| Objective:To study the regulation of transarterial chemoembolization(TACE)sequential pirfenidone on the expression of autophagy-related proteins Beclin1,LC3 and P62 in rabbit VX2 liver cancer,and to explore its effect on the level of autophagy and tumor growth.Methods:(1)34 clean-grade New Zealand rabbits were selected to make rabbit VX2 animal models.Among them,32 rabbits showed a single tumor on the left lobe of the liver,whose size was within a predetermined range,which met the experimental requirements.The experimental tumor-bearing rabbits were divided into four groups A,B,C,and D by random number table method,with 8 rabbits in each group.Group A is a control group;Group B is a pure TACE group;C is a pure pirfenidone group;Group D is a TACE sequential pirfenidone group;(2)Group A is a blank control group,with no treatment for normal feeding In group B,the right femoral artery was punctured and the nerve micro-guide wire was introduced to the left hepatic artery to inject 2 mg/kg pirarubicin+0.2 ml/kg lipiodol mixed emulsion;group D was given pirfenidone dose 7 days after the operation by the same method Stomach administration 200 mg/kg/d.Group C was intragastrically administered with a dose of200 mg/kg/d pirfenidone dissolved in 0.2 ml/kg saline for 15 days after tumor implantation for 7 consecutive days.In the experimental rabbits,ultrasound and CT of the upper abdomen were performed 14 days after the tumor mass was implanted to check the liver tumor growth and lipiodol deposition.(3)7 days after the completion of the treatment,the experimental rabbits were anesthetized and killed,and the tumor tissue of the left lobe of the liver was cut out.HE staining was used to observe experimental rabbit liver cancer tissues and cell morphology;Western Blot was used to detect the expression of HIF-1α,autophagy-related proteins Beclin1,LC3 and P62 in liver cancer tissues.SPSS24.0 software was used for statistical analysis.The tumor volume and growth of experimental rabbits were expressed as mean ± standard deviation(x ± S).The growth and relative expression of detected protein were expressed by one-way analysis of variance,and each group was paired with each other.LSD-T test was used for comparison;P<0.05 was considered as statistically significant.Results:(1)After 14 days of implantation,ultrasound monitoring showed that the tumor in the left lobe of the liver of the experimental rabbits(n=32)showed a hypoechoic nodule with less clear edges and regular shapes.The spectral Doppler showed abundant short bands around and inside the tumor.The high-speed and low-impedance blood flow signal in the shape and point of the rod confirms the success of the vaccination.The tumor volume increase in group D was significantly less than that in groups A,B,and C,and the difference was statistically significant(t=-5.060,P<0.001;t=-2.444,P=0.021;t=-2.374,P=0.025).(2)The upper abdominal CT plain scan + enhanced 14 days after the tumor mass implantation indicated that the tumor showed a nodular density reduction shadow on the plain scan image,the boundary was not clear,and the shape was irregular.Arterial liver tumors showed significant ring enhancement.In the portal vein and the delayed phase,the tumor enhancement gradually weakens,and the boundary between the tumor and the surrounding liver parenchyma is clear.(3)Pathological HE staining showed a large number of tumor cells and a small amount of extruded sinusoid tissue in group A under light microscope.In group B,a small amount of necrosis was seen under the light microscope,the tissue structure disappeared,the interstitial fibrous tissue proliferated,and the nuclear chromatin was deep.In group C,a large number of tumor cells and a small amount of squeezed sinusoid tissue can be seen under the microscope.Under the microscope of group D,large areas of unstructured red-stained necrosis were seen,with scattered tumor cells and a little interstitial fibrous tissue.(4)Western Blot detection showed that the expression level of HIF-1α was significantly higher in groups B and D compared with group A(t=6.526,P<0.001;t=7.578,P<0.001);the expression level of P62 protein was at Compared with group A,the expressions of group B and group D were reduced respectively(t=-5.982,P<0.001;t=-2.872,P=0.021),group D was higher than group B(t=-3.109,P=0.014);The expression level of Beclin1 protein was higher in group B and D compared with group A(t=10.629,P < 0.001;t=6.078,P < 0.001),group D was lower than group B(t=4.552,P=0.002)).The expression level of LC3-II protein was higher in group B and D compared with group A(t=6.681,P<0.001;t=0.254,P=0.035),group D was lower than group B(t=-4.323,P =0.003).Conclusions:TACE can induce autophagy induced by hypoxic microenvironment.Compared with TACE alone or pirfenidone alone in treating rabbit VX2 liver cancer,TACE sequential pirfenidone has more significant tumor growth inhibition.Pirfenidone can inhibit autophagy levels. |
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