| ObjectiveTo explore the mechanism of liver cancer generation and development based on bone marrow stem cells transforming into liver cancer stem cells, and to expound the effect and mechanism of Diwu Yanggan capsule on the occurrence and development of liver cancer, then rich the scientific connotation of “the liver governing growth”(“marrow stem cells differentiate into liver cells”,“marrow stem cells fail to differentiate into liver cells” and “tonifying the kidney to regulate liver regeneration and repair by affecting stem cells and their microenvironment”).MethodsTo establish liver cancer models in rats by Solt-Farber method. SPF 80 male SD rats were randomly divided into model group, Sorafenib group, Diwu Yanggan treatment group(DWYG group), BMSC group, and the normal group. Before the study began, the interior cleaning and disinfection was the first should be done. After adding fodder and water and adaptive feeding the rats of 1 week. Then began to establish liver cancer models in rats. Specifically as follows: ① On the first day of model establishment, rats were givenintraperitoneal injection of diethylnitrosamine(DEN) by 50mg/kg dose of as a start agent in the model group, Sorafenib group, and DWYG group. Two weeks later, a intragastric administration with was given to rats for 7 days according to a dose of 15 mg/kg/d. At the end of the 3rd week, liver partial hepatectomy(PHx) was conducted to rats in the model group, Sorafenib group, and DWYG group. First, experimental animals were given anhydrous ether inhalation by anesthesia. Second, incising the skin and subcutaneous fat along the middle of the abdomen of rats in order to exposed the liver. Third, after binding the root of the liver according to the standard method of hepatectomy, removaling the left hepatic lobe and the middle of the hepatic lobe(accounting for about 68% of the whole liver), ligation and excision of the left hepatic lobe and the middle of the(accounting for about 68% of the whole liver), and then transplanting the red fluorescent protein-labeled bone marrow stem cells(RFP-BMSCs) to the right hepatic lobe of the three groups rats in five different positions. Each position injected with 0.1ml RFP-BMSCs 0.1ml(105 cells). Layered suture and disinfected the wound surface. Three days after surgery, it should be continued to give 2-AAF for 16 weeks. During the period of establishing the model, DWYG group was given aqueous solution of Diwu Yanggan capsule according to a dose of 750mg/kg/d as a intervention, and Sorafenib group was given aqueous solution of Sorafenib according to a dose of 80mg/kg/d as a intervention. The model group was given saline as a control. ② On the first day of model establishment, rats were given intraperitoneal injection of saline in the BMSC group as a control. Rats were administered saline, when other groups need to a intragastric administration. At the end of the 3rd week, rats inthe BMSC group were not accepted liver partial hepatectomy(PHx) but incising the skin and subcutaneous fat along the middle of the abdomen of rats and gently fliped the liver.Then transplanting the red fluorescent protein-labeled bone marrow stem cells(RFP-BMSCs) to the right hepatic lobe of rats in the BMSC group. ③ On the first day of model establishment, rats were given intraperitoneal injection of saline by 50 mg/kg dose in the normal group. The rats in the normal group were only given saline, when other groups need to a intragastric administration. The group were not accepted liver partial hepatectomy(PHx) as well as bone marrow stem cells.In short, the model group was dealed with DEN ip, 2-AAF ig, PHx, RFP-BMSCs and NS ig). Sorafenib group was treated with DEN ip,2-AAF, PHx,RFP-BMSCs and Sorafenib ig. DWYG group was dealed with DEN ip,2-AAF, PHx, RFP-BMSCs and DWYG ig. The BMSC group was treated with NS ip, RFP-BMSCs and NS ig. The normal group was treated with NS ip and NS ig.After the completion of modeling rats in each group were sacrificed, liver tissue was excised in rats with 4% paraformaldehyde-fixed, paraffin- embedded, then do 5μm thick serial sections for HE staining for histological testing. Then randomly selected within each slice 3-4 400 magnification in each group, and using semiquantitative scoring method to analysis and evaluation the pathological degree of liver cancer. We scored separately according to Edmondson-Steiner grading standards and percentage of positive cells, analysising the pathological degree with average credit pathological liver of rats in different groups. Cut the cancer tissue in rats,then deal with Carnoy’s fixative(ethanol: acetic acid = 3: l), do 5μm thick serial sections,Feulgen staining in order to detect hepatocyte nuclear DNA content. The liver