Effects of Periplaneta americana Extract on Autophagy and Invasive Metastasis of Subcutaneous Transplanted Hepatocellular Carcinoma in Nude Mice
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摘要:
目的 研究美洲大蠊提取物CII-3和脱脂膏对肝癌耐药细胞BEL-7402/5-FU自噬和侵袭转移的影响以及初步的作用机制。 方法 对接种肝癌敏感细胞BEL-7402和肝癌耐药细胞BEL-7402/5-FU的移植瘤裸鼠，通过索拉菲尼、CII-3和脱脂膏药物处理后，观察给药后裸鼠血清生化指标，裸鼠瘤块变化和心、肝、肾、脾等脏器指数的变化，以及肝脏和瘤块病理学改变，同时观察肿瘤组织细胞中的自噬相关因子和侵袭转移相关因子的变化。 结果 CII-3和脱脂膏能使裸鼠体重呈下降趋势，并抑制肿瘤的生长，明显减轻瘤重（P < 0.01）；CII-3能提高脾脏指数和心脏指数（P < 0.05）；与耐药组相比，CII-3组裸鼠血清CR有升高趋势（P < 0.05）；索拉菲尼和CII-3处理组能诱导肿瘤组织坏死，作用优于脱脂膏组；与耐药组相比，除AKT外（P > 0.05），CII-3和脱脂膏给药组均能明显抑制自噬和侵袭转移相关因子（P < 0.05）。 结论 美洲大蠊提取物CII-3和脱脂膏均能抑制肝癌耐药细胞的自噬和侵袭转移，同时具有较好的抑瘤作用。其对裸鼠肝癌移植瘤的肝肾损伤有待进一步观察。 Abstract:Objective To investigate the effects and preliminary mechanisms of the extract CII-3 and defatted ointment of Periplaneta americana on autophagy, invasion and metastasis of drug-resistant hepatocellular carcinoma cell line BEL-7402/5-FU. Methods The transplantation tumors of liver cancer-sensitive cells BEL-7402 and liver cancer-resistant cells BEL-7402/5-FU were treated with Sorafenib, CII-3, and defatted ointment drugs. Then, the serum biochemical indicators of the nude mice, changes in tumor size, and changes in organ indices such as heart, liver, kidney, and spleen were observed after drug administration. The histopathological changes in the liver and tumor were also observed, along with changes in autophagy-related factors and invasion and metastasis-related factors in tumor tissue cells. Results CII-3 and the deffated cream reduced the body weight of nude mice and inhibited tumor growth, significantly reducing tumor weight （P < 0.01）. CII-3 increased the spleen index and heart index （P < 0.05）; compared to the drug-resistant group, the CII-3 group showed an upward trend in serum CR （P < 0.05）. Sorafenib and CII-3 treatment induced tumor tissue necrosis, with better effects than the deffatede ointment group; compared to the drug-resistant group, both the CII-3 and deffatede ointment groups significantly inhibited autophagy and invasion-related factors, except for AKT （P < 0.05）. Conclusion Periplaneta americana extract CII-3 and deffatede ointment can inhibit the autophagy, invasion and metastasis of drug-resistant hepatocellular carcinoma cells, and also have a good anti-tumor effect. Further observation is needed for its liver and kidney damage on nude mice with hepatocellular carcinoma transplanted tumors. -
图 1 CII-3和脱脂膏对皮下移植瘤裸鼠体重、肿瘤体积及瘤重的影响
