| Research objectivesObserve the influence of New Bitongling,the Chinese herbal compound with the functions of dispelling cold and removing dampness and dispelling stasis and freeing the network vessels,on the cardiac abnormalities of EAM rat.Reveal the relationship between the changes of cardiomyopathy and heart function of EAM rat and the changes of TLR4/MAPK/NF-?B signal pathway and CAMK ?ignal pathway related to inflammation.Expound the regulatory mechanism of New Bitongling towards TLR4/MAPK/NF-?B signal pathway and CAMK? signal pathway in myocardial cells,identify the molecular target for TCM treatment and provide experimental evidence for TCM treatment of rheumatoid arthritis(RA)heart disease.Research methodsChoose the female Lewis rats of SPF grade and divide them into nonnal group,EAM group,Methotrexate group,and New Bitongling high-dose,medial-dose and low-dose groups.Except for the normal group,the experimental autoimmune myocarditis(EAM)is prepared with polypeptide immuning rats in the other five groups.The drug administration is conducted 10 days after immunization and continues for 4 weeks.The rats are fed with the crude drug in the dose of 2.75g/kg,5.5g/kg and 11g/kg respectively in New Bitongling high-dose,medial-dose and low-dose groups according to the rat weight;the rats in Methotrexate group are fed with the Methotrexate suspension in the drug concentration of 0.1mg/ml according to the dose of 10ml/kg,once a week;and the rats are fed with sterilized drinking water of the same volume in nonnal and EAM groups,once a day.Observe the general situation of rats during intervention period,measure the weight and evaluate the arthritis index of rats,and measure the thickness of their toes with a vernier caliper.Evaluate the cardiac structure and function through ultrasonic cardiogram(UCG)after the treatment of 28 days.Collect blood after anesthetizing rats and collect tissues and organs after killing them.Detect the levels of myocardial enzyme and troponin based on serology;detect the contents of IL-17,IL-6 and TNF-a serum pro-inflammatory cytokines based on Enzyime-Linked ImmunoSorbent Assay(ELISA);observe the histopathological changes in cardiac muscle and synovial membrane at ankle joints based on hematoxylin-eosin staining(HE staining)and collagenous fiber staining method(Masson trichrome staining);detect the apoptosis rate of myocardial cells based on terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL staining);detect the expression of apoptosis genes of Bcl-2,Bax and Caspase-3 cardiac muscles based on immunohistochemistry;detect the levels of TLR4,p38MAPK,NF-KB/p65.TNF-a and IL-6 mRNA cardiac muscles based on Quantitative Real-time PCR method(qRT-PCR method);and detect the protein expression I of TLR4,My D88,p-p38MAPK(phosphorylation p38MAPK)and NF-KB/p-p65(phosphorylation p-p65)in total proteins of cardiac muscle,the protein expression of CAMK?,PLB,p-PLB and SERCA2 in calcium pumps of cardiac muscle,the protein expression of p-p38MAPK,NF-?B/p-p65 and IRF3 in nucleoproteins of cardiac muscle,and the protein expression of TLR4,p-p38MAPK(phosphorylation p38MAPK)and NF-KB/p-p65(phosphorylation p-p65)in synovial tissues based on Western Blotting(WB)method.Research results1.Evaluation of rat models in EAM group:? The rats in EAM group have the phenomena of hypokinesia,tiredness and irritability;the weight gain is obviously slower that that of rats in normal group;and all rats suffer from ankle swelling and foot ulcers.?The ultrasonic examination after 37 days:compared with the rats in normal group,the left ventricular internal dimension(LVIDs)and left ventricular volume(LVVs)for rats in EAM group are enlarged(P<0.01),indicating the enlargement of left ventricular cavity;some rats have the phenomenon of hydropericardium;and the cardiac systolic and diastolic functions of peak mitral inflow velocity at early diastole(E)/peak mitral inflow velocity at late diastole(A),fraction shortening(FS)and left ventricular ejection fraction(LVEF)are reduced,indicating the degression of cardiac systolic and diastolic functions.? HE staining:the cardiac muscle fibers of rats in EAM group are arranged loosely;a large amount of inflammatory cell infiltration are found in mesenchymes and muscle bundles;there are small vessels expanding and congesting in mesenchymes;the flocculent fibers are observed around