Protective Effect Of The Dendrobium Candidum From Yunnan On The Liver Fibrosis In CCl₄-induced Rats

Objective:To observe the effects of different dosage forms of dendrobium candidum from Yunnan（DCY）on the hepatic histopathology,liver function and the blood biochemical indexes of liver fibrosis of the rats induced by CCl₄,and the effect of candidum polysaccharide（DCP）on the proliferation and apoptosis of the activated hepatic stellate cell line LX2（HSC-LX2）in vitro culture,for studying the anti-hepatic fibrosis mechanism of dendrobium candidum.Methods:（1）Experiment 1:the 86 SD rats were selected and randomly divided into the normal group（n=15）and the model group（n=71）.The model rats were injected subcutaneously with CCl₄to induce the liver fibrosis three times weekly,while the normal rats were treated with the equivalent normal saline,and all ones were weighed.After 5 weeks of continuous injection,the five normal and the five model rats were randomly killed,their livers were removed and sliced for the pathological detection under microscope.And then the eight normal rats were randomly selected as the negative control group（NG）,and the other 64 model rats were equally divided into eight groups:the low concentration group（DDGl,0.15g/m L）,the medium concentration group（DDGm,0.45g/m L）,and the high concentration group（DDGh,0.75g/m L）of the DCY powder,the low concentration group（FDGl,0.15g/m L）,the medium concentration group（FDGm,0.45g/m L）,and the high concentration group（FDGh,0.75g/m L）of the DCY fresh-squeezed juice,the colchicine group（CG,0.002%）and the model group（MG）.Each rat of the drug groups was administrated 3ml by gastric lavage once a day,the rats of MG and NG were treated with the equivalent normal saline synchronously.After 4 weeks,the rats were decapitated after weighing their body masses,and then under the sterile condition we cut open their abdominal cavities immediately for collecting the blood from the abdominal artery and removing their livers after observing the color and texture.Then the livers were weighed to calculate the liver index（LI）;their tissue slices were used to observe the histopathology changes under light microscope by the hematoxylin-eosin（HE）and Masson stainings;the expression of transforming growth factor-β1（TGF-β1）was tested by the immunohistochemistry in liver tissues;the expressions of the liver function indexes ALP,AST and GPT in serum were detected by the automatic biochemistry instrument,and the expressions of the liver fibrosis indexes HA,LN and PCIII in serum by the enzyme-linked immunosorbent assay（ELISA）.（2）Experiment 2:HSC-LX2 cells in logarithmic phase were cultured in vitro and implanted into the 6-well plates with a cell density of 5×10⁴/well.The cells were divided into negative control group and DCP group for cell wound scratch assay.In DCP group,the polysaccharides from Dendrobium candidum（400ug/ml）was joined into the culture plates for detecting the cell mobility of HSC-LX2 at 2 h,6 h,12 h,24 h and 48 h.In the meantime,HSC-LX2cells were implanted into 6-well plates with a cell density of 1×10³/well,all holes were divided into the 6 groups（n=3）:negative control group（NG）,25ug/m L DCP group（DCPG-25）,50ug/m L DCP group（DCPG-50）,100ug/m L DCP group（DCPG-100）,200ug/m L DCP group（DCPG-200）,400ug/m L DCP group（DCPG-400）.After the DCP treatment for 24 hours,the inhibition ratio of DCP on HSC-LX2 was calculated with a colorimetric MTT assay.Using the same methods as above to culture and implant HSC-LX2 cells into the 6-well plates with a cell density of 5×10⁴/well,all holes were divided into the 4 groups（n=3）:negative control group（NG）,100ug/m L DCP group（DCPG-100）,200ug/m L DCP group（DCPG-200）,400ug/m L DCP group（DCPG-400）.After cell culture for 12 hours,the different concentrations of DCP were added into the holes of the corresponding groups with 1.5 ml each hole.After 24 hours,the cell morphology was observed under the inverted microscope and the cell apoptosis rate was detected by the flow cytometry.Results:（1）Experiment 1:（1）The general status of rats and the liver appearance:During the model preparation,the MG rats were in poor general condition and showed a slow increase in body weight.After the intervention of DCY with two dosage forms,the general conditions of rats in DDG,FDG and CG were improved.The liver of NG rats was mostly divided into 6 lobes with a smooth brown-red surface and soft texture,but the liver of MG rats was dark red with the diffuse small nodules on their surface and a tough texture,and the liver of rats in DDG,FDG and CG was tougher and grainy,but it was significantly improved compared with MG.（2）The change of LI:Compared with NG,the LI of MG rats was significantly increased（P<0.01）,but LIs of CG,DDG and FDG were higher than that of NG,which was no significant difference（P>0.05）.Compared with MG,the LIs of DDGl,DDGm,DDGh,FDGl,FDGm,FDGh and CG were significantly decreased（P<0.05 or P<0.01）.Compared with CG,the LIs of DDGl,DDGm,DDGh,FDGl,FDGm and FDGh were higher,and the difference was not statistically significant（P>0.05）.Compared with DDGh,the LI of DDGl did not decrease significantly without statistical significance（P<0.05）,but compared with FDGh,the LI difference of FDGl and FDGm has no statistical significance（P>0.05）.