Effects On Of Liver Targeting Administration Of TGF-β₁ SiRNA On TGF-β/Smad Signaling Pathway In Rats With Acute Liver Damage

Objectives (1) Establish a improved method of the rat hepatic artery cannula model for the drug liver targeting administration; (2) Construct an acute liver damage animal model for priming TGF-β/Smad signal conduction pathway in hepatic tissue of rats; (3) Investigate the intervention effect of the combination of transforming growth factor beta 1 (TGF-β₁) and transforming growth factor beta 1 receptor(TGF-β₁ R) siRNA against TGF-β/Smad signal conduction pathway; (4)compare the differences of transfection effects under four modes in which exogenous gene enter target cell, and establish and evaluate the benefit of hepatic artery catheterization administration.Methods (1) Establish the improved model of rat's hepatic artery catheterization and detaining in vitro: To improve the Lindell HA catheterization, a specially-made conduit with 0.5mm internal diameter (fabricated from epidural anaesthetic tube) was deployed and insertted into hepatic inherent artery debouchement through arteria gastroduodenalis under the operation microscope(10×), then the tube was dwelt and fixed.(2) In order to induce an acute liver damage animal model for priming TGF-β/Smad signal conduction pathway in hepatic tissue of rats, 25% of carbon tetrachloride (CCl₄) contained in arachis oil liquor was given to the rats with HA catheterization or with sham operation twice by an intraperitoneal injection on the next day and the fifth day after the operation, respectively. The dosage of CCL₄ is 6ml per kg. At mean time, the rats were fed by water and common grains feeder. After 48 hours of the second CCl₄ intake, the rats were sacrificed and the samples were taken for testing.(3) The intervention of TGF-β₁ and TGF-βR siRNA on TGF-β/Smad signal conduction pathway: The first, three recombinant plasmids were constructed, which could transcribe siRNA targetting to TGF-β₁, TβRâ… and TβRâ…¡, respectively. Second, thirty six rats were used to experiment and divided into 6 groups randomly, that is model comparing group (group M), TGF-β₁ siRNA intervention group (group T), TGF-β₁ siRNA and TpRI siRNA intervention group(group T+R₁), TGF-β₁ siRNA and TβRâ…¡siRNA intervention group (group T+R₂), TGF-β₁, TβRâ… and TβRâ…¡siRNA intervenention group (group T+R₁+R₂) and blank group (group N). Except blank group, the rats were treated twice by CCL₄ on day 1 and day 5, respectively, then 50μg specific siRNA plasmid DNA in 2ml Ringer' solution per rat were injected to the rat in group T, T+R₁,T+R₂, and T+R₁+R₂ by vena caudalis injection at 0.5 hours after the second CCL₄ administration. 50μg plasmid DNA with nonspecific siRNA was given to the rats in group M at 0.5 hours after the second CCL₄ administration. No any processing was done to the rats in blank group. Finally, the rats were killed at 48 hours after siRNA injection and the samples were taken for testing.(4) The inhibition effect of TGF-β₁ siRNA on acute hepatic injury and hepatic fibrosis dependent on the plasmid DNA introduction approaches: 30 rats with acute hepatic injury were divided into 5 groups, that was comparing group (group m), vena caudalis straight injection group (group v), liposome package group (group L) and hydrodynamics group (group H), and HA catheterization group (group A). After 0.5 hours of the second CCL₄ administration, 50μg plasmid DNA with TGF-β₁ siRNA in 2ml Ringer's solution was given to the rats in group V by vena caudalis injection and to the rats in group A by HA catheterization. The mixture of 50μg plasmid DNA with TGF-β₁ siRNA and 250μg LipofectamineTM 2000 in 2ml Ringer's solution was given to the rats in group L by vena caudalis injection. Large volume(10% of rat body weight) of solution containing 50μg plasmid DNA with TGF-β₁ siRNA was injected to the rats in group H by rapid vena caudalis injection in 10 seconds. For the rats in group M, 50μg plasmid DNA with nonspecific siRNA in 2ml Ringer's solution was given by vena caudalis injection. The rats were killed at 48 hours after siRNA injection and the samples were taken for testing.(5) The following tests were done for evaluation of the effects of TGF-β1,TβRâ… ,and TβRâ…¡siRNA on TGF-β/Smad signal conduction pathway: (1) serum ALT, AST, HA; (2)the pathologic changes of liver tissue by HE staining. (3)Quantitative detection of mRNA ofα-SMA,â… ,â…¢type collagen, TGF-β₁,Smad₃,PCNA and TGF-αby RT-PCR, and (4) The expression ofα-SMA,collagenâ… andâ…¢,TGF-β₁,Smad₃,PCNA and TGF-αin liver tissue by Immunohistochemistry technology.Result:(1)In the experiment of constructing detaining HA catheterization on 20 rats, there were 17 rats getting successfully detaining HA catheterization, and the achievement ratio is 85%. The detaining HA catheterization was failure in 3 rats because 2 had blood vessel broken during the operation and 1 died after the operation.(2) In the priming experiment of TGF-β/Smad signal conduction pathway in rat's hepatic tissue, the levels of ALT, AST and HA were apparently higher in group M, and group A than that in group N (P＜0.01). The results showed there were much more inflammatory changes in the rat liver tissue in group M and group A, such as obvious inflame, apomorphosis, cellular necrosis and fibroplasias. Gene expression ofα-SMA,â… type,â…¢type collagen,TGF-β₁,Smad₃ in liver tissue of the rats treated by CCL₄ were much stronger than that in normal control, as showed both by mRNA detection by RT-PCR (P＜0.01)and by protein expression detected by Immunohistochemistry technology (P＞0.05). However, above data showed no any differences between the group M and group A. So that, the TGF-β/Smad signal conduction pathway could be activitied successfully in the hepatic tissue of the rats with acute liver injury, whether cannula detainment or sham operation had be done.(3) Gene expression ofα-SMA,â… type,â…¢type collagen,TGF-β₁,and Smad₃ could be reduced by transfection of plasmid DNA with TGF-β₁,TGF-β₁RI,and TGF-β₁Râ…¡siRNA. The effect was much better in combination use of TGF-β1,TGF-β₁Râ… ,and TGF-β₁Râ…¡siRNA than that in single use of TGF-β₁ targeting siRNA.(4)The signal conduction TGF-β/Smad pathway in the rats' hepatic tissue could be effectively blocked by tansfection of the plasmid DNA with TGF-β₁targeting siRNA. This effect was much more intensive in the group L and group A than that in the group group V and group H (P＜0.05).Conclusion1. the improved technique approach of HA catheterization and detainment in vitro is straightforward, litter hepatic injury, and the higher achievement ratio.2. The acute liver injury rat model could be used to evaluate the drugs and methods against liver fibrosis because in this rat model it is clearly showed priming TGF-β/Smad signal conduction pathway in liver tissue,which is the key point to induce liver fibrosis.3. The combination use of TGF-β₁,TGF-β₁Râ… and TGF-β₁Râ…¡siRNA had obviously synergistic effect on the intervention of the TGF-β/Smad signal conduction pathway.4. The transfection of siRNA plasmid by HA catheterization is a safe, convenient and efficient.