| ObjectiveThis study probe the mechanism of EGb761 to inhibit hepatocarcinogeneses induced by AFB1 through the observation of the expressions of heparan sulfate class of proteoglycans (MXR7), cyclooxygenase (COX-2) and cyclin-dependent kinase inhibitory protein (PT6) in liver cells of each group animals.Methods71 rats were randomly divided into 3 groups : (1) AFBi group: 28 rats were fed normal diet and were injected AFBi in intraperitoneal at the beginning of 3 weekth, 1 ~ 3 times / weekly, 1OOug ~ 200ug/kg/time. was stoped to injecte at the 47 weekth. (2) AFB1 + EGb761 Group: 29 rats were fed with feed containing 2 g/kg EGb761, each rat eating 40 mg ~ 100 mg/day. AFB1 was injected as what done in AFB1 group; (3) Control group: 14 rats were fed normal diet neither treated with AFB1 nor with EGb761. The animal experiment ended at 64th-week and the surviving animals were sacrificed by cervical dislocation. The tumor and liver tissues specimens were collected and formalin-fixed and then paraffin-embedded, for HE and immunohistochemistry staining to detecte the MXR7, P16, COX-2 protein expression respectively.Results1.Carcinogenesis induced by AFB1At the end of 64-week , AFB1 group had 25 effective animals, AFB1 + EGb761 group was 26 and control group was 12. PHC incidence was 76% (19/25) In AFB1 Group and was significantly higher than that in AFB1 + EGb761 group( 26.92%, 7/26, P = 0.001). Control group had not tumor development.2. Effect of EGb761 on the expression of MXR7 proteinPositive expression of MXR7 protein showed brown collor located in the cytoplasm of positive cells. They were scattered or diffused distribution. The expression levels of MXR7 protein in AFB1 group were significantly higher than that in AFB1 + EGb761 group ( 5.133± 2.725 vs 1.488±1.178, P <0.001) and control group ( 5.133±2.725 vs 1.200±0.902 , P <0.001).3. Effect of EGb761 on the expression of P16 proteinP16 protein expression was brownish yellow located in the cytoplasm of positive cells. They were scattered or diffused distribution. The expression level of p16 protein in AFB1 group were significantly lower than that in AFB1 + EGb761 group (1.533±0.856 vs 2.456±1.014 , P = 0.03 ) and the control group(1.533±0.856 vs 4.000±1.206 , P <0.001 )4. Effect of EGb761 on the expression of COX-2 proteinCOX-2 protein expression was brownish yellow located in the cytoplasm of positive cells and was scattered or diffused distribution. The expression of COX-2 protein levels were 3.305±0.566 in AFB1 group and 2.704±0.431 in AFB1 + EGb761 group. There was not significant difference (P = 0.783), But there was significant difference compared with control group(3.305±0.566 vs1.077±0.261, P = 0.04) .Conclusions1. EGb761 can inhibit or delay liver cell proliferative lesions,hyperplastic nodules to the formation of precancerous lesions that significantly reduce or delay,inhibit or delay the development of precancerous lesions to cancer.2. EGb761 can down MXR7 protein expression possibly through inhibition classic Wnt/β- catenin pathway activity,to reduce the occurrence and development of liver cancer in rats.3. EGb761 can interfere AFB1 with cell cycle induced liver cancer, inhibition of DNA synthesis, which suggesting that inhibition of cancer cell proliferation is the cellular basis EGb761 one play.4. EGb761 may be applied as a chemopreventive agent for human AFB1-related PHC.
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