| Primary liver cancer（PLC）is one of the common malignant tumors in China.It has a high incidence,high mortality rate,high metastasis and high recurrence.It is difficult to treat,has a poor prognosis.The chronic inflammation and the inactivation of tumor suppressor genes is an important factor leading to the development of liver cancer.Recent studies have found that some tumor suppressor genes can also be expressed in immune cells.This led to further thinking:whether it can screen out some genes that have both anti-inflammatory and anti-cancer functions,and affect the development of cancer,especially inflammation-related cancer,by targeting the same signaling pathway molecules in immune cells and tumor cells.Clinical studies have found that low levels of serine/threonine-protein kinase 4（STK4）are negatively correlated with high levels of IL-1 receptor associated kinase 1（IRAK1）in peripheral blood of patients with liver cancer,and tumor tissue have a large amount of macrophage infiltration.IRAK1 inhibitors can significantly reduce the development of chronic inflammation and liver cancer due to STK4 deficiency.Therefore,STK4/IRAK1 can be used as a new target to block the development of inflammatory liver cancer.This paper mainly studies whether saikosapoins（SS）-b2 inhibits the occurrence and development of primary liver cancer through STK4/IRAK1/nuclear factor-κB（NF-κB）pathway.Chapter 1 Inhibition of saikosaponin-b2 on DEN-induced primary liver cancer in miceObjectiveThe aim of this study was to investigate the effect of saikosapoins（SS）-b2 on the development of Primary liver cancer（PLC）,and its regulation on STK4/IRAK1/NF-κB signaling pathway.MethodsDEN-induced mouse primary liver cancer model was established in healthy male BALB/c mice,and randomly divided into control group（NS）,model group(DEN 50mg·kg-1),SS-b2 groups(DEN+SS-b2 1.5 mg·kg-1,3 mg·kg-1 and 6 mg·kg-1)and doxorubicin group(DEN+DOX 1 mg·kg-1).Morphological changes of liver tissue were observed in mice at 4,12,and 19 weeks after DEN induction.The levels of AFP,AST,ALT and LDH in serum of liver cancer mice were detected at the end of the 19th week.The liver,spleen,lung and thymus were excised and weighed.Ki67 staining was used to observe the effect of SS-b2 on the proliferation of hepatocarcinoma cells in mice.The mRNA expression levels of STK4 and IRAK1 in peripheral blood macrophages of liver cancer mice were detected by qPCR.The mRNA expression levels of IL-1β,IL-6 and TNF-αin liver tissues of liver cancer mice were detected by qPCR.Western blot and immunohistochemistry were used to detect the protein expression of STK4,IRAK1 and NF-κB in liver tissue.Results1.In the 4th week of DEN-induced mice,the serum aminotransferase levels of AST,ALT increased significantly in the model group.HE staining showed that the normal structure of the mice in the model group was destroyed,the boundary of the lobule was unclear,the hepatic cord disorder was disordered,the hepatocytes were swollen,the nucleus was dissolved and vacuolated,and the symptoms of liver injury were obvious.In the 12th week of DEN-induced mice,the liver surface of the mice had granular hyperplasia,and the interstitial mass of collagen fibers proliferated,showed obvious liver fibrosis lesions in the model group.In the 19th week of DEN-induced mice,most of the liver surface of the mice were distributed with gray nodules of different sizes in the model group,the typical columnar and ductal carcinoma nests were found in the liver tissue,and the liver cancer cells were arranged in a cord or mass and infiltrated into the surrounding liver tissue,with a large infiltration of inflammatory cells.SS-b2 could significantly improved the liver fibrosis at 12 weeks,and could reduced the number and size of cancer nests in the liver of mice for 19weeks,showed obvious anti-cancer effect.2.SS-b2 could significantly reduced the levels of AST,ALT and LDH in the serum of 19-week liver cancer mice,and significantly reduced the liver index,which had a certain protective effect on liver function of liver cancer mice.3.At the 19th week,the level of AFP in the serum was significantly decreased of SS-b2 treatment group,and the immunohistochemistry results showed that SS-b2could significantly reduce the positive expression of Ki67 in liver tissue,and had obvious anticancer activity.4.The results of qPCR showed that the mRNA expression levels of STK4 was significantly decreased,and the mRNA expression levels of IRAK1 was significantly increased in peripheral blood macrophages of mice in the 19-week liver cancer model group.Western blot and immunohistochemistry showed that the protein levels of STK4 was down-regulated in liver tissue of model group,and the protein levels of IRAK1 was highly expressed.SS-b2 could increased the expression level of STK4mRNA and protein,decreased the expression level of IRAK1 mRNA and protein.5.The expression of NF-κB protein and the expression of IL-1β,IL-6 and TNF-αmRNA in liver tissues of 19-week liver cancer mice were significantly increased,and SS-b2 could significantly reduce the expression of NF-κB protein and the mRNA levels of the above cytokines.ConclusionSS-b2 could inhibited the development of primary liver cancer,and its mechanism may be related to its regulation of STK4/IRAK1/NF-κB pathway.Chapter 2 Regulatory effect of saikosaponin-b2 on STK4/IRAK1/NF-κB pathway in LPS-stimulated RAW 264.7 macrophagesObjectiveTo study the regulation of SS-b2 on the STK4/IRAK1/NF-κB pathway in LPS-stimulated RAW 264.7 macrophages,and further elucidate the molecular mechanism of SS-b2 affecting the development of primary liver cancer.MethodsMTT assay and Griess assay were used to detect the cell viability and NO secretion of RAW 264.7 macrophages stimulated by different doses of LPS(0、0.125、0.25、0.5、1、2、4、8、16μg·mL-1),SS-b2(0、2.5、5、10、20、40、80、160、200μg·mL-1)and Dexamethasone(DEX,0、0.1、0.2、0.4、0.8、1.6μg·mL-1)for 24 h,and the LPS modeling conditions,SS-b2 and DEX doses were screened.qPCR was used to detect the effect of SS-b2 on the expression of IL-1β,IL-6and TNF-αmRNA in LPS-stimulated macrophages.The expression of STK4,IRAK1and NF-κB in macrophages was detected by Western blot.Results1.The results of MTT and Griess showed that 1μg·mL-11 LPS stimulation was the optimal dose to induce RAW 264.7 macrophage inflammation.And the therapeutic doses of SS-b2(15,30 and 60μg·m L-1)and DEX(1μg·mL-1)were determined.2.SS-b2 could significouldtly reduced the expression levels of inflammatory cytokines IL-1β,IL-6 and TNF-αmRNA in RAW 264.7 macrophages.3.Western blot analysis showed that SS-b2 significouldtly increased the protein expression levels of STK4 and decreased the expression levels of IRAK1 and NF-κB.ConclusionSS-b2 could regulated the STK4/IRAK1/NF-κB pathway in RAW 264.7macrophages,inhibited the expression of transcription cytokines by LPS-stimulated,further played the role of treating primary liver cancer. |
