,respectively,which were significantly reduced,with significant differences(P<0.05).3.2 The concentration of ethanol was screened by MTT methodMTT results showed that after stimulation with ethanol of different concentrations for 24 hours,the inhibition rates of cells at ethanol concentrations of 100,200,300,400,500,and 600 mmol·L-1 were 23.58±4.35%,36.08±3.39%44.83±6.76%93.21±2.26%95.43±0.79%,96.16±0.74%.Compared with the blank group,it was significantly inhibited and had a significant difference(P<0.05).According to the analysis of the results,when the ethanol concentration was 200 mmol·L-1,the cell suppression rate was 36.08%,so the ethanol concentration of 200 mmol·L-1 was the appropriate modeling concentration.3.3 Effects of rhoifolin on the activity of ethanol-induced LO2 cells.MTT results showed that compared with the 100%survival rate of the blank control group,the model group was 64.28±9.12%,with a significant difference(P<0.05).The cell survival rate after treatment with 25 μmol·L-1 of rhoifolin was 71.69±2.86%,which was increased compared with the model group,but there was no significant difference(P>0.05).The cell survival rate was 86.11±4.83%and 82.91±5.53%when the rhoifolin was 50 μmol·L-1 and 100 μmol·L-1 and compared with the model group,it was significantly increased with significant difference(P<0.05).3.4 Protective effect of rhoifolin on ethanol-induced LO2 cells and its mechanism.After the LO2 cells were treated with rhoifolin and ethanol for 24 hours,LDH,AST and ALT were measured.The LDH,AST,and ALT of the blank group were 2034.91±89.50 U/gprot,11.83,10.87,the model group was 3294.48±117.55 U/gprot,22.00,42.08,the model group was higher than the blank group,with a significant difference(P<0.05).LDH,AST,ALT in the 25 μmol·L-1 group of rhoifolin decreased compared with the model group,but ALT and AST were significantly different(P<0.05),and LDH was not significantly different(P>0.05).LDH,AST,ALT in the 50μmol·L-1 and 100 μmol·L-1 groups of rhoifolin were all reduced compared with the model group,with significant differences(P<0.05).RT-PCR results showed that the expression levels of TNF-α,IL-6,IL-10,Bax,Caspase-3,and CYP2E1 mRNA in the model group were 2.20±0.10,2.53±0.28,1.70±0.12,2.56±0.26,2.10±0.14,2.82±0.28,significantly higher than the blank group(P<0.05).The mRNA expression of Bcl-2 was 0.696±0.055,which was significantly lower than that of the blank group(P<0.05).The expression levels of TNF-α,IL-6,IL-10,and Bax mRNA in the 25μmol·L-1 group of rhoifolin were lower than the model group,but there was no significant difference(P>0.05).The expression level of Bcl-2 mRNA was increased compared with the model group,but there was no significant difference(P>0.05).Caspase-3 and CYP2E1 mRNA were significantly increased compared with the model group,with a significant difference(P<0.05).The mRNA expression levels of TNF-α,IL-6,Bax,Caspase-3,and CYP2E1 in the 50μmol·L-1 group of rhoifolin were significantly lower than those in the model group(P<0.05).The expression level of Bcl-2 mRNA was significantly higher than that of the model group,with a significant difference(P<0.05).The mRNA expression level of IL-10 was lower than that of the model group,but there was no significant difference(P>0.05).The expression levels of TNF-α,IL-6,IL-10,Bax,Caspase-3,and CYP2E1 mRNA in the 100 μmol·L-1 group of rhoifolin were significantly higher than those in the model group(P<0.05).The expression level of Bcl-2 mRNA was significantly higher than that of the model group(P<0.05).Conclusion:1.The rhoifolin prepared by percolation combined with recrystallization is stable and reliable,which could be used as a qualitative and quantitative control and meet the requirements of experimental animals and cells.2.Animal experiments show that the high-dose,middle-dose and the low-dose of rhoifolin,and the total flavonoid had a certain protective effect on mice with alcoholic liver injury.They could alleviate the pathology in the model mice,reduced the liver index and improved the expression levels of ALT,AST,TG,MDA,SOD,and GSH in mice.3.In the cell experiment,the effect of rhoifolin on blank LO2 cells was small;as the ethanol concentration increased,the growth inhibition of LO2 cells increased.Rhoifolin had a value-added effect on ethanol-induced LO2 cells.Flow cytometry showed that rhoifolin inhibited ethanol-induced apoptosis of LO2 cells.RT-PCR showed that rhoifolin could inhibit the expression of apoptosis factors Bax and Caspase-3 mRNA,and promote the expression of Bcl-2 mRNA.Rhoifolin could reduce the LDH,AST and ALT expression levels of LO2 cells induced by ethanol and restored ethanol-induced CYP2E1 mRNA expression in LO2 cells.Rhoifolin could inhibit the expression of TNF-α,IL-6,IL-10 mRNA of inflammatory factors produced by ethanol-induced LO2 cells.This study proved that rhoifolin could play a protective role against alcoholic liver injury from different levels of animals,tissues,cells,molecules and so on.