tissue should be frozened in liquid nitrogen. Immunohistochemical staining was given for detecting PCNA, CD34, ABCG2, and Thy-1 expression in liver tissue. Western blot was used to detect of EMT-related protein E-cadherin, Vimentin, TGF-β expression as well as related protein express of Ras/Raf /Mek/Erk signaling pathway and JAK/STAT signaling pathway. Quantitative Real-time PCR was used to detect E-cadherin, Vimentin, TGF-β, Ras/Raf/Mek/Erk, and JAK/STAT signaling pathway m RNA expression. All data are used SPSS19.0 statistical software for statistical analysis, the results are Mean±SD(mean±standard deviation). Multiple groups were compared using ANOVA, p<0.05 was considered statistically significant.Results1 The results of visually observed of the liver: compared with the normal group, the liver in the model group showed a irregular shape, hepatic lobes insufficiency, rough surface, uneven, hard surface and cut surface of diffuse liver nodules withdifferent sizes, or bleeding, necrosis and other features. And the above pathological manifestations significantly reduced after treating with Diwu Yanggan capsule or Sorafenib.2 Histopathological Results(HE staining): compared with the normal group, we can see in the model group tumor cell of the model group growth exuberant, with a high cell density, disorganized, nuclear-cytoplasmic ratio, nuclear staining deep, nuclear atypia, inflammatory cell infiltration and other pathological features. And the above pathological manifestations significantly reduced after treating with Diwu Yanggan capsule or Sorafenib.The results of the pathological degree analysis by semi-quantitative scoring method: the average credit in the model group(10.90±2.71) was significantly higher than the normal group(0.00 ± 0.00), the difference was statistically significant(p<0.05); the DWYG group(8.00±2.43) compared with the model group(10.90±2.71), the difference was statistically significant(p<0.05); the average credit in the sorafenib group(8.20±3.11) was lower than that in the model group(10.90±2.71), the difference was statistically significant(p<0.05); there was no statistical significance between the DWYG group(8.00±2.43) and sorafenib group(8.20±3.11),p>0.05.3 The effect of Diwu Yanggan capsules on hepatocyte tonuclear DNA content by Feulgen staining: IOD values in the model group(14297.24±620.84) was significantly higher than the normal group(2901.33±84.64), the difference was statistically significant(p<0.05); the DWYG group(8554.73±135.86) compared with the model group(14297.24±620.84), the difference was statistically significant(p<0.05); the DWYG group(8554.73 ± 135.86) compared with the sorafenib group(8965.99 ± 338.65), the difference was statistically significant(p<0.05); there was no statistical significance between the normal group(2901.33±84.64) and BMSC group(3346.42±181.37), p>0.05.4 The effect of Diwu Yanggan capsules on PCNA expression(Immunohistochemistry): OD values in the model group(0.5790±0.0303) was significantly higher than the normal group(0.2821±0.0138), the difference was statistically significant(p<0.05); the DWYG group(0.4397±0.0181) compared with the model group(0.5790±0.0303), the difference was statistically significant(p<0.05); the DWYG group(0.4397 ± 0.0181) compared with the sorafenib group(0.4553 ± 0.0208), the difference was not statistically significant(p > 0.05); there was no statistical significance between the normal group(0.2821±0.0138) and BMSC group(0.2934±0.0223), p>0.05.5 The effect of Diwu Yanggan capsules on liver cancer stem cells(1) The effect of Diwu Yanggan capsules on the expressions of relevant markers ABCG2, CD34 and Thy-1(Immunohistochemistry): OD values of ABCG2 in the model group(0.3276±0.0210) was significantly higher than the normal group(0.1930±0.0045), the difference was statistically significant(p<0.05); the DWYG group(0.2423±0.0144) compared with the model group(0.3276±0.0210), the difference was statistically significant(p<0.05). the DWYG group(0.2423±0.0144) compared with the sorafenib group(0.2514±0.0203), the difference was not statistically significant(p>0.05); there was no statistical significance between the normal group(0.1930±0.0045) and BMSC group(0.1964±0.0126), p>0.05.OD values of CD34 in the model group(0.5880±0.0290) was significantly higher than the normal group(0.4077±0.0086), the difference was statistically significant(p<0.05); the DWYG group(0.4857±0.0312) compared with the model group(0.5880±0.0290), the difference was statistically significant(p<0.05). the DWYG group(0.4857±0.0312) compared with the sorafenib group(0.4648±0.0239), the difference was not statistically significant(p>0.05); there was no statistical significance between the normal group(0.4077±0.0086) and BMSC group(0.3980±0.0098), p>0.05.OD values of Thy-1 in the model group(0.5887±0.0361) was significantly higher than the normal group(0.4121±0.0134), thedifference was statistically significant(p<0.05); the DWYG group(0.4787±0.0228) compared with the model group(0.5887±0.0361), the difference was statistically significant(p<0.05); the DWYG group(0.4787±0.0228) compared with the sorafenib group(0.4790±0.0274), the difference was not statistically significant(p>0.05); there was no statistical significance between the normal group(0.4121±0.0134) and BMSC group(0.4085±0.0220), p>0.05.