Study On The Induction Of Anti-tumor In Immune Cells By Aqueous Extract Of Pu-Erh Ripe Tea Through Modulating The Expression Of HSPA1A

In recent years,the prevalence of cancer has been increasing year by year and has become an important factor threatening human life health.Among the traditional treatments for cancer,surgery,radiotherapy and chemotherapy still dominate.However,these therapies are costly,prone to recurrence and have many side effects.In our laboratory,we previously examined the expression of several genes in human peripheral blood leukemia T cells(Jurkat)using gene chip technology while studying the function of Pu-Erh tea,and found that HSPA1 A was upregulated more than 14-fold.Therefore,based on this,this paper will study the specific mechanism of enhancing anti-tumor immune response through HSPA1 A by extracting the active ingredients of ripe Pu-Erh tea,and lay the theoretical foundation for the development and utilization of Pu-Erh tea.In this paper,we used Western-blotting and q PCR experiments to detect the expression levels of HSPA1 A in gastric cancer cells(SGC7901)and macrophages(RAW 264.7)under the effect of ripe tea aqueous extract of Pu-Erh tea at protein and m RNA levels,and the results showed that HSPA1 A was significantly up-regulated,which was consistent with the basis of our previous experiments.After that,the HSPA1 A promoter was cloned into p GL3-Basic vector and a luciferase expression plasmid containing HSPA1 A promoter was constructed to further verify the regulation of HSPA1 A in cells by Pu-Erh tea,and the results further proved the above conclusion.On this basis,the HSPA1 A promoter-regulated luciferase expression plasmid was used to transfect gastric cancer cells and treated with different concentrations of aqueous extracts of Pu-Erh tea at the same time.The results showed that the optimal treatment conditions were when the concentration of Pu-Erh tea extract was 220 ?g /m L and the action time was 12 h.To screen out the core functional region of HSPA1 A promoter,we adjust the length of HSPA1 A promoter sequence and constructed HSPA1 A plasmids containing different length promoters.p HSPA1A-Luc4(total length 527 bp,which contains-771 nucleotides to +214 nucleotides(relative to the HSPA1 A transcription start site,The TSS sequence was obtained from EPD(https://epd.epfl.ch/))was found by luciferase assay.Corresponding to the promoter is the core promoter region of HSPA1 A.In order to detect the levels of inflammatory factors and key factors in the signaling pathway related to HSPA1 A in macrophages,the supernatant of mouse breast cancer cells(4T1)after the action of Pu-Erh tea was added to RAW 264.7 cells in this paper.q PCR revealed that Pu-Erh tea extracts could significantly up-regulate the expression of pro-inflammatory factors IL1?,IL1?,IL6 and IL12 in macrophages,and inhibit the level of the anti-inflammatory factor IL13;after suppressing HSPA1 A,IL1?,IL1?,IL6 and IL12 were down-regulated and IL13 was up-regulated.The changes in the expression levels of these immune factors indicated that HSPA1 A has an important role in the promotion of immune cell activation and its anti-tumor immune response by Pu-Erh tea.On the other hand,we found that Pu-Erh tea significantly inhibited the expression of JNK2 and p38 and had a significant inhibitory effect on TLR4-mediated My D88/NF-?B signaling pathway,which further improved the anti-apoptotic ability of macrophages and enhanced their immune protective effects.Finally,this paper examined the HSPA1 A binding on the surface of macrophages after the effect of PuErh tea by flow cytometry using HSPA1 A antibody labeled with FITC,and the results showed that the HSPA1 A binding on the surface of macrophages was significantly increased after tumor supernatant stimulation.Although Pu-Erh tea extracts reduced this binding to some extent,it was still significantly higher than the normal level overall.This phenomenon may be related to the inhibition of TLR4-mediated My D88/NF-?B signaling pathway on the one hand,and the antioxidant activity of Pu-Erh tea extracts and the acidification of lysosomes on the other.In conclusion,the research in this paper further reveals the detailed mechanism by which ripe tea extracts of Pu-Erh tea enhance the anti-tumor immune response by regulating the expression of HSPA1 A,which lays a certain research foundation for the development and utilization of Pu-Erh tea.