Substituting Sheep Placenta For Hominis Placenta In Fufang Biejia Ruangan Tables:Anti-fibrosis Effects And Mechanisms

Objective:Hominis placenta?HP?,an important component of Fufang Biejia Ruangan Tables?FBRT?,is in shortage and is restrained in production.As a result,production of FBRT is limited.To resolve this issue,this study is designed to investigate the pharmacodynamics effects of FBRT where HP was substituted by Sheep placenta?SP?,and to compare SP with HP in their immune regulation function.This study also provides an insight into the research on other traditional Chinese herbal medicines that are in shortage or endangered state.Methods:This study investigates the pharmacodynamics effects of FBRT where HP was removed or substituted by SP in perspective of cytology and whole-animal level,and compares SP with HP in their immunology efficacy.The full text is divided into five parts:1.Preliminary cytological study on FBRT where HP was removed or substituted by SPMale SD rats were taken to prepare the drug-containing serum of the corresponding group.The prepared drug-containing serum samples were added to the cultured hepatic stellate cells?HSC-T6?system.After 24 hours,the inhibition rate of each group was detected by MTT assay,and the cell apoptosis was detected by flow cytometry.2.Effects of HP removed or substituted by SP in FBRT on the acute liver injury in miceKM mice were randomly divided into the following nine groups:?I?the normal control group,?II?the model control group,?III-VI?the dose groups where the HP of FBRT?0.8436,0.8745,0.9510,and 1.1193g/kg?were substituted by SP by 0.5:1,1:1,2:1,and 4:1,respectively,?VII?the dose group where the HP was removed from FBRT?0.7662g/kg?,?VIII?the original FBRT?0.8590g/kg?dose group,and?VIIII?the bifendate-treated group?0.2g/kg?.The normal and model control groups were given equal capacity of 0.5%CMC-Na solution by gavage,and the other groups given corresponding dose of the test solution once a day for 14 days.After 2 hours of administration,the mice in the normal control group were given soybean oil solution by intraperitoneal injection,whereas the other groups were injected intraperitoneally with 0.1%CCl₄ soybean oil solution to induce acute liver injury.At the end of the modeling,the changes of serum biochemical,lipid peroxidation,and liver histopathology were measured in each group.3.Effects of HP removed or substituted by SP in FBRT on CCl₄-induced hepatic fibrosis in ratsSD rats were randomly divided into control group and experimental group,the SD rats in the control group were given soybean oil solution by subcutaneous injection,whereas the SD rats in the experimental group was subcutaneously injected with 40%CCl₄ soybean oil solution to construct a liver fibrosis model in rats.The subcutaneous injections were conducted by twice a week and lasted for 6 weeks.After the completion of the modeling,except for?I?the control group,the survived experimental rats were regrouped and randomly divided into the following nine groups:?II?the model control group,?III-VI?the dose groups where the HP of FBRT?0.5840,0.6054,0.6584,and 0.7749g/kg?were substituted by SP by 0.5:1,1:1,2:1,and 4:1,respectively,?VII?the dose group where the HP was removed from FBRT?0.5305g/kg?,?VIII?the original FBRT?0.5947g/kg?dose group,and?VIIII?the colchicine-treated group?0.1mg/kg?.At the 7th week,the normal and model control group were given 0.5%CMC-Na solution by gavage,whereas the other groups were given corresponding dose of the experimental solution until the end of the 12th week.At the end of the administration,the rat liver and spleen index,serum biochemistry,hydroxyproline?Hyp?,lipid peroxidation,and four indexes of liver fibrosis?HA,LN,PC-III,and C-IV?were measured;the changes of HE staining and Masson's staining in right hepatic tissue of rats were observed,and the expression of?-smooth muscle actin??-SMA?in hepatic tissue was observed by immunohistochemistry.The remaining liver tissue was used for the subsequent study of the mechanism of action.4.Study on the mechanism of FBRT where HP was substituted by SP on hepatic fibrosis in ratsBased on the above results,the changes of inflammatory mediators?IL-6,IL-1?and TNF-??and hepatic fibrogenic factors?CTGF and PDGF-??in hepatic tissue of liver fibrosis rats were detected by Elisa method.RT-PCR method was used to detect gene expression changes of type I collagen?Col-I?,type III collagen?Col-III?,?-smooth muscle actin??-SMA?,transforming growth factor-??TGF-??and liver-fibrosis-related matrix metalloproteinase family related factors.Western-blot quantitative method was used to detect liver fibrosis-related Smad2/3,p-Smad2/3,Akt and p-Akt expression.The above were designed to explore the possible mechanism of FBRT where HP was substituted by SP on hepatic fibrosis.5.Evaluation of immunoregulatory consistency between SP and HP?KM mice were randomly divided into the following eight groups:?I?the normal control group,?II?the model control group,?III-V?the?1.46,0.73 and0.37g/kg?three SP dose groups and?VI-VIII?the?1.46,0.73 and 0.37g/kg?three HP dose groups.The normal control group was given equal capacity of 0.5%CMC-Na solution vy gavage,and the other groups gavage corresponding dose of the test solution twice a day for 7 days.At the end of administration,except for the normal control group,the remaining groups were given Indian ink by tail vein injection.The blood from the fundus venous plexus of the mouse was taken at 1 and 10 min after injection,and the optical density of the mouse