The Inhibiting Effect And Mechanism Of Extracts Of The Oleogum Resin From Boswellia Serrate (BSE) On The Hepatic Granuloma And Fibrosis Of Mice Infected With Schistosoma Japonicum

Schistosomiasis remains a serious public health problem in China.The primarycause of death in schistosomiasis is formation of liver egg granulomas and hepaticfibrosis, which cannot be cured by chemotherapy. Hepatic egg granulomas and fibrosisare main pathological manifestation caused by inflammation. Extracts of the oleogum resinfrom Boswellia serrate (BSE) have anti-inflammatory and immunomodulatory activity.BSE has been used in traditional medicine for the treatment of various inflammatorydiseases. Studies provide evidence for clinical therapeutic efficacy of BSE, especially inrheumatoid arthritis and chronic inflammatory bowel disease, and the side effect is light.The new studies have shown that BSE regulate NF-κB signal transduction throughinhibiting inhibitor of nuclear factor kappa-B kinase (IKK) activity. Rencently, some studies demonstrated that boswellic acids extract attenuated pulmonary fibrosis andcolonic fibrosis. Thus, the studies described above suggest that BSE may be valuable inthe fiborsis treatments of clinical diverse diseases. However, little is known regardingthe role of boswellic acids in S. japonicum egg-induced liver granuloma and fibrosis, wewill investigate the effect and its mechanism of a watersoluble complex withcyclodextrin (CD) preparation of BSE, BSE-CD, and our results will provide theoreticaland practical supportion for controling schistosomal hepatic fibrosis. Our study includedthe following two parts: Fist, the role and mechanism of the oleogum resin fromBoswellia serrate on the hepatic granuloma of mice infected with Schistosomajaponicum; secondly, the role and mechanism of the oleogum resin from Boswelliaserrate on the fibrosis of mice infected with Schistosoma japonicum.The inhibiting effect and mechanism of extracts of the oleogum resin fromBoswellia serrate on the hepatic granuloma of mice infected with SchistosomajaponicumWe established mice model, C57BL/6mice were infected with S. japonicumcercariae to form liver granulom. The animals were divided into5groups: BSE-CD(280mg/kg), BSE-CD (140mg/kg), cyclodextrin (280mg/kg), infected mice and normalmice. Mice were injected intraperitoneally with BSE-CD (280mg/kg) or BSE-CD (140mg/kg) every day for3weeks starting at28days after infection, respectively. Controlmice were given intraperitoneally injections of cyclodextrin (280mg/kg) alone everyday for3weeks starting at28days after infection. Another set of mice did not receiveany treatment. Mice were sacrificed at24h after the last injection. The effect ongranulomatous inflammation and liver injury were detected after being treatment ofBSE. The effects of BSE-CD on the IKK-NF-κB signal transduction in liver tissue weredetected by immunohistochemistry and EMSA assay, the effects of BSE-CD on thecytokine in liver tissue were analyzed by RT-qPCR, at last, we used western blot,RT-qPCR and ELISA to investigate expression level of MMP-9, LTB4and PGE2. Our results showed that BSE-CD (280mg/kg) treatment significantly improvedhepatic gross appearance. Morphometric analysis showed that the average granulomasize in BSE-CD treatment treated mice reduced significantly compared with the infectedmice and cyclodextrin (280mg/kg) mice (P <0.05). Accordingly, BSE-CD treatmentmice displayed attenuated liver injury, as indicated by a considerable decrease in serumaspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels (P <0.05).We analyzed egg burden in the individual livers, no relevant differences could beobserved between the four groups (P>0.05). Our results showed that phospho-IKKstaining intensities in hepatic granulomna from BSE-CD (280mg/kg)-treated animalswere clearly reduced in comparison to the control mice (P <0.05). EMSA data showedthat BSE-CD (280mg/kg) treatment suppressed NF-κB signal transduction in livertissue (P <0.05). Furthermore, MCP-1, TNF-α and VEGF expression which dependenton NF-κB of liver in BSE-CD (280mg/kg)-treated mice were decreased in real timePCR assay (P <0.05), and no difference in the expression of IL-1, IL-6, IL-13andTGF-β in the livers of between the four groups mice. On the other hand, ELISA andwestern blot data showed early BSE-CD (280mg/kg) and BSE-CD (140mg/kg)treatment dramatically decreased expression of MMP-9(P <0.05), and ELISA datashowed early BSE-CD (280mg/kg) treatment dramatically decreased expression ofMMP-9, LTB4and PGE2(P <0.05).In conclusion, treatment with BSE-CD significantly reduced liver granuloma size,at the same time, we found NF-κB system was inhibited, then MCP-1, TNF-α andVEGF expression was decreased. Furthermore, expression of MMP-9, LTB4and PGE2was decreased. Therefore, we speculate that BSE-CD treatment can attenuate S.japonicum egg-induced hepatic granulomas, which might be partly due to suppressedNF-κB passway and decreased expression of MCP-1, TNF-α, and VEGF. Reduction ofbiochemical mediators such as MMP-9, LTB4and PGE2may also play a role in thetreatment effect of BSE-CD. This study explores BSE-CD in treatment of S. japonicum egg-induced hepatic granuloma a liver and its mechanism， and may provide atheoretical and practical method to treat schistosomal hepatic granuloma.The inhibiting effect and mechanism of extracts of the oleogum resin fromBoswellia serrate on the hepatic fibrosis of mice infected with SchistosomajaponicumWe established mice model, C57BL/6mice were infected with S. japonicum cercariaeto form liver fibrosis. The animals were divided into5groups: BSE-CD (280mg/kg),BSE-CD (140mg/kg), cyclodextrin (280mg/kg), infected mice and normal mice. Inorder to decrease the mortality of infected mice, each group, except for the control, wasorally given praziquantel (PZQ,500mg/kg/day) on the42nd day (2days of successiveadministration) after infection. Then mice were injected intraperitoneally with BSE-CD(280mg/kg) or BSE-CD (140mg/kg) every day for3weeks starting at7week afterinfection, respectively. Control mice were given intraperitoneally injections ofcyclodextrin (280mg/kg) alone every day for3weeks starting at7week after infection.Another set of mice did not receive any treatment. Mice were sacrificed at24h after thelast injection. The effect on liver fibrosis were observed by Masson staining after beingtreatment of BSE; the HA (hyaluronic acid) and LN (laminin) were detected by radioimmunoassay, The effects of BSE-CD on theα-SMA in liver tissue were detected byimmunohistochemistry assay, the effects of BSE-CD on the cytokine in liver tissue wereanalyzed by RT-qPCR. The hepatic stellate cell (HSC)-T6was cultured in vitro, thenthe cell growth and proliferation were assessed via MTT assay, flow cytometry andHoechst33258staining were used to analyze the apoptosis and apoptosis rate ofHSC-T6.Our results showed that BSE-CD treatment significantly improved hepatic grossappearance. Morphometric analysis showed that the average fibrosis size in BSE-CD(280mg/kg) treatment mice reduced significantly compared with the the infected miceand cyclodextrin (280mg/kg)(P <0.05). BSE-CD treatment mice did not display a considerable decrease in serum HA and LN levels. Immunohistochemistry (IHC)staining demonstrated that the content of α-SMA in the BSE-CD (280mg/kg) treatmentmice was dramatically decreased compared with the the infected mice and cyclodextrin(280mg/kg) mice (P <0.05). MCP-1, TNF-α and VEGF expression which dependent onNF-κB of liver in BSE-CD (280mg/kg)-treated mice were decreased in real time PCRassay (P <0.05). and no difference in the expression of IL-1, IL-6, IL-13and TGF-β inthe livers of between the four group mice. BSE could significantly inhibit theproliferation of HSC dose-dependently compared with the control group (P <0.05).Flow cytometry and Hoechst33258staining result showed that BSE-CD couldsignificantly induce the apoptosis dose-dependently compared with the control groupafter BSE-CD application for48h (P <0.05).In conclusion, treatment with BSE-CD significantly reduced liver fibrosis size, atthe same time, we found α-SMA expression was inhibited, then MCP-1, TNF-α andVEGF expression was decreased. Furthermore, we found BSE could inhibit the HSC-T6proliferation and induce HSC-T6apoptosis. Therefore, we speculate that BSE-CDtreatment can attenuate S. japonicum egg-induced hepatic fibrosis, which might bepartly due to suppressed NF-κB passway and decreased expression of MCP-1, TNF-α,and VEGF, then the HSC activation were inhibited. Inhibiting the HSC-T6proliferationand inducing HSC-T6apoptosis may also play a role in the treatment effect of BSE-CD.