Study On Anti-tumor Mechanism Of Abnormal Savda Munziq In Vivo Based On Transplanted S-180and EAC Tumor Model

Objective:A number of anti-tumor agents including alkyl-ting agents, anti-tumor antibiotics,anti-metabolites and various other agents such as platinum derivatives, procarbazine andemetine have been administered alone or in a variety of combinations by different routesto treat carcinoma, but the predictable response rate is no more than20%. Additionally,adverse drug reactions are frequent. Some current trials have been investigating the use ofplant derivatives and much attention has been paid to Chinese herb since it has littletoxicity comparatively and is not susceptible to drug resistance and it plays the role ofanti-tumor by multiple means. Multi-drug resistance （MDR） is the protection of tumorcell population against numerous drugs which differ in structure and in actingmechanisms on the tumor cells. MDR is also one of the major causes of failure ofchemotherapy of human malignancies, which is related to many active mechanisms.Often several different mechanisms are switched on in the cells, but usually one majormechanism would operate. The active mechanisms are summarized as follows:1)activation of trans-membrane proteins refluxing different chemical substances from thecells （P-glycoprotein is the most known efflux pump）;2) activation of the enzymes ofglutathione detoxification system; alteration of the genes and the proteins involved intothe control of apoptosis （especially P53and Bcl-2）; changes of activity and quantity oftopoisomerases. Study on multi-drug resistance of tumor cell and to look forchemotherapy drug with effectiveness and little toxic have long been an interesting topicof research.It is believed in Traditional Uighur medicine （TUM） that Abnormal Savda is apathological production resulted from the combustion of different body fluids known as Savda, Sapra, Belghem and Kan, which is believed to be the leading cause of cancers,and about90%of cancers are attributed to Abnormal Savda in clinical reports by TUM.Bahki Yusup and Tursun Tohti Haji, two of the most famous Uighur physicians, haveapplied the ASMq-TF in treatment of cancers based on the traditional therapy, whichdemonstrated remarkable clinical results, there by they gain the honor of “highly skilledphysician” by the local people.Abnormal savda munziq （ASMq）（National Invention Patent No: C082130082.8） isan Uighur medical compound preparation，which consists of Cordia dichotoma, Ziziphusjujuba Mill, Glycyrrhiza uralensis Fisch, Anchusa italica Retz, Foeniculum vulgare Mill,Euphorbia humifusa Willd and other herbs, which are used to treat tumor, diabetes andcardiovascular disease caused by abnormal ASMq. Recent studies have shown that ASMqcan scavenge free radicals and protect DNA oxidative damage and mitochondrialoxidative damage from Hydroxyl free radical, resulting in inhibiting the growth ofHepG2in-vitro and its synthesis of protein, DNA and RNA significantly.This study aims to explore the inhibitive effect of abnormal savda munziq on twokinds of transplanted tumor, protective effect of abnormal savda munziq onchemotherapy by reducing toxicity, synergistic attenuation of abnormal savda munziq onfluorouracil and further identify anticancer effect, the anti-fatigue and hypoxia resistanceeffect of abnormal savda munziq in vivo by hypoxia and pole climbing experiment.Methods:1．Establishment of transplanted S-180tumor model：180mice were randomized to6groups: normal control group, tumor control group, high-dose group of ASMq, middledose group of ASMq, low dose group of ASMq, cyclophosphamide （CY） control group.Indexes are measured in batch. Mouse tumor inhibition rate, spleen index, thymus indexand the index of liver are measured. Variation of mouse serum TNF-α, IL-1β, IL-2, IFN-γ,NK, LPO are observed. Pathological conditions of Tumor in mice spleen and other organsare examined by HE staining. By the MTT method, the proliferation of spleenlymphocytes in mice and variation of serum lymphocyte subsets such as CD₃⁺, CD₄⁺,CD₈⁺, CD₄⁺/CD₈⁺are identified.2．