| BackgroundLiver fibrosis is the common pathological basis for diverse liver injuries. The current study shows that liver fibrosis can be reversed, but liver cirrhosis can not be reversed. Therefore it is much academic attention to prevent, inhibit or reverse hepatic fibrosis. TanshinoneⅡA (Tsn) is a major fat-soluble of Salvia miltiorrhiza Bunge and a natural antioxidant. It was proofed Tsn with antifibrotic effect by experiment and clinic. Researches of Lixin Liu et al discovered that Tsn was able to effectively inhibit the activated hepatic stellate cell (HSC) proliferating in vitro experiments. However, the mechanism of Tsn with antifibrotic effect is not clear. The central link of liver fibrosis is activation and proliferation of HSC, and producing extracellular matrix (ECM). Transforming growth factor-β1 (TGF-β1) is one of the important cytokines, which result in hepatic fibrosis. TGF-β1/Smad3 signaling pathway plays an important role in liver fibrosis. Researches of instructor Lixin Liu et al discovered that insulin-like growth factor binding protein related protein1 (IGFBPrP1) is a new pathogenic factor in the mechanism of liver fibrosis. In order to determine whether the mechanism of Tsn with antifibrotic effect relates to TGF-β1/ Smad3 signaling pathway and the expression of IGFBPrP1, we conducted the study.Objective:To explore the new mechanism of Tsn on experimental hepatic fibrosis in mice, by investigating the changes of expression of both IGFBPrP1, ECM and TGFβ1/Smad3 signaling pathway in hepatic fibrotic mice treated with Tsn.Methods:Hepatic fibrosis was induced by intraperitoneal injection of the thiacetamide (TAA). 36 male Kunming mice were randomly divided into five groups: normally control group (N group), 4-week model group (TAA4-week group), Tsn prevented group (TAA4+Tsn4-week group), 6-week model group (TAA6-week group) and Tsn treated group (TAA6+Tsn3-week group). Besides normally control group, the rest groups were injected with TAA 200mg/kg, 3 times a week, lasting 4 weeks and 6 weeks. Normally control group was injected with saline. TAA4+ Tsn4-week group was injected with Tsn 20 mg/kg, 3 times a week, lasting 4 weeks, before injecting with TAA one day. After injected TAA 3 weeks, TAA6+Tsn3-week group was started to inject with Tsn 20 mg/kg, 3 times a week, lasting 3 weeks. The serum levels of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) were detected. The histopathology changes of liver were observed with HE staining and the area density of Collagen of liver specimens was assessed by Masson staining. Immunohistochemistry method was used to observe the expression ofα-smooth muscle actin (α-SMA), Collagen I (Collagen I), fibronectin (FN), TGF-β1, Smad3 and IGFBPrP1 in liver; terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) was used to observe the hepatocyte apoptosis; Western blot method was used to observe the contents of FN, Smad3 and IGFBPrP1 in liver.Results:1 The results of the serum levels of ALT and LDH: Compared with TAA4-week group, the serum levels of ALT and LDH in TAA4+Tsn4-week group decreased significantly (32.19±8.43 vs 138.78±15.93, 56.63±9.44 vs 136.01±12.20, P<0.05). Compared with TAA6 group, the serum levels of ALT and LDH in TAA4+Tsn4-week group also decreased significantly (56.94±10.01 vs 177.29±14.26, 99.42±10.91 vs 298.01±25.11, F=298.20, P<0.05).2 The results of HE and Masson staining: With the extension of model time, edema and necrosis of the hepatocyte in model groups gradually deteriorate and area density of Collagen increased gradually. Compared with TAA4-week group and TAA6-week group, injury of liver in TAA4+Tsn4-week group and TAA6+Tsn3-week group improved obviously and area density of Collagen decreased(6.48±0.58 vs 1.18±0.11, 14.73±1.00 vs 3.86±0.35, H=27.87, P<0.05)3 The results of immunohistochemical staining: The positive expression is the brown and tan coloring. The expression ofα-SMA, Collagen I, FN, TGF-β1, Smad3 and IGFBPrP1 in normal control group of mice liver tissue showed yellow and light brown coloring, weak positive expression. The expression in TAA4-week group and TAA6-week group was stained gradually deepen, showing brown yellow or brown shading, strong positive expression. Compared with TAA4-week group and TAA6-week group, the staining in TAA4+Tsn4-week and TAA6+ Tsn3-week group was light.4 The results of immunohistochemical staining of image analysis: The IOD of Collagen I, FN,α-SMA, TGF-β1, Smad3 and IGFBPrP1 in TAA4+Tsn4-week group decreased significantly than in TAA4-week group (0.87±0.18 vs 4.33±1.05, 3.37±0.53 vs 8.16±0.79, 0.84±0.12 vs 5.51±0.59, 0.82±0.10 vs 6.44±1.08, 0.38±0.08 vs 3.02±0.55, 0.70±0.08 vs 5.97±0.48, H=30, F=298.53, H=27.88, F=525.68, H=27.05, H=27.87, P<0.05). The IOD of Collagen I, FN,α-SMA, TGF-β1, Smad3 and IGFBPrP1 in TAA6+Tsn3-week group decreased significantly than in TAA6-week group (3.34±0.78 vs 9.86±2.01, 5.33±0.57 vs 17.58±1.52, 3.59±0.39 vs 9.83±1.32, 5.52±1.28 vs 13.09±2.67, 2.44±0.50 vs 9.70±0.93, 4.84±0.47 vs 10.33±1.31, H=30, F=298.53, H=27.88, F=525.68, H=27.05, H=27.87, P<0.05).5 The results of TUNEL staining: Compared with TAA4-week group (7.21%±0.79%), hepatic apoptosis index in TAA4+Tsn4-week group (7.21%±0.79%) reduced significantly (t=12.76, P<0.05).6 The results of Western blot: The place of 220KD, 58KD, 31KD and 43KD represented FN, Smad3, IGFBPrP1 andβ-actin respectively. Compared with the normal control group, the contents of FN, Smad3 and IGFBPrP1 in liver tissue of TAA4-week group and TAA6-week group increased, especially in TAA6-week group. Compared with TAA4-week group and TAA6-week group, the contents of FN, Smad3 and IGFBPrP1 in TAA4-week group and TAA6-week group reduced significantly.7 The results of Western blot image analysis: The gray value of FN, Smad3 and IGFBPrP1 in TAA4-week group was significantly increased than TAA4+Tsn4-week (1.18±0.14 vs 0.77±0.06, 0.64±0.06 vs 0.40±0.03, 1.07±0.15 vs 0.87±0.14, P<0.05). The gray value of FN, Smad3 and IGFBPrP1 in TAA6-week group was significantly increased than TAA6+Tsn3-week (1.43±0.12 vs 1.13±0.09, 0.75±0.06 vs 0.54±0.07, 1.55±0.12 vs 1.10±0.11, F=73.91, F=46.96, F=47.1, P<0.05).Conclusion:1 TanshinoneⅡA not only improved liver function but also inhibited the activated HSC, decreased the expression of Collagen I and FN, and protected the hepatocytes.2 The new mechanism of TanshinoneⅡA with antifibrotic effect maybe related to influencing TGF-β1/Smad3 signaling pathway.3 The new mechanism of TanshinoneⅡA with antifibrotic effect maybe related to down- regulating the expression of IGFBPrP1.
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