(2) The effect of Diwu Yanggan capsules on bone marrow stem cells and liver cancer stem cell-related markers double positive expressions(immunefluorescence):Double positive expressions of ABCG2/CD34 in the model group(0.3136±0.0294) was significantly higher than the normal group(0.1954±0.0110), the difference was statistically significant(p<0.05); the DWYG group(0.2188±0.0114) compared with the model group(0.3136 ± 0.0294), the difference was statistically significant(p<0.05); the DWYG group(0.2188±0.0114) compared with the sorafenib group(0.2359±0.0179), the difference was not statistically significant(p > 0.05); there was no statistical significance between the normal group(0.1954±0.0110) and BMSC group(0.1944±0.0066), p>0.05.OD values of ABCG2/CD45 in the model group(0.3089±0.0113) was significantly higher than the normal group(0.1996±0.0104), the difference was statistically significant(p<0.05); the DWYG group(0.2377±0.0163) compared with the model group(0.3089±0.0113), the difference was statistically significant(p<0.05); the DWYG group(0.2377±0.0163) compared with the sorafenib group(0.2328±0.0157), the difference was not statistically significant(p>0.05); there was no statistical significance between the normalgroup(0.1996±0.0104) and BMSC group(0.1891±0.0090), p>0.05.OD values of ABCG2/Thy-1 in the model group(0.2953±0.0146) was significantly higher than the normal group(0.1995±0.0082), the difference was statistically significant(p<0.05); the DWYG group(0.2320±0.0148) compared with the model group(0.2953±0.0146), the difference was statistically significant(p<0.05); the DWYG group(0.2320±0.0148) compared with the sorafenib group(0.2458 ± 0.01360), the difference was not statistically significant(p > 0.05); there was no statistical significance between the normal group(0.1995±0.0082) and BMSC group(0.1980±0.0076), p>0.05.6 The effect of Diwu Yanggan capsules on molecular mechanisms related indicators of liver cancer(1) The effect of Diwu Yanggan capsules on EMT related indicators: Western Blot detection of EMT-related markers showed: the protein expression of epithelial cell marker E-cadherin in model group(0.35±0.03) was significantly lower than the the normal group(1.16±0.10),the difference was statistically significant(p<0.05); the protein expression of E-cadherin in DWYG group(0.72±0.04) was higher than in the model group(0.35±0.03), the difference was statistically significant(p<0.05); the DWYG group(0.72±0.04) compared with the sorafenib group(0.62±0.07), the difference was not statistically significant(p>0.05); there was no statistical significance between the normal group(1.16±0.10) and BMSC group(1.22±0.09), p>0.05.And the protein expression of mesothelial cell marker Vimentin(1.52±0.06) and regulatory factor TGF-β(1.76±0.10) in model group was significantly higher than in the normal group(0.36±0.03; 0.43±0.04),the difference was statistically significant(p<0.05); the protein expressions of Vimentin(0.93±0.08) and TGF-β(0.93±0.05) in DWYG group were lower than in the model group(1.52 ± 0.06; 1.76 ± 0.10), the difference was statistically significant(p<0.05); the protein expression of Vimentin in DWYG group(0.93±0.08) compared with the sorafenib group(1.08±0.11), the difference was statistically significant(p<0.05); there was statistical significance in the protein expression of TGF- βbetween the normal group(0.43±0.04) and BMSC group(0.30±0.03), p<0.05.Real-time PCR results showed that: the m RNA expression of epithelial cell marker E-cadherin in the model group was significantly lower than the normal group; the expressions of E-cadherin in DWYG group was higher than in the model group, the difference was statistically significant(p<0.05). The m RNA expression of Vimentin in the model group was significantly higher than that in the normal group, the statistical difference was significant(p<0.05); And the m RNA expressions of Vimentin in DWYG group was lower than in the model group, the difference was statistically significant(p<0.05); there was no statistical significance between the normal group and BMSC group, p>0.05;there was no statistical significance between the normal group and BMSC group, p>0.05.