Establishment of transplanted S-180tumor model:60mice were randomlydivided into6groups: normal control group, tumor control group,5-fluorouracil （5-FU）group, high dose of5-FU ASMq group, middle dose of5-FU ASMq group, and low doseof5-FU ASMq group. Variations of mouse tumor inhibition rate, indexes of thymus, spleen and liver, mouse serum SOD, MDA, GSH-PX are observed.3．Establishment of transplanted EAC tumor model:180mice were randomlydivided into six groups, normal control group, tumor control group, high-dose group ofASMq, middle dose group of ASMq, low dose group of ASMq, cyclophosphamide （CY）control group. Observation the mouse tumor inhibition rate, spleen index thymus index,and the index of liver, mouse TNF-α, IL-1β, IL-2, IFN-γ, NK, LPO content in serum andHE staining examination of tumor in mice spleen and other organs pathologicalconditions. Determination of the proliferation of murine spleen lymphocytes by MTTmethod and mouse serum lymphocyte subsets in CD₃⁺, CD₄⁺, CD₈⁺, CD₄⁺/CD₈⁺.4．Establishment of the transplanted EAC tumor model:60mice were randomlydivided into6groups: normal control group, tumor control group,5-fluorouracil （5-FU）group, high dose of5-FU ASMq group, middle dose of5-FU ASMq group, and low doseof5-FU ASMq group. Variations of mouse tumor inhibition rate, index of spleen, thymus,and liver, mouse serum SOD, MDA, GSH-PX are observed.5．Establishment of chemotherapy drug toxicity model:50mice were randomlydivided into5groups: normal control group, tumor control group，AF(amycin and5-fluorouracil （5-Fu） group, AF⁺ASMq high dose group, AF⁺ASMq middle dose group,and AF⁺ASMq low dose group. Spleen, liver and thymus index，mouse serum ALT, AST,TP content, SOD in mouse heart, MDA, GSH-PX content are observed and pathologicconditions of mouse heart, liver and other viscera are examined by HE staining methods.6．Establishment of transplanted S-180tumor model and EAC tumor modelrespectively: ASMq effect on mice serum SOD, MDA, GSH-PX indexes, as well as onmouse tumor inhibition rate, spleen index, thymus index and liver index are evaluated;ASMq effect on mice under normal pressure hypoxia tolerance test and polele test areevaluated.Results:1．Comparison between each dose group of ASMq and cyclophosphamide: Thespleen/weight ratio increased significantly in each dose group of ASMq （P＜0.05）. Theinhibition rate of each dose group of ASMq and cyclophosphamide were49.5%、64.26%、43.43%and50.88%respectively, in which inhibitory effect was observed. But there wereno significant differences between the cyclophosphamide and ASMq high does group.Compared with tumor model group, serum IL-1β level varied in ASMq every dose groupsignificantly （P＜0.05）, the content of IL-1β was the highest in ASMq middle dose group. Compared to the group of tumor model and positive control, Serum IL-2contentincreased in every dose group of ASMq significantly （P＜0.05）, and ASMq middle dosegroup was the highest. The serum TNF-α content was higher in every dose group ofASMq than in the tumor model group and in cyclophosphamide, there was significantdifference （P＜0.05）, which was the highest in ASMq middle dose group. Compared withtumor model group, serum IFN-γ content varied in every dose group of ASMqsignificantly （P＜0.05）, the content of IL-1β was the highest in ASMq middle dose group.Compared to tumor model group and cyclophosphamide, Serum IFN-γ content increasesin every dose group of ASMq, there was significant difference （P＜0.05）, Serum IFN-γwas the highesin in ASMq middle dose group. The serum LPO content in every dosegroup of ASMq were all lower than tumor model group and cyclophosphamide, there wassignificant difference （P＜0.05）, and serum LPO was the lowest in ASMq middle dosegroup. Compared with tumor model group, the serum NK content varied significantly inevery dose group of ASMq, there was significant difference （P＜0.05）, the content of NKwas the highest in ASMq middle dose group. In ASMq high dose group, CD₃⁺, CD₈⁺andCD₄⁺was reduced apparently, the CD₄⁺/CD₈⁺ratio increased and The CD₄⁺/CD₈⁺ratiowas close to the normal value. In ASMq middle dose group, CD₈⁺decreased, CD₃⁺, CD₄⁺CD₈⁺and CD₄⁺was reduced apparently, the CD₄⁺/CD₈⁺ratio