blood was measured by a spectrophotometer.The changes of clearance index and phagocytic index of mice in each group were analyzed,and the effects of test drugs on the removal of carbon particles in mice were observed.?Under the same animal grouping and administration conditions described above,a mouse model of delayed-type hypersensitivity?DTH?induced by 2,4-dinitrofluorobenzene?DNFB?was used to observe the change of ear swelling and swelling rate for mice in each group.Based on the above test results,the feasibility of mutual replacement between SP and HP was preliminarily evaluated from the perspective of immunology.Results:1.Preliminary cytological study on FBRT where HP was removed or substituted by SPThe serum pharmacological test results show that the inhibitory rate of rat serum containing drugs on HSC-T6 cells gradually is increased in a concentration-dependent manner,and the inhibition rate of the cells is high under the condition of 10%drug-containing serum.Compared with original FBRT,the cell inhibition rate and apoptotic rate are significantly reduced in FBRT where HP was removed,whereas the cell inhibitory rate and apoptotic rate are significantly increased in FBRT where HP was substituted by SP?SP:HP=0.5:1 and 1:1?.2.Effects of HP removed or substituted by SP in FBRT on the acute liver injury in miceCompared with original FBRT,removing HP in FBRT decreases the liver protection,transaminase,anti-lipid peroxidation and liver-pathological improvement,but there is no significant difference?P>0.05?.Compared with the model,substituting SP for HP in FBRT decreases the liver index and the serum levels of AST,ALT,T-Bil,ALP,and MDA,but increases ALB,TP,SOD and GSH-Px contents.In addition,hepatocyte edema,ballooning,and inflammatory cell infiltration are all reduced in mice?P<0.01 or 0.05?.The results show a significant liver protection.Compared with original FBRT,substituting SP for HP?SP:HP=0.5:1 and 1:1?in FBRT shows an increasing trend in liver protection?P>0.05?.3.Effects of HP removed or substituted by SP in FBRT on CCl₄-induced hepatic fibrosis in ratsCompared with the model,removing HP in FBRT decreases the spleen index and serum levels of AST,ALT,T-Bil,ALP,MDA,LN,and C-IV,but increases ALB and SOD levels.In addition,liver fibrosis area and?-SMA immunohistochemistry positive expression area are decreased?P<0.01 or 0.05?,showing a certain anti-fibrotic liver fibrosis activity.Substituting SP for HP?SP:HP=0.5:1?in FBRT reduces the following indexes,including the liver index and spleen index,serum levels of AST,ALT,T-Bil,GLO,ALP,Hyp and MDA,four indexes of liver fibrosis?HA,LN,PC-III,and C-IV?,ALB,TP and SOD,hepatocyte inflammatory cell infiltration,hepatic fibrosis area,and?-SMA immunohistochemistry positive expression area?P<0.01 or 0.05?.However,it increases GSH levels and significantly improves the pathological lesions of liver tissue,showing a significant anti-fibrotic effect.Compared with original FBRT,removing HP in FBRT has a weaker anti-fibrotic effect in rats,whereas substituting SP for HP?SP:HP=0.5:1?in FBRT has a certain degree of anti-fibrosis activity.4.Study on the mechanism of FBRT where HP was substituted by SP on hepatic fibrosis in ratsCompared with the model,substituting SP for HP?SP:HP=0.5:1?in FBRT decreases the levels of IL-6,IL-1?,TNF-?,CTGF and PDGF-?in the liver tissue,and decreases the mRNA expression of Col-I,Col-III,?-SMA,TGF-?,MMP-2 and TIMP-1.However,the expression of MMP-1 mRNA is increased and the relative expressions of p-Smad2/3 and p-Akt are down-regulated in the liver?P<0.01 or 0.05?.Compared with the original FBRT,substituting SP for HP?SP:HP=0.5:1?in FBRT significantly reduces the relative expression of Smad2/3,p-Smad2/3,Akt and p-Akt in liver tissue?P<0.01?.It shows that the mechanism of FBRT where HP was substituted by SP?SP:HP=0.5:1?anti-hepatic fibrosis is still through inhibiting the expression of inflammation-related and pro-fibrogenic factors,reducing collagen content,regulating the balance of MMPs and TIMPs in liver and liver tissue,inhibiting the activation and proliferation of HSCs,and inhibiting the expression of TGF-?/Smad2/3 and Akt signaling pathway.They eventually reverses the course of hepatic fibrosis.5.Evaluation of immunoregulatory consistency between SP and HPCompared with the model control group,the clearance index and phagocytic index of mononuclear macrophages in mice of the 1.46 g/kg and 0.37g/kg SP dose groups are significantly increased,but the ear swelling and ear swelling rate of mice are significantly decreased?P<0.01 or 0.05?.The 0.37 g/kg HP dose group shows a significant decrease in ear swelling and ear swelling?P<0.01 or 0.05?.The results show that SP may have the function of enhancing non-specific immune function and inhibit delayed-type hypersensitivity,and its efficacy is slightly stronger than that of HP.Conclusions:Under the conditions of this experiment,in aspects of the inhibition proliferation of HSC-T6 cells,anti-mouse acute liver injury and anti-hepatic fibrosis,the pharmacological efficacy of FBRT after removing HP was significantly weaker,while FBRT where HP was substituted by SP is better than the original prescription.Among them,substituting SP for HP?SP:HP=0.5:1?in FBRT is the best alternative.Therefore,SP can be considered as substitute medicine for HP in FBRT prescription.SP has certain substitutability to the HP in terms of immune regulation,and it also lays the foundation for the further development of the SP,which is HP's new Chinese herbal medicine substitutes.