| BackgroundInflammation is closely connected with malignancy. Chronic infection, as well as inflammation, is deemed to be one of the important factors which could result in the occurrence and the development of malignancy. Clinical research shows that, the incidence of hepatocellular carcinoma has a positive correlation with the incidence rate of chronic hepatitis. The inflammation which is resulted from chronic viral hepatitis, alcohol liver, liver cirrhosis, and many other chronic hepatic injuries can accelerate the occurrence and the development of liver cancer, however, the explicit mechanism of how it is to be like still hasn’t been clear completely. The chronic inflammation in body often generates some cytokines which can cause immunological tolerance, and these cytokines have a big contribution to the occurrence and the development of cancer. Despite this, lipopolysaccharidef(LPS) plays an important role in the process of chronic inflammation. Lipopolysaccharide, located at the outermost layer of cytoderm, covering mucopeptides of cytoderm, is one of the constituent components of cytoderm which is in gram-negative bacterium. By adjusting the generation of the regulators of inflammation, lipopolysaccharide can accommodate congenital immunity, stimulate the generation of inflammatory factors and oxygen radicals, then accelerate the occurrence and the development of cancer. Lipopolysaccharide often gives rise to systemic inflammatory response and also leads to multiple organ failure, cardiac involvements, and cardiac functional damage, so LPS can be used to simulate the toxic reaction of cancer cells caused by inflammatory response.Resveratrol has an extensive function on physiology and Pharmacology, such as anti-oxidation, anti-inflammation, anti-cancer, anti-aging and so on. There are numerous researches having showen that resveratrol can alleviate arthritis and the inflammatory response and the immune response of autoimmune diseases. PurposeOur research focuses on studying how LPS affects the proliferation, invasion and metastasis of human liver cancer cells, observing the gene expression changes of PPAR-γ, TGF-β, P21, and Bcl-2. And we also study the role of Resveratrol in the proliferation, invasion and metastasis of hepatoma cell line induced by LPS. Methods1. MTT colorimetric method was used to test the impacts of varied concentrations(0 μg/ml, 1 μg/ml, 2.5 μg/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml, 40 μg/ml) of LPS on the proliferation of Hep G2 and SMMC7721 at different time.2. Scratch test was used to observe the impacts of varied concentrations(0 μg/ml, 1 μg/ml, 2.5 μg/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml, 40 μg/ml) of LPS on cell migration of Hep G2 and SMMC7721.3. Transwell chamber was used to test the impacts of varied concentrations(0 μg/ml, 1 μg/ml, 2.5 μg/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml, 40 μg/ml) of LPS on invasion of Hep G2 and SMMC7721.4. Real-time fluorescent quantitative PCR was used to test the expression level of relevant m RNA induced by varied concentrations(0 μg/ml, 1 μg/ml, 2.5 μg/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml, 40 μg/ml) of LPS in Hep G2 and SMMC7721.5. The above mentioned methods were used to observe the impacts of varied concentrations of Resveratrol(50 μmol/L, 100 μmol/L, 200 μmol/L) on the proliferation, invasion, metastasis, and expression level of relevant m RNA of SMMC7721 induced by 20 μg/ml LPS. Results1. MTT analysis showed that LPS could accelerate the proliferation of SMMC7721 by the concentration- and time-dependent manner. The difference between groups was statistically significant(p<0.01). Low concentration of LPS neither accelerated nor suppressed the proliferation of SMMC7721. The difference between groups was not statistically significant(p>0.05). High concentration of LPS(40 μg/ml) suppressed both Hep G2 and SMMC7721.2. Scratch test showed that after the affecting of varied concentrations of LPS with 24 h, the capacity of cell repair of SMMC7721 enhanced dramatically; and with the increase of concentration, so was the capacity of metastasis; when LPS was 20 μg/ml, the enhancement was at its peak. As to Hep G2, LPS didn’t work on it.3. Transwell test showed that, after the intervention of LPS, the number of metastasis cells of SMMC7721 increased dramatically comparing with the control group, and the higher concentration of LPS the more number of metastasis cells(p<0.01). However, after the intervention of varied concentration of LPS on Hep G2, the number of metastasis cells of Hep G2 hadn’t changed. The difference hadn’t a statistical significant(p>0.05).4. q PCR showed that, after the affecting of varied concentrations of LPS with 48 h on SMMC7721, the expression level of Bcl-2 and TGF-β both raised with different degree(p<0.01), while the expression levels of PPAR-γ and P21, along with the NC group, both descended with different degree. The difference had a statistical significant(p<0.01). After the affecting of LPS on Hep G2, the expression level of m RNA of PPAR-γand TGF-βdescended somehow(p<0.01), there was no distinct difference between control group and P21 and Bcl-2 m RNA(p>0.05).5. The affecting of varied concentrations of resveratrol on SMMC7721, along with 20 μg/ml LPS, MTT showed that, varied concentrations of Resveratrol could suppress the proliferation of SMMC7721 induced by LPS with different degree, and with the increase of concentration of Resveratrol, the effect of suppressing enhanced(p<0.01). Scratch test showed that, Resveratrol could suppress the metastasis of SMMC7721 induced by LPS, and Transwell test showed that, the number of invasion cells of SMMC7721 with varied concentrations of Resveratrol as well as with induced by LPS, decreased comparing with control group, and the higher concentration of Resveratrol the less number of invasion cells(p<0.01). And q PCR showed that, after the affecting of varied concentrations of Resveratrol with 48 h on SMMC7721, the expression level of Bcl-2 and TGF-β both descended with different degree(p<0.01), while the expression level of PPAR-γ and P21 both raised with different degree comparing with the group which only had LPS, and The difference had a statistical significant(p<0.01). Conclusions1. LPS promotes the proliferation and invasion and metastasis of SMMC7721.2. LPS increases the m RNA expression levels of Bcl-2 and TGF-β and descreases the expression levels of PPAR-γ and TGF-β in SMMC7721.3. LPS has no effects on the proliferation, invasion, and metastasis of Hep G2, while LPS can change the expression levels of PPAR-γ and TGF-β.4. Resveratrol can suppress the proliferation, invasion, and metastasis of SMMC7721 induced by LPS.5. The mechanism of the inhibition of resveratrol on proliferation, invasion, and metastasis of SMMC7721 induced by LPS is likely related to increasing the expression levels of PPAR-γ and P21 and decreasing the expression levels of bcl-2 and TGF-β. |
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