increased. The CD₄⁺/CD₈⁺ratio was close to the normal value. There was no significant improvement in the ASMqlow dose group. Comparison between normal control and tumor model group, the SOD，GSH-PX, MDA content in the liver of both ASMq and tumor groups were significantlydifferent. The difference was significant （P＜0.05）. The SOD，GSH-PX content in theliver of ASMq every dose group increased, there was significant difference （P＜0.05）,ASMq middle dose group was the highest. The MDA content in mouse liver, of everydose group of ASMq were all lower than tumor model group and cyclophosphamidegroup, there was significant difference （P＜0.05）. ASMq middle dose group was thelowest. After examinations of mouse liver, spleen, thymus and other organs by HEstaining methods, Pathological conditions are all shown normal, necrosis and liquefactionof transplanted S-180tumor mouse tumor was observed.2．The inhibition rates of5-Fu-ASMq high dose group,5-Fu-ASMq middle dosegroup, low dose group of5-Fu⁺ASMq And5-fluorouracil group were52.26%,66.41%,44.87%and48.02%. There was significant difference between normal control group and5-Fu group in SOD, GSH-PX content in serum（P＜0.05）, SOD, GSH-PX contentdecreased significantly in every dose group of ASMq, In ASMq middle dose group, the highest content of SOD GSH-PX was close to the normal value. The MDA value ofwhich was lowest there was significantly reduced comparing with the5-Fu control group,the difference was significant （P＜0.05）. Compared to tumor model group with, serumSOD, GSH-PX content in the model group, both ASMq high and low dose groups weresignificantly lower. The difference was significant （P＜0.05）, in which the ASMq middledose group maximum has been close to or exceed the normal values.3． Compared with the spleen/weight ratio each group of ASMq andcyclophosphamide, ASMq every dose group significantly increased, there was significantdifference （P＜0.05）, in which ASMq middle dose group. The inhibition rate of eachdose ASMq and cyclophosphamide were49.5%、64.26%、43.43%and50.88%respectively, which inhibitory effect was observed, but there has no difference betweenthe groups of cyclophosphamide and ASMq. Compared with the serum IL-1βcontent,ASMq every dose group significantly changed, there was significant difference （P＜0.05）,the content of IL-1βwas the highest in which ASMq middle dose group Compared withthe serum IL-1βcontent between the group of tumor model and positive control, ASMqevery dose group significantly increased, there was significant difference （P＜0.05）, inwhich ASMq middle dose group was the highest, The serum TNF-αcontent in ASMqevery dose group were all higher than tumor model group and cyclophosphamide, therewas significant difference （P＜0.05）,. in which ASMq middle dose group was the highest,Compared with the serum IFN-γ content, ASMq every dose group significantly changed,there was significant difference （P＜0.05）, the content of IL-1βwas the highest in whichASMq middle dose group. Compared with the serum IFN-γ content in tumor modelgroup and cyclophosphamide, ASMq every dose group of relative to rise, there wassignificant difference （P＜0.05）, in which ASMq middle dose group was the highest, Theserum LPO content in ASMq every dose group were all lower than tumor model groupand cyclophosphamide, there was significant difference （P＜0.05）, in which ASMqmiddle dose group was the lowest, Compared with the serum NK content, ASMq everydose group significantly changed, there was significant difference （P＜0.05）, the contentof NK was the highest in which ASMq middle dose group ASMq high dose group, CD₃⁺、CD₈⁺and CD₄⁺was reduced apparently, the CD₄⁺/CD₈⁺ratio increased, and TheCD₄⁺/CD₈⁺ratio was close to the normal value. ASMq middle dose group, CD₈⁺decreased, CD₃⁺、CD₄⁺CD₈⁺and CD₄⁺was reduced apparently, the CD₄⁺/CD₈⁺ratioincreased, elevated CD₄⁺/CD₈⁺ratio increased, and this ratio was close to the normalvalue. There was no significant improvement with the ASMq low dose group. Compared with, the SOD, GSH-PX, MDA content in the liver of both ASMq and tumor groups weresignificantly difference. The difference was significant （P＜0.05）.Compared with theSOD，GSH-PX content in the liver of tumor model group, ASMq every dose group ofrelative to rise, there was significant difference （P＜0.05）, in which ASMq middle dosegroup was the highest, Compared with the MDA content in mouse liver, ASMq everydose group were all lower than tumor model group and cyclophosphamide, there wassignificant difference （P＜0.05）, in which ASMq middle dose group was the lowest, HEstaining methods of mouse liver, spleen, thymus and other organs Pathological conditionsare all normally, transplanted S-180tumor mouse tumor necrosis and liquefaction.4．