Experimental Study Of The Proliferation Of HSC Actived By Leptin And Its JAK₂-STAT₃ Signal Transduction Pathway Treated By Baoganning.

ObjectiveTo investigate the effect of the compound prescription named Baoganningmaded of chinese traditional medicine on proliferation of hepatic stellate cells (HSCs)actived by leptin and its JAK-STAT signal transduction pathway about signalfactor:OB-Rb,JAK₂,STAT₃ and their phosphorylations,we try to clarify themechanisms that Baoganning resist haptic fibrosis.Methods1.Preparation for drug serum: 20 Wistars mouses were at random divided intofour group(six per group) that were fed with regular dosage baoganning (wascomposed of Milkvetch Root, Peach Seed, Baical Skullcap Root, WhitebackleafMallotus Root, Turtle Shell, Dan-Shen Root, RadixBupleuri, White Peony Root,Immature Bitter Orange,1.77g/ml),double dosage baoganning(3.54g/ml),Colchicine(0.05g/ml) and saline for seven days respectively. They were normalmouse serum,serum of baoganning regular dosage, serum of baoganning doubledosage and serum of Colchicine. 2. Cell culture: HSC-LX2 was cultured at 5%CO₂,37℃circumstance withRPMI-1640 which included 10% fetal bovine serum(FBS). The cells are continuouslypassaged every two days. Different groups were treated with drug serum and regularmouse serum.3.The dosage of leptin secreted by HSC-LX2 at 6,12,18,24h by enzyme-linkedimmunosorbent assay(ELISA).4.0,10,50,100,200 or 400ug/l leptin improves the proliferation of HSC-LX2at 6,12,18,24h,and the effect of Baoganning drug-serum on HSC-LX2 growthpromoted by 100ng/ml leptin was detected by the methylthiazolyl tetrazolium(MTT)assay.5.The expression of OB-Rb,JAK₂,STAT₃ and phosphor-JAK₂, phosphor-STAT₃at different time was detected by WB.Results1.Concentration of leptin secreated by HSC-LX2 at different time with theONE-way:F=271.363,P=0.000,LSD was applied between every two groups. Therewere distinguished difference among 12,24,36,48h concentrate of leptin HSCsecrected(P=0.000).With the increasing time,the dosage of leptin secreted byHSC-LX2 decreased, significant difference was observed between 6,12,18,24h(P＜0.01).2.Results of the proliferation of HSC-LX2 actived by different concentrationalleptin at different time by MTT with repeated measuresshow:F=193917.2,P=0.000,At the same time,Compared with control regular group,leptin 10ng/ml groups were different(P=0.031),there were distinguished differencesamong other groups (P=0.000);There were no differences between leptin 10ng/ml andleptin 50ng/ml groups(P=0.300),and between leptin 200ng/ml and leptin400ng/ml(P=0.391);Compared with leptin 100ng/ml group, leptin 10ng/ml groupshad distinguished differences(P=0.001),leptin 50ng/ml groups had differences (P=0.009), similarly, leptin 200ng/ml groups P=0.012 and leptin 400ng/ml groupsP=0.028.This said that at the same with dosage leptin promoted the growth ofHSC-LX2, when leptin was 10-400μg/L dose.Then at the different time,the OD dataof HSC-LX2 proliferation at the same group had distinguished differences(F=1419.662, P=0.000).3.Results of the proliferation of HSC-LX2 treated baoganning by MTT with theONE-way:F=22016.44,P=0.000,LSD was applied between every twogroups.Baoganning regular dosage and double dosage significantly inhibit HSC-LX2proliferation compared with normal group(P=0.000), Colchicine could also inhibit theproliferation of HSC-LX2 compared with normal group(P=0.000).Compared withColchicine, baoganning regular dosage and double dosage significantly inhibitHSC-LX2 proliferation (P=0.000).However, no significant difference was observedbetween Baoganning regular dosage and double dosage group(P=0.077).4.The level of OB-Rb on Baoganning treated HSC-LX2 for different times byWB:Compared to the blank control, the serum with baoganning ingredients decreasedthe level of OB-Rb at 6h, returned at 12,18,24h, but Colchicine couldn't lower thanOB-Rb. After treatment with different drug serum groups ingredients for 6h,baoganning regular dosage and double dosage significantly inhibit OB-Rb than othergroups.5.The expression of JAK₂ on different serum groups treated HSC-LX2 fordifferent times by WB: Compared to the blank control, JAK₂ begain to descend at 6hafter the serum with baoganning ingredients treat HSC-LX2 and reached to the lowestlevel. After treatment with ingredients of different drug serum groups for 6h,baoganning regular dosage and double dosage significantly inhibit JAK₂ than othergroups.6. The expression of P-JAK₂ on different serum groups treated HSC-LX2 afterdifferent times by WB: Compared to the blank control, P-JAK₂ begain to descend at6h after baoganning ingredients treated HSC-LX2;Then P-JAK₂ reached to the lowestlevel at 12h. After treatment with different drug serum ingredients for 12h,baoganning and double dosage regular ingredients could significantly inhibit P-JAK₂ than other groups.7. The expression of STAT₃ on different serum groups treated HSC-LX2 fordifferent times by WB: Compared to the blank control, STAT₃ begain to descend at 6hafter the serum with baoganning ingredients treat HSC-LX2; STAT₃ reached to thelowest level at 24h. After treatment with different drug serum groups ingredients for24h, baoganning regular dosage and double dosage significantly inhibit STAT₃ thanother groups.8. The expression of P-STAT₃ on different drug serum groups treated HSC-LX2for different times by WB: Compared to the blank control, P-STAT₃ begain todescend at 6h after baoganning ingredients treated HSC-LX2; Then P-STAT₃ reachedto the lowest level at 12h. After treatment with serum groups containedsaline,Baoganning and Colchicine ingredients, baoganning regular ingredients anddouble dosage significantly could inhibit the level P-STAT₃ than other groups.Conclusion1.HSC-LX2 can secrete leptin by itself, but the leptin decreased with theincreasing time.2.With time and dosage,leptin increased the proliferation of HSC-LX2 gradually.3.Baoganning could inhibit the proliferation of HSCs-LX2 actived by leptin.4.The inhibition effects of Baoganning on proliferation HSC-LX2 may bepartly explained by Baoganning's down-regulation of OB-Rb.5.The one of mechanisms of the inhibition effects of Baoganning onproliferation HSC-LX2 may be Baoganning's down-regulation of JAK₂ and STAT₃of phosphorylations.6. The one of mechanisms of the inhibition effects of Baoganning onproliferation HSC-LX2 may be Baoganning's down-regulation of JAK₂ and STAT₃.7.At the experiment,we founded that JAK₂,STAT₃ could be activedcontinuously.