The Mechanism Of The Berberine Attenuates Irinotecan-induced The Injury Of Intestinal Immunity

Background: In the immune system, intestinal is believed to be the biggest immuneorgan because it has a large number of immune cells and the largest surface contactedwith antigen. The area of intestinal mucosa is large and constantly facing a high load ofantigens. And approximately70%of the immunocyte are present in the gut and arecontinuously discriminating between harmless and pathogenic antigens. They are thefirst line of defense pathogenic and the function of them is not only in the intestinalmucosa, but also involved in the systemic immunity.In an effort to control cancer and sustain remission, adjuvant chemotherapy havebeen used to eliminate microscopic tumor and extend life expectancy in patients.However, parts of the chemotherapy agents caused serious damage to the intestinalmucosa leading to the diarrhea which is the dose limiting toxicity of the agents, and oneof the most representative drug among them is irinotecan (CPT-11). Irinotecan (CPT-11)is a semi synthetic derivative of camptothecin. CPT-11can bind to thetopoisomeraseⅠ-DNA complex which prevent relegation of the DNA strand. This leadsto double-strand DNA breakage and cell death that is the primary mechanism ofantitumour activity of CPT-11. CPT-11is one of the most commonly usedchemotherapy agents in metastatic colorectal cancer and also has certain effect on lungcancer, breast cancer, gastric cancer, ovarian cancer and so on. The common side effects of CPT-11are late diarrhea, neutropenia, acute cholinergic syndrome, nausea andvomiting and others: such as hair loss, fever and so on. Among them the late diarrhea isthe main reason of dose limiting toxicity of CPT-11. The late diarrhea often occurs in24hours after CPT-11administration, the incidence is about60-87%. According to thetoxicity of NCI standard, the severe diarrhea of grade3/4account for about20-39%.However, the mechanism of irinotecan induced diarrhea is still unclear, manyscholars believed that irinotecan is to coverted by hepatic or gastrointestinalcarboxylesterases to its active metabolite7-ethyl-10-hydroxycamptothecin (SN-38).SN-38is further processed by glucuronyltransferase to become SN-38glucuronide(SN-38G), a less toxic form of SN-38. However, SN-38G is transported to the intestineduring bile excretion, and is able to be hydrolysed by β-glucuronide to return to thetoxic form, SN-38. SN-38is responsible for the damage to the intestinal mucosa,characterized by stubby and bare of villi, necrosis of intestinal epithelial cell,destruction of normal mucosal architecture and also inflammatory cellular infiltrationwere noted which caused chemotactic of macrophage and synthesis of cyclooxygenase2(COX-2) and prostaglandin E2(PGE2). These caused the inflammation and ulcer inintestinal, leading to the decrease of absorption in intestine and diarrhea. In addition, thePGE2drived by COX-2also can induce the expression of indoleamine2,3-dioxygenase(IDO). IDO is a heme monomer protein, mainly secreted by the placental trophoblastcells, peripheral blood monocytes/macrophages and dendritic cells. Some reasearchershave showed that IDO is the downstream of PGE2, and both IDO and PGE2are relatedto the progress and poor prognosis of colorectal cancer. The mechanism is that IDO candegrade the lubricious ammonia acid which is required for the T cells activated andsuppress the proliferation of T cells, including apoptosis of them and reduce the produceof IL-12. These inhibit the Th1type of anti-tumor immune response and increased theexpression of regulatory T cells (Treg) and suppressive cytokines of IL-10. Theproduction of IL-10can promote the Th cells shift to Th2, driving Th2type immunityand weakened Th1type of anti-tumor immunity which formed the immunosuppressivemicroenvironment. The antitumor immunity is suppressed in this immunosuppressive microenvironment, which leads to the immunologic escape of tumor. So if the injury ofintestine induced by SN-38is not protected, the immunosuppressive microenvironmentis formed and destroy the intestinal immune system which weaken the ability ofanti-tumor, promoting the metastasis and recurrence of tumor. Thus, to seek methods ofprotecting intestinal mucosa from the damage of SN-38, inhibiting the proinflammatoryand immunosuppressive cytokines, regulating the immunity microenvironment andrepairing the intestinal immune system is vital to prevent metastasis