In the5-FU high-dose ASMq group,5-FU medium-dose ASMq group,5-FUlow-dose ASMq group and5-FU group, the values of tumor inhibition rate were42.41%、58.09%,41.58%and50.83%, respectively, showing that tumor function had beenweakened. Compared with the model group, the thymus/body weight ratio was clearlyreduced in the5-FU medium-dose ASMq group and5-FU low-dose ASMq group.Compared with the5-FU group, the spleen/body weight ratio was clearly increased in the5-FU high-dose ASMq group,5-FU medium-dose ASMq group, and5-FU low-doseASMq group, and the difference was significant （P＜0.05）. The5-FU ASMq dose groupshad reduced levels of SOD and GSH-PX compared with the5-FU control group, and thedifference was significant （P＜0.05）. In the5-FU medium-dose ASMq group, SOD andGSH-PX levels were high. With respect to MDA values, the5-FU medium-dose ASMqgroup had the lowest values and in comparison with the model control group, thedifference was significant （P＜0.05）. With respect to SOD and GSH-PX levels, comparedwith the model group, the5-FU high dose ASMq group had lower levels, and thedifference was significant （P＜0.05）.5．Compared with the AF model group,serum ALT, AST content, in every dosegroup of ASMq significantly decreased, there was significant difference （P＜0.05）, whichwas the lowest in ASMq middle dose group, ALT, AST content were close to the normalvalue. The TP value of ASMq middle dose group is the highest, compared to the AFmodel control group, which had significantly increased, the difference was significant （P＜0.05）. Compared with the normal control group, serum ALT, AST content in abnormalsavda munziq every dose group significantly decreased, there was significant difference（P＜0.05）, TP value of which in the ASMq middle dose group was the highest. Comparedwith the AF model group, SOD, GSH-PX content in the heart, significantly increased inASMq every dose group. The difference was significant （P＜0.05）, in which ASMq middle dose group had the highest content of SOD, GSH-PX and which were close to thenormal values. Compared with the AF model group, MDA value of ASMq with middledoses was significantly lower; the difference was significant （P＜0.05）. Compared withthe normal control group SOD, GSH-PX content in the heart, ASMq high and low dosegroups were significantly lower. The difference was significant （P＜0.05）, in which theASMq middle dose group maximum has been close to or exceed the normal values.ASMq cannot only improve the spleen index, but also reduce the serum levels of ALT,AST content and improve the mouse heart SOD, GSH-PX content, and protect the heartand liver in preventing necrosis.6．Experiments of transplanted tumor S-180mice model for climbing, hypoxiatolerance: Compared with cyclophosphamide group, ASMq high dose group, mediumdose group, low dose group showed significantly higher spleen/body weight ratio; thedifferences were significant （P＜0.05）. Compared with tumor model group, the abnormalsavda munziq middle dose group and low dose group have obvious liver/body weightratio changes, the difference was significant （P＜0.05）. Compared with cyclophos-phamide group, in terms of the content of SOD, GSH-PX in serum, every dose group ofabnormal Savda Munziq is increased significantly, and is close to the normal level; thedifference was significant （P＜0.05）. The dose of MDA in abnormal savda munziq is thelowest, compared with tumor model group; the difference was significant （P＜0.05）.Compared tumor model group with normal controls, the time of climbing was decreasedsignificantly （P＜0.05）. Compared with cyclophosphamide, every dose group ofabnormal savda munziq can improve the time of mice climbing, particularly in abnormalsavda munziq dose for which the effect is significant （P＜0.05）. Compared with tumormodel group, every dose group of abnormal savda munziq can enhance the time of micehypoxia tolerance, and there is significant difference （P＜0.05）. Compared with thenormal control group, every dose group of abnormal savda munziq can enhance the timeof hypoxia tolerance; especially for the dose of abnormal savda munziq the effect is mostremarkable.7．