(2) The effect of Diwu Yanggan capsules on Ras/Raf/Mek/Erk signaling pathway:Western Blot showed the protein expressions of Raf-1、Mek1、Erk1 and VEGF in model group(0.91±0.05; 1.81±0.10; 1.49±0.04; 2.31±0.15) were significantly higher than the normal group(0.40±0.05; 0.70±0.03; 0.55±0.11; 0.59±0.05), the difference was statistically significant(p<0.05); the protein expressions of Raf-1、Mek1、Erk1 and VEGF in DWYG group(0.79±0.02; 1.10±0.05; 0.95±0.09; 1.76±0.24) were lower than in the model group(0.91±0.05; 1.81±0.10; 1.49±0.04; 2.31±0.15), the difference was statistically significant(p<0.05); the protein expression of Mek1 in DWYG group(1.10±0.05) compared with the sorafenib group(1.25±0.05), the difference was statistically significant(p<0.05);the protein expressions of Raf-1、Erk1 and VEGF in the DWYG group(0.79±0.02; 0.95±0.09; 1.76±0.24) compared with the sorafenib group(0.77±0.05; 0.98±0.08; 1.66±0.13), the difference was not statistically significant(p > 0.05); there was no statistical significance between the normal group(0.40±0.05; 0.70±0.03; 0.55±0.11; 0.59±0.05) and BMSC group(0.36±0.04;0.72±0.06;0.53±0.12;0.77±0.07;)in the protein expression of Raf-1、Mek1、Erk1 and VEGF, p>0.05.Real-time PCR results showed that: the m RNA expressions of Raf-1、Mek1 and Erk1 in model group were significantly higher than the normal group, the difference was statistically significant(p<0.05); the m RNA expressions of Raf-1、Mek1 and Erk1 in DWYG group were lower than in the model group, the difference was statistically significant(p<0.05);the DWYG group compared with the sorafenib group, the difference was not statistically significant(p>0.05); there was no statistical significance between the normal group and BMSC group, p>0.05.(3) The effect of Diwu Yanggan capsules on JAK/STAT signaling pathway:Western Blot showed the protein expressions of JAK2, STAT3 andSTAT5 in model group(1.40±0.04; 1.27±0.09; 1.58±0.04) were significantly higher than the normal group(0.47±0.03; 0.44±0.06; 0.63±0.04),the difference was statistically significant(p<0.05); the protein expressions of JAK2, STAT3 and STAT5 in DWYG group(1.01±0.05; 0.85±0.03; 0.95±0.09) were lower than in the model group(1.40 ± 0.04; 1.27 ± 0.09; 1.58 ± 0.04), the difference was statistically significant(p<0.05);the protein expressions of JAK2 in the DWYG group(1.01±0.05) compared with the sorafenib group(1.12 ± 0.09), the difference was statistically significant(p<0.05); there was no statistical significance between the normal group(0.47±0.03; 0.44±0.06; 0.63±0.04) and BMSC group(0.49±0.04;0.40±0.09;0.62±0.05), p>0.05.Real-time PCR results showed that: the m RNA expressions of JAK2 and STAT3 in model group were significantly higher than the normal group, the difference was statistically significant(p<0.05); the m RNA expressions of JAK2 and STAT3 in DWYG group were lower than in the model group, the difference was statistically significant(p<0.05);the DWYG group compared with the sorafenib group, the difference the m RNA expressions of JAK2 was statistically significant(p<0.05); there was no statistical significance between the normal group and BMSC group, p>0.05.Conclusion1 Diwu Yanggan capsules can inhibit Solt-Farber rat model of liver cancer’s occurrence and development to a certain extent, and the effect of not less than sorafenib.2 “Marrow stem cells failing to transforming into liver cancer stem cells” are involved in the occurrence and development of liver cancer is the biological basis of “Suishishenggan”. On the contrary,“marrow stem cells transforming into liver cancer stem cells” are involved in the liver regeneration and repair is the biological basis of “Suishenggan”. Diwu Yanggan capsules had an effect on inhibiting “marrow stem cells failing to transforming into liver cancer stem cells” as well as promoting “marrow stem cells transforming into liver cancer stem cells”. The study had riched the scientific connotation of “marrow stem cells differentiate into liver cells ” as well as “ marrow stem cells fail to differentiate into liver cells”.3 Diwu Yanggan capsules reflect the treatment rule of “tonifying the kidney to regulate liver regeneration and repair by affecting stem cells and their microenvironment”, and it can promote the liver regeneration microenvironment to delay the process of liver cancer by the following molecular mechanisms:(1)via inhibiting “marrow stem cells failing to transforming into liver cancer stem cells”,and promoting “marrow stem cells transforming into liver cancer stem cells”.(2)via regulating the imbalance of EMT/MET.(3)via regulating the JAK/STAT signaling pathway.(4)via regulating the Ras/Raf/Mek/Erk signaling pathway. |
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