and recurrence oftumor.Berberine an isoquinoline alkaloid, which is extract from rhizoma coptis is presentin several plants, such as Hydrastis canadensis (goldenseal), Berberis aquifolium(Oregon grape), and berberis vulgaris (barberry). The berberine is one of the mostcommonly used herbal medicines for the treatment of bacteria-associated diarrhea,intestinal parasitic infections and ocular trachoma infections for several decades.Several studies revealed that berberine had anti-inflammatory activity and inhibited theproduce of proinflammatory and immunosuppressive cytokines, such as TNF-α, IL-1β,IL-6and PGE2. At the same time, the berberine also inhibited the β-glucuronide whichproduce SN-38in intestine. Thus based on the mechanism above, we deduced berberinemay not only reduce the diarrhea and the injury of intestine induced by CPT-11, but alsoinhibit the Th2type immunity and the formation of immunosuppressivemicroenvironment, promoting the Th1type anti-tumor immunity and improving theanti-tumor immune of T cells which prevent the recurrence and metastasis of tumor.Objective:1. To establish the diarrhea model induced by CPT-11and detect thechanges of pathology in intestinal.2. To investigate whether the intestinal immune system is damaged bythe injury of intestinal induced by CPT-11and then affect the systemic immunity,leading to the recurrence and metastasis of tumor.3. To verification whether berberine alleviate the injury of intestinal immunity induced by CPT-11and explore it’s molecular mechanismMethods:1. We establish the diarrhea model induced by CPT-11of subcutaneousimplanted tumor mice and assess the injury of intestinal by score of diarrhea and HEstaining.2. The effect on immune system by the injury of intestinal induced byCPT-11:①Enzyme-linked immunosorbent assay(ELISA) was used to deteced theexpression of TNF-α, IL-1β, IL-6, PGE2and IL-10in the tissue extracts and blood.②Western blot was used to deteced the expression of IDO in intestine.③Flow cytometrywas used to deteced Treg in Peyer’s Patches and blood.3. Berberine alleviate the injury of intestinal immunity induced byCPT-11:①Assess the injury of intestinal by score of diarrhea and HE staining.②Enzyme-linked immunosorbent assay(ELISA) was used to deteced the expression ofTNF-α, IL-1β, IL-6, PGE2and IL-10in the tissue extracts and blood.③Western blotwas used to deteced the expression of IDO in intestine.④Flow cytometry was used todeteced Treg in Peyer’s Patches and blood.Results:1. The optimal dose of CPT-11induced diarrhea was100mg/kg/day, for3consecutive days, the incidence of onset diarrhea was72%, mortality was40%, andcompared to the control group, the CPT-11treatment group caused severe damage ofmucosal surface in intestine, characterized by stubby and bare of villi, necrosis ofepithelial cells in intestine, destruction of normal mucosal architecture andinflammatory cellular infiltration.2. The expression of proinflammatory cytokines such as TNF-α, IL-6, IL-1βof CPT-11treatment group were increased and Th1type anti-tumor immune cytokineIL-12was decreased. The expression of immunosuppression cytokines including PGE2,IL-10and IDO of CPT-11treatment group were all increased. In the CPT-11treatmentgroup, Treg was increased in the Peyer’s Patches and blood. 3. Compared to the CPT-11treatment group, the berberine+CPT-11treatment group showed less damage of epithelial cells and intestinal glands. Theinflammatory cellular infiltration of berberine+CPT-11treatment group was alsodecreased and diarrhea was alleviated. In berberine+CPT-11treatment group, theexpression of TNF-α, IL-6, IL-1β were decreased and along with the IL-12increased,the immunosuppression cytokines PGE2, IL-10and IDO were all decreased. The Tregwas decreased in the Peyer’s Patches and blood of the berberine+CPT-11treatmentgroup.Conclusions：1. The injury of intestinal induced by CPT-11increased the intestinalinflammatory cytokines and immunosuppressive cytokines, forming theimmunosuppressive microenvironment.2. The immunosuppressive microenvironment may reduce theantitumor immunity and then affect the systemic immunity, leading to the recurrenceand metastasis of tumor.3. Berberine alleviate the injury of intestinal immunity induced byCPT-11and inhibit the formation of immunosuppressive microenvironment, increasingthe antitumor immunity. Berberine is not only reduced the toxicity of chemotherapydrug in intestine, but also prevented the recurrence and metastasis of tumor.