Experiments of transplanted EAC tumor mice model for climbing, hypoxiatolerance test: Compared with cyclophosphamide group, the abnormal savda munziq highdose group, middle dose group, and low dose group have increased spleen/body weightratio, the difference was significant （P＜0.05）. Compared with tumor model group, theabnormal savda munziq middle dose group and the low dose group have obviousliver/body weight ratio changes; the difference was significant （P＜0.05）. Compared the model group tumor with normal control group, the climbing time was decreasedsignificantly （P＜0.05）. Compared with cyclophosphamide group, every dose group ofabnormal savda munziq can improve the mice climbing time, particularly for the dose ofabnormal savda munziq the effect is significant （P＜0.05）. Compared the content of SOD,GSH-PX in serum of cyclophosphamide group, every dose group of abnormal savdamunziq was increased, in which the content of SOD, GSH-PX of middle dose group isclose to the normal value, and cyclophosphamide group was increased significantly, thedifference was significant （P＜0.05）. The MDA value of abnormal savda munziq is lowerthan tumor model group. Compared with the normal control group, every dose ofabnormal savda munziq group can enhance hypoxia tolerance in mice; especially themiddle dose abnormal savda munziq is the best. Compared with tumor model, every dosegroup of abnormal savda munziq was increased; the difference was significant （P＜0.05）.Compared with verapamil, the abnormal savda munziq high and low dose groups weredecreased, and the difference was significant （P＜0.05）.Conclusion:1．Tumor is an immunodeficiency disease, which is mainly caused by immunesystem disorders, decreased anti-tumor activity factor and loss of function. Activating thebody independent anti-tumor immune mechanisms and strengthening the activity ofcytokines are important aspects of cancer treatment. This study found that ASMq canimprove immune system function, enhance antioxidant enzyme activity in immuneorgans, and regulate the lymphocyte subsets levels and cytokines to reach the purpose oftumor suppression. Performance of experiments in transplanted S-180and EAC tumormodels in mice showed decreased mass volume, decreased weight, and necrosis andliquefaction in the tumor, by thus the anti-tumor rate increased.2． The ASMq has a good antagonism on the toxicity side effects of thechemotherapy drug doxorubicin and5-fluorouracil （AF）, it can protect the liver and heartand reduce the damage of chemotherapeutic drugs on the liver and heart. It can reduceserumin ALT, AST content and improve the level of TP. It increases the content of themouse model of heart tissue SOD and GSH-PX, and reduces the level of MDA.Abnormal black Munziq can improve the content of serum SOD and GSH-PX in5-fluorouracil Reducing Toxicity Model and reduce the level of MDA.3．The ASMq has a function of anti-oxidation, hypoxia and anti-fatigue effect invivo. Compared with the other control group, ASMq can significantly improve the general living conditions of the tumor model mice, active state and mouse fur luster,while weight is not significantly reduced.4．It was found that compared to ASMq high dose group and low dose group, interms of suppressing the transplanted S-180and EAC tumor, the protective effect of toxicside effects induced by the chemotherapy drug doxorubicin and5-fluorouracil （AF）,5-fluorouracil efficiency attenuation, anti-oxidation, hypoxia and anti-fatigue effect inclimbing the pole and hypoxia experiment, ASMq middle dose group shows better effect...