Effect Of Lianhua Decoction On TLR4 / MyD88 Signal Transduction Pathway In Ishikawa Cells Of Human Endometrial Carcinoma Co - Cultured With TAMs

[Objective]Tumor-associated macrophages (TAMs) are a type of immune inflammatory cells, which are infiltrating within the tumor tissues, and play a decisive role in tumorigenesis and progression. The researchers have found that a large number of TAMs infiltrating in the organization of endometrial carcinoma (EC), which were associated with poor prognosis in patients. As the advantage of traditional Chinese medicine (TCM) anti-tumor prominent, anti-tumor Chinese medicine gradually become a hot research topic. However, the research evidence of TCM compound effective "groups" and monomer compounds directly targets on TAMs was few, and the role of TAMs in EC tumorigenesis and progression was also not clear, as well as the molecular mechanism of EC. After years of clinical experience in the treatment of EC, professor Guo Zhiqiang, a famous TCM Gynaecology doctor in Dongzhimen hospital, Beijing university of Chinese medicine, had summarized a effective prescription-Lianhua decotion. On the basis of the prescription or by adding or subtracting some herbal medicines, the clinical effect is remarkable. Firstly, this study used immunohistochemical method to observe TAMs existence and distribution characteristics in pathological tissues of endometrial hyperplasia and EC patients, and discussed the correlation between TAMs and EC progression. Then we induced in vitro TAMs activation model to clear the influence of TAMs on the proliferation of human EC Ishikawa cells. Finally we mainly by research of extracted TCM prescription-Lianhua decotion compound effective "groups" and monomer compounds, which may have anti-tumor effects, with TAMs co-cultuled with Ishikawa cells, to screen for Lianhua decotion compound effective "groups" and monomer compounds inhibition of TAMs, to preliminary investigate traditional Chinese medicine in vitro resistance function and molecular mechanism of EC, and to provide clinical and experimental evidence study of TAMs targeting on anti-EC traditional Chinese medicine or TCM monomer compounds in the future.[Method]1 Using immunohistochemical method to detecte the distribution characteristics ofCD163, CD206 and distribution of NF-κB p65 expression in paraffin section of clinical endometrial hyperplasia and EC patients.2 The TAMs activation model using human EC cells Ishikawa and U937 human macrophages in vitro co-cultured system was induced; the model established or not was tested by Flow cytometry detecting the expressions of CD163 and CD206 phenotype molecules; the proliferation effect of TAMs on Ishikawa cells was observed by MTT method.3 MTT assay was applied to observed evaluate the proliferation effect of TAMs on Ishikawa cells. Ishikawa cells were cultured with different concentrations of Lianhua decotion compound effective "groups" and monomer compounds and harvested for 24 and 48hours by measuring OD value to screen the optimal concentration and action time.4 The expressions of CD163 and CD206 molecular phenotype were detected by Flow cytometry; the expression of IL-10 in supernatant fluid was detected by Elisa and the expression of NF-κB p65 was detected by Western Blot. Meanwhile, the expressions of TLR4 /MyD88 signal transduction pathways was detected by real-time RT-PCR[Result]1 CD163 and CD206 expressed in the cytoplasm in different levels of endometrial hyperplasia tissues, and were scattered flakes or spotty distribution in EC tissue nests and stromal tissues. As the endometrial simple hyperplasia (ESH), endometrial complex hyperplasia (ECH), endometrial atypical hyperplasia (EAH) and EC disease progress, the expressions of CD163 and CD206 tended to increase (p<0.05).2 The expressions ofCD163 and CD206 in EC tissues with≥ 1/2 muscular infiltration were both higher than the group that<1/2 muscle layer infiltration (p<0.05). EC patients with high blood pressure or with the proportion of obesity disease at the same time were slightly higher than those with EH (p<0.05). However, the proportion of different degrees of EH with the two diseases had no obvious difference (p>0.05).3 As EC progressing, the expression of NF-κB p65 showed a trend of increasing, the difference was statistically significant (p<0.05). The expression of NF-κB p65 in the organization of EC Ⅰ was lower than Ⅱ period tissues (p< 0.05). The expression of NF-κB p65 in EC tissues with>1/2 muscular infiltration was higher than the group that<1/2 muscle layer infiltration (p<0.05).4 Compared with U937 blank group, the expressions of CD163 and CD206 in TAMs group were higher(p<0.05). After TAMs and Ishikawa cells were co-cultured and separately Ishikawa cells cultured 24h and 48hours, the proliferation rate of co-cultured was higer than the Ishikawa group, the differences were statistically significant (p<0.05).5 After different concentrations of Lianhua decoction flavonoids, hedyotis diffusa flavonoids (HDF), and scutellaria barbata flavonoid (SBF) compared with that of the untreated cells interventing on Ishikawa cells 24 and 48hours, the inhibition rates within a certain range were showing a dose-dependent tention. Proliferation of Ishikawa cells was obviously suppressed by Lianhua decoction flavonoids HDF and SBF at the concentrations of 800μg/ml,800μg/ml and 400 μg/ml respectively for 24 hours (P<0.001). However, after different concentrations of Lianhua decoction polysaccharide, hedyotis diffusa polysaccharides (HDP), scutellaria barbata polysaccharides (SBP) and astragalus polysaccharide (AS) interventing on Ishikawa cells 24h and 48 hours, the inhibition rates were less than 15% or even negative (P>0.05).6 The expressions of CD206, CD163 and IL-10 in TAMs control group were significantly higer than U937 blank group (p<0.05).However, the expressions ofCD206, CD163 and IL-10 in TAMs control group were lower than Lianhua decoction flavonoids, HDF and SBF groups(p<0.05). Besides, compared with other two groups, the expressions of CD206, CD163 and IL-10 in Lianhua decoction flavonoids decreased significantly (p<0.05).7 The expression of NF-κB p65 in TAMs control group was significantly higer than U937 blank group, the difference was statistically significantly (p<0.05). Lianhua decoction flavonoids, HDF and SBF groups can significantly decrease the expression of NF-κB p65 protein(p<0.05). Lianhua decoction flavonoids group took down the biggest, and the HDF group followed (p<0.05).8 The expressions of TLR4 and MyD88 in TAMs control group all were significantly higer than U937 blank group (p<0.05), but were lower than Lianhua decoction flavonoids, HDF and SBF groups, the differences were statistically significant (p<0.05). The downregulation degree of TLR4/MyD88 in Lianhua decoction flavonoids group spented more than HDF and SBF groups, the difference was statistically significant (P<0.05).[Conclusion]1 The degree of TAMs infiltrating in endometrial tissues gradually increases along with EC progress. TAMs may be can promote the progression of EC.2 Successfully established the tumor-associated macrophages model in vitro TAMs and Ishikawa cells were co-cultured. TAMs may be can promote the proliferation of human endometrial carcinoma Ishikawa cells.3 Lianhua decoction flavonoids, HDF and SBF playing the role of inhibiting TAMs activation may be through by blocking TLR4/MyD88 signal transduction pathway, which has the effect of resistance to EC.