（3）Liver histopathological changes:HE staining showed that in NG rats the morphology of liver cells was normal with the media round nuclei,and the radial hepatocyte cords were arranged tightly,but in MG rats,the liver cells were arranged disorderly with the fat vacuoles widely formed in the cells,a large number of pseudolobuli were formed,the infiltration of inflammatory cells was present in the enlarged portal area.However,the liver injury of CG,DDG and FDG rats was significantly improved.Masson staining showed that in NG rats the morphology of liver cells was normal,the hepatocyte cords were regular without the obvious fat vacuoles,and there was no obvious collagen fiber deposition in the portal area.However,The arrangement of hepatocyte cords in MG rats was disordered,a large number of the pseudolobular formation in liver tissue and the collagen fibers deposited in the portal area were seen.The liver injury of CG,DDG and FDG rats was significantly relieved,the formation of pseudolobules was reduced,and the fibrosis degree of portal area was decreased.With higher doses of each form of DCY,the pathological changes of rat liver tissue improved more obviously,which may be dose-dependent.（4）Immunohistochemical staining:Compared with NG,the expression of TGF-β1 in the liver tissue of MG,DDG1 and FDG1 rats was significantly increased（P<0.01）.Compared with MG,the expression of TGF-β1 in each group of DDGm,DDGh,FDGm and FDGh was significantly decreased（P<0.01）,but compared with CG,the expression of TGF-β1 in DDGl,FDGl,DDGm and FDGm was significantly different（P<0.01）.Comparing DDGl,DDGm with DDGh,and comparing FDGl,FDGm with FDGh,their differences in the expression of TGF-β1 was statistically significant（P<0.01）.The results showed that DCY can decrease the expression level of TGF-β1 in rat liver tissue,and it may be dose-dependent.（5）The expressions of ALP,AST and GPT in serum:Compared with NG,the expressions of ALP,AST and GPT in MG rats was significantly increased（P<0.01）,and the expressions of the three in DDGl,DDGm,DDGh,FDGl,FDGm and FDGh rats were all decreased compared with MG（P<0.01）,and then Compared with CG,the expression differences of them in DDGl,DDGm,DDGh,FDGl,FDGm and FDGh rats have no statistical significance（P>0.05）.Comparing DDGl,DDGm with DDGh,and comparing FDGl,FDGm with FDGh,there were no significant differences in the expression of ALP,AST and GPT（P>0.05）.（6）The serum biochemical indexes of liver fibrosis:Compared with NG,the expression of HA,LN,and PC-III in serum of MG rats was significantly increased（P<0.01）.Compared with MG,the expressions of HA,LN,and PC-III in rat serum of DDGl,DDGm,DDGh,FDGl,FDGm,and FDGh all decreased（P<0.05）.Compared with CG,The expressions of the three in serum of DDGm,DDGh,FDGm and FDGh rats were not statistically different（P>0.05）,while in DDGl and FDGl rats they were significant differences（P<0.01 and P<0.05）.However,it is worth noting that the comparison may be dose-dependent in different dose groups of DCY interference groups,that is,there are significant differences only in the expression of LN when comparing DDGl with DDGh and comparing FDGl with FDGh（P<0.01）.（2）Experiment 2:（1）General situation:After the passage of HSC-LX2 adheres to the wall,the light microscope showed that the cells grow vigorously,the cytoplasm was transparent,the cells spread in a star shape,the nucleoli were clear,and the cell fragments were few.（2）Scratch test:The cell migration rates of the treatment groups was significantly decreased,which indicated that DCP can play an effect of anti-liver fibrosis by inhibiting the migration of HSC-LX2.（3）MTT assay:With the increase of DCP concentration,the inhibition rates of it on the proliferation of HSC-LX2 gradually increased,and the difference was statistically significant（P<0.01）when comparing each concentration group with NG.When comparing between any two ones in DCPG-25,DCPG-50,DCPG-100,DCPG-200 and DCPG-400 groups,the inhibition rates of DCP on the proliferation of HSC-LX2 was significantly lower,and the difference was statistically significant（P<0.05）.The results showed that DCP could inhibit the proliferation of HSC-LX2 in a dose-dependent manner.（4）Flow cytometry:As the DCP concentration increased,the growth density of HSC-LX2 gradually decreased,and the cell apoptosis rate gradually increased,which was positively correlated with DCP concentration.the difference was statistically significant（P<0.01）when comparing each concentration group with NG.Compared with DCPG-400,the apoptosis rates of DCPG-100 and DCPG-200 was lower with significant statistical difference（P<0.01）.The results showed that DCP could significantly promote the apoptosis of HSC-LX2 in a dose-dependent manner.Conclusion:Both formulations of DCY could improve the general state,liver function of the rats with liver fibrosis,the insum biochemical indexes of liver fibrosis and the liver pathological change,and decrease the expression of TGF-β1 in liver tissue;DCP could inhibit the proliferation of the activated HSC-LX2 and promote its apoptosis.As the concentration of DCP increase,most of the detection indicators in vivo and in vitro are significantly improved.Therefore,Dendrobium candidum has the effect of anti-liver fibrosis in a potential dose dependent manner.