Anti-fibrotic Effects Of The Masson Pine Pollen Aqueous Extract On Experimental Hepatic Fibrosis

Objective To observe the effect of masson pine pollen extracts on experimental liver fibrosis, and to investigate the mechanisms and approaches via the experiment results in vivo and vitro, so that we can utilize masson pine pollen resources scientifically and reasonably and provide experimental basis for seeking safe, effective, economic and convenient nutritional components for liver fibrosis.Methods: The study was divided into three parts:1. Nutrients analysis of masson pine pollen original powder(MPPOP), masson pine pollen aqueous extract(MPPAE) and masson pine pollen ethanol extract(MPPEE) were executed, including key nutrients such as crude protein, crude fat, crude fiber, carbohydrate, and 9 kinds of minerals and trace elements, 18 kinds of amino acid content and several main vitamins content. We evaluated the nutrition characteristics of MPPOP, MPPAE, and MPPEE, in order to lay the foundation for analyzing their physiological functions and action mechanism.2. Via cell culture in vitro, oxidative damage model of human liver cell line L-02 by CCl4 had been built, in order to observe the effects of different concentrations of MPPAE on the cell proliferation activity, cell morphology and functional changes after the injury. Meanwhile, the inhibition effects and effective way of MPPAE on hepatic stellate cell line LX-2 model stimulated by conditional medium of liver cell injury were observed.3. Rats model of experimental liver fibrosis had been built via peritoneal injection of CCL4. At the same time, different concentrations of MPPAE were given by gavage to observe the effects and main functional ways on animal models of experimental liver fibrosis comparing with MPPOP.Results:1. The nutrients analysis of masson pine pollen and its extracts showed that:1) The most abundant nutrients were carbohydrate and crude fiber in MPPOP. The crude fiber content decreased significantly in MPPAE and MPPEE because it was unsolved in inorganic and organic media. Carbohydrate was obviously enriched in MPPEE, on the contrary, crude proteincontent increased in MPPAE. For water-soluble property of protein, it was almost all lost in MPPEE.2) There were plenty of minerals and trace elements in masson pine pollen, especially for potassium, calcium, phosphorus and magnesium. Except the iron, elements content of MPPAE are 1.6-3.5 times higher than MPPOP, opposite, it was significantly reduced 1.7 to 13.4 times in MPPAE.3) Masson pine pollen contains all essential amino acids human body need. Among them, some amino acids with immune activity, such as glycine, arginine,and branched chain amino acid were rich, and moreover their content in MPPAE were 2.0- 12.8 times more than MPPOP and enormously fit for the nutrient requirements of patients with chronic liver disease. The content of amino acid decreased 1.1-115.4 times in MPPEE just as the content of protein.4) The total polyphenols and flavonoids content in masson pine pollen reached 380 mg/100 g and 364 mg/100 g respectively, and the total polyphenols content in MPPAE were 5.2 times higher than MPPOP. The transcendental antioxidant activity might play the main roles of anti-fibrosis.5) MPPOP contains a variety of vitamins, but most vitamins were lost after extraction process in MPPAE and MPPEE. The content of liposoluble vitamin A in MPPEE got enrichment and enhanced 100 times, while other vitamins were extremely low.2. The effect of MPPAE on cell injury model in vitro revealed that:1) The MPPAE have no obvious protective effects on the proliferation activity of L-02 cell damaged by CCl4, but have obvious positive effects on the CCl4 induced liver cell morphology and fatty degeneration;2) The activity of ALT and AST increased after CCl4 damaging on L-02 cells, MPPAE can inhibit the changes, and has a dose effect relationship;3) After damaged by CCl4, the SOD activity and MDA content of L-02 cells were increased significantly, reached to 3.90 times and 6.51 times as the normal group. The main reason is the SOD compensatory increase in oxidative stress, at the same time oxidative damages in the liver cells produce large amounts of lipid peroxide. MPPAE can effectively inhibition the abnormal increase of SOD and MDA. The content of MDA drop about 34.8- 87.5% and the SOD activity fell to 68.4% with good concentration effect relationship. May be it related to the rich content of polyphenolic compounds;4) In the appropriate dose, L-02 cell conditioned medium could stimulate LX-2 cell activation and proliferation. The intervention of MPPAE can effectively inhibit the proliferation andpromote the apoptosis of activated LX-2 cells, thus can reduce the production of ECM and inhibite fibrosis progress;5) After stimulated by L-02 conditioned medium, LX-2 cell was activated andthe content of type I and type III collagen increased, the expression of matrix metalloproteinase MMP-1 and MMP-2 did not change significantly, the expression of TIMP-1 was significantly increased about 85.2%; MPPAE intervention can reduce the content of collagen, upregulate the expression of MMP-1 and MMP-2. At the same time MPPAE played a significant regulatory role on the unusually high expression of TIMP-1. Therefore MPPAE regulate the content of I, III type collagen is through down-regulation of TIMP-1 expression, so as to reduce the inhibitory effect of TIMP-1 on MMPs, indirectly enhanced ECM catabolism.3.The observation of MPPAE and MPPOP on CCl4 induced rat liver fibrosis model in vivo indicated that:1) The experimental hepatic fibrosis model of rats was successfully built via intraperitoneal injection of CCl4 for 8 weeks;2) MPPAE intervention can effectively ameliorate the experimental hepatic fibrosis model rats symptoms: had positive effects on ascites formation and growth and liver cell morphology changes. Result of collagen fibers staining showed that the collagen area decreased from 20.14% to 8.23%;3) The activity of the liver transaminase ALT and AST in liver fibrosis model of rats is higher than that of the normal group by 6.86 times and 4.42 times, APPAE significantly inhibited the raise, the high dose group can decreased the activity by 64.3% and 58.1%.4) The content of fibrin and collagen in serum and liver tissue were significantly increased in rat model of hepatic fibrosis, the serum hyaluronic acid(HA) and liver hydroxyproline content were increased 1.85 times and 1.81 times, but the intervention of MPPAE decreased 23.7% and 23.2%;5) After injuried by CCl4, the SOD activity in rats liver had no significant changes, while the MDA content was increased and the GSH-Px activity was decreased about 36%. MPPAE can significantly improve activity of SOD and GSH-Px. GSH-Px level can be increased by 23.3%. At the same time the accumulation of peroxide can be reduced effectively by application of MPPAE;6) The MPPAE could regulate fibrosis related factors such as TGF- β 1,PDGF and NF- κ B to express higher m RNA in the animal model so as to inhibit hepatic stellate cells(HSC) activation, control collagen production, while enhance the expression of matrix metalloproteinase(MMPs) in order to promote the degradation of extracellular matrix(ECM).Conclusion: MPPAE had most kinds of nutrients and the content was abundant. According to the experimental observations in vitro and in vivo, it makes a great contribution to the inhibition of fibrosis of the cells in vitro and hepatic fibrosis animal model, improvement of the status and function of liver cell. The mechanism is the cooperation of improving the antioxidant capacity, inhibiting peroxide accumulation, regulating the expression of fibrosis related factors and protein, and promoting the apoptosis of activated HSC etc. So we think MPPAE can be an ideal nutritional supplements for antifibrosis of liver.