A：CII-3和脱脂膏对皮下移植瘤裸鼠体重的影响；B：CII-3和脱脂膏对皮下移植瘤裸鼠瘤体积的影响；C：CII-3和脱脂膏对皮下移植瘤裸鼠瘤重的影响。与敏感组相比较，*P < 0.05， **P < 0.01；与耐药组比较，#P < 0.05， ##P < 0.01；与索拉非尼组相比较，△P < 0.05，△△P < 0.01。
Figure 1. Effects of CII-3 and defatted ointment on body weight，tumor volume and tumor weight of nude mice with subcutaneously transplanted tumor
图 2 CII-3和脱脂膏对移植瘤裸鼠肝脏组织病理学的影响（HE染色）
Figure 2. Effects of CII-3 and defatted ointment on liver histopathology of nude mice with transplanted tumor （HE staining）
图 3 CII-3和脱脂膏对移植瘤裸鼠肿瘤组织病理学的影响（HE染色）
Figure 3. Effects of CII-3 and defatted ointment on tumor histopathology in nude mice with transplanted tumor （HE staining）
表 1 自噬相关因子引物序列表
Table 1. Primers sequence of autophagy related factors
名称 Forward Reverse 鼠ATG5 TGTGCTTCGAGATGTGTGGTT ACCAACGTCAAATAGCTGACTC ATG7 TGACCTTCGCGGACCTAAAGA CCCGGATTAGAGGGATGCTC 鼠BECLIN AGGCGAAACCAGGAGAGAC CCTCCCCGATCAGAGTGAA 鼠PIK3C3 AACAACCGTGTCGCTCTTTG GAACCATCTGCCTCCACGTTA 鼠LC3B GTCCTGGACAAGACCAAGTTCC CCATTCACCAGGAGGAAGAAGG 鼠P62 CCGGCTGATTGAGTCCCTC CCCCGATGTCGTAATTCTTGG GAPDH AGGTCGGTGTGAACGGATTTG TGTAGACCATGTAGTTGAGGTCA 
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表 2 侵袭转移相关因子引物序列表
Table 2. Primer sequence of invasion and metastasis related factors
名称 Forward Reverse 鼠MMP2 CAAGGATGGACTCCTGGCACAT TACTCGCCATCAGCGTTCCCAT 鼠MMP9 GCTGACTACGATAAGGACGGCA TAGTGGTGCAGGCAGAGTAGGA 鼠mTOR GGCACACATTTGAAGAAGCAG CTCGTTGAGGATCAGCAAGG 鼠AKT CCTTTATTGGCTACAAGGAACGG GAAGGTGCGCTCAATGACTG 鼠4EBP1 GGGGACTACAGCACCACTC GTTCCGACACTCCATCAGAAAT GAPDH TGGCCTTCGTGTTCCTAC GAGTTGTGTTGAAGTCGCA
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表 3 CII-3和脱脂膏对皮下移植瘤裸鼠脏器指数的影响（
$\bar x \pm s $ ，n = 12）Table 3. Effects of CII-3 and defatted ointment on organ index in nude mice with subcutaneously transplanted tumor （
$\bar x \pm s $ ，n = 12）组别 心脏指数（mg/g） 肝脏指数（mg/g） 脾脏指数（mg/g） 肺脏指数（mg/g） 肾脏指数（mg/g） 空白组 6.02 ± 0.80 56.73 ± 7.30 6.02 ± 0.80 9.03 ± 1.60 17.84 ± 2.10 敏感组 5.98 ± 0.90 60.50 ± 8.89 5.98 ± 0.90 9.12 ± 2.12 18.16 ± 1.17 耐药组 6.67 ± 1.55 63.74 ± 8.09 6.67 ± 1.55 9.68 ± 2.79 17.21 ± 3.17 索拉非尼组 6.67 ± 1.01 69.42 ± 8.81▲*# 6.67 ± 1.01 11.43 ± 3.52 17.45 ± 4.59 CII-3组 7.12 ± 1.55▲*# 58.14 ± 10.6△ 7.12 ± 1.55▲* 11.54 ± 2.97 18.06 ± 3.54 脱脂膏组 6.08 ± 0.71 52.67 ± 8.10#△ 6.08 ± 0.71 10.37 ± 3.14 20.85 ± 3.38△ F 3.45 9.31 3.45 3.31 3.53 P 0.021 < 0.01 0.021 0.025 0.019 与空白组比较，▲P < 0.05，▲▲P < 0.01；与敏感组比较，*P < 0.05，**P < 0.01；与耐药组比较，#P < 0.05，##P < 0.01；与索拉非尼组比较，△P < 0.05，△△P < 0.01。
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表 4 CII-3和脱脂膏对皮下移植瘤裸鼠生化指标的影响（
$\bar x \pm s $ ，n = 12）Table 4. Effects of CII-3 and defatted ointment on biochemical indexes of nude mice with subcutaneously transplanted tumor （
$\bar x \pm s $ ，n = 12）组别 AST（U/L） ALT（U/L） CR（μmol/L） BUN（mmol/L） 空白组 481.22 ± 62.98 13.99 ± 2.00 19.61 ± 2.75 5.34 ± 0.41 敏感组 660.91 ± 210.46▲ 12.27 ± 0.53 13.95 ± 4.25▲ 3.90 ± 0.51 耐药组 500.51 ± 83.87* 21.91 ± 5.26▲** 14.93 ± 2.35 4.88 ± 0.97 索拉非尼组 498.53 ± 140.13* 8.42 ± 2.34▲## 22.66 ± 4.13*# 4.83 ± 1.80 CII-3组 656.83 ± 106.84▲#△ 17.93 ± 1.88**△ 24.88 ± 4.59▲*# 3.33 ± 0.54▲#△ 脱脂膏组 491.06 ± 62.76# 12.81 ± 1.83# 19.92 ± 7.61* 4.64 ± 1.13 F 10.13 62.34 17.62 10.83 P < 0.01 <0.01 <0.01 <0.01 与空白组比较，▲P < 0.05，▲▲P < 0.01；与敏感组比较，*P < 0.05，**P < 0.01；与耐药组比较，#P< 0.05，##P < 0.01；与索拉非尼组比较，△P < 0.05，△△P < 0.01。
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表 5 CII-3和脱脂膏对移植瘤裸鼠自噬相关因子mRNA的影响 （
$\bar x \pm s $ ，n = 12）Table 5. Effects of CII-3 and defatted ointment on autophagy related factor mRNA in nude mice with transplanted tumor （
$\bar x \pm s $ ，n = 12）组别 ATG5 ATG7 BECLIN LC3B PIK3C3 P62 敏感组 0.08 ± 0.01 0.04 ± 0.01 0.05 ± 0.03 0.03 ± 0.02 0.03 ± 0.00 0.04 ± 0.01 耐药组 1.00 ± 0.00** 1.00 ± 0.00** 1.00 ± 0.00** 1.00 ± 0.00** 1.00 ± 0.00** 1.00 ± 0.00** 索拉菲尼组 0.30 ± 0.14*# 0.03 ± 0.00## 0.02 ± 0.01## 0.16 ± 0.04**## 0.10 ± 0.02**## 0.1 ± 0.01**## CII-3组 0.16 ± 0.05## 0.05 ± 0.02## 0.12 ± 0.05*##△ 0.13 ± 0.06**## 0.04 ± 0.01##△ 0.08 ± 0.02*## 脱脂膏组 0.27 ± 0.12*## 0.07 ± 0.02## 0.12 ± 0.06*##△ 0.33 ± 0.01**##△△ 0.14 ± 0.03**##△ 0.24 ± 0.06**##△△ F 300.26 16442.05 1974.64 2171.41 10020.07 3148.02 P < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 与敏感组比较，*P < 0.05，**P < 0.01；与耐药组比较，#P < 0.05，##P < 0.01；与索拉非尼组比较，△P < 0.05，△△P < 0.01。
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表 6 CII-3和脱脂膏对移植瘤裸鼠侵袭转移相关因子mRNA的影响（
$\bar x \pm s $ ，n = 12）Table 6. Effects of CII-3 and defatted ointment on the mRNA of invasion and metastasis related factors in nude mice with transplanted tumor （
$\bar x \pm s $ ，n = 12）组别 MMP2 MMP9 mTOR AKT 4EBP1 敏感组 0.01 ± 0.00 0.01 ± 0.02 0.04 ± 0.01 0.02 ± 0.01 0.23 ± 0.08 耐药组 1.00 ± 0.00** 1.00 ± 0.00* 1.00 ± 0.00** 1.00 ± 0.00 1.00 ± 0.00** 索拉非尼组 0.02 ± 0.01**## 0.18 ± 0.08# 0.04 ± 0.01## 0.11 ± 0.06 1.80 ± 0.30**## CII-3组 0.01 ± 0.01## 0.00 ± 0.00# 0.08 ± 0.02## 0.17 ± 0.10 0.15 ± 0.06# 脱脂膏组 0.04 ± 0.02## 0.03 ± 0.01# 0.15 ± 0.30**##△ 0.20 ± 0.32 0.33 ± 0.15# F 26096.68 2171.29 154.83 156.90 325.86 P < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 与敏感组比较，*P < 0.05，**P < 0.01；与耐药组比较，#P < 0.05，##P < 0.01；与索拉非尼组比较，△P < 0.05，△△P < 0.01。
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