small vessels and among muscle bundles;and there are a small amount of myocardial steatosis.? Masson trichrome staining:the distance between muscle fibers gets farther for rats in EAM group;and the collagenous fibers around myocardial matrix and blood vessels are proliferated significantly.? TUNEL staining:the Tunel stained positive cells in myocardial cells of rats in EAM group are increased obviously compared with those of rats in normal group.? Expression of apoptosis genes in Bcl-2,Bax and Caspase-3 cardiac muscles based on immunohistochemistry:compared with the rats in blank group,the expression of Bcl-2 is down-regulated and that of Bax and Caspase-3 is up-regulated for rats in EAM group.? Hypersensitive troponin ?(hs-cTnI)detected based on serology:the hs-cTnI of rats in EAM group is increased obviously,being positive.?TNF-a,IL-6 and IL-17 serum cytokines detected based on ELISA:compared with the rats in normal group,the contents of TNF-a.IL,6 and IL-17 inflammatory cytokines are increased(P<0.05)for rats in EAM group.In conclusion,it can be verified that the EAM molding is successful according to symptoms of rats and imageological and pathological assessments.2.The influence of New Bitongling on myocardial inflammation,mvocardial damage and heart function? Compared with the rats in EAM group,the rats in all New Bitongling groups have much higher E/A,EF and FS(P<0.05,0.01)and the rats in Methotrexate group have much higher E/A and EF(P<0.05,0.01),which indicates that the cardiac systolic and diastolic functions of rats in EAM group can be improved for rats in all New Bitongling groups and Methotrexate group after drug administration.LVVs and LVIDs are decreased obviously in New Bitongling medial-dose and high-dose groups(P<0.05,0.01)and the difference of LVIDs and LVVs has no statistical significance in New Bitongling low-dose group and Methotrexate group.It's indicated that the left ventricle enlargement can be inhibited in New Bitongling medial-dose and high-dose groups after drug administration.Compared with the rats in normal group,the difference of E/A,EF and FS and that of LVVs and LVIDs have no statistical significance for rats in New Bitongling medial-dose and high-dose groups.But the rats in New Bitongling low-dose group and Methotrexate group have lower EF and FS,and larger LVIDs and LVVs(P<0.05).It's indicated that the left ventricular cavity size and left ventricular function are close to normal levels in New Bitongling medial-dose and high-dose groups after drug administration,while in New Bitongling low-dose group and Methotrexate group,the left ventricular cavity size is shrank and the left ventricular function is not recovered to normal level,but it is improved slightly.?HE staining:the histopathological changes of four treatment groups are between those of normal group and EAM group.Thereinto,the amount of inflammatory cell infiltration in mesenchymes and muscle bundles of New Bitongling medial-dose and high-dose groups is much lower than that of EAM group,and the degeneration and necrosis of myocardial tissues are alleviated.?Masson trichrome staining:the proliferation of collagenous fibers in New Bitongling medial-dose and high-dose groups and Methotrexate group is reduced obviously compared with that of EAM group,and the fibrosis of myocardial tissues is alleviated.? TUNEL staining:the myocardial cells of positive staining in all New Bitongling groups are obviously less than those of EAM group,and the difference is most obvious in New Bitongling high-dose group.?The chemical staining of immune tissues:compared with EAM group,the Bcl-2 expression of Methotrexate group is down-regulated,while that of all New Bitongling groups is up-regulated,showing a dose dependency at the same time.The Bax expression of all New Bitongling groups and Methotrexate group is down-regulated,of which there is an obvious difference in New Bitongling high-dose group.The Caspase-3 expression of Methotrexate group is down-regulated,and that of New Bitongling low-dose and medial-dose groups trends to be down-regulated.And the down-regulated expression is of significant difference in New Bitongling high-dose group.?Hypersensitive troponin I detected based on serology:the concentration of hs-cTnI in New Bitongling medial-dose group is lower than that of EAM group(P<0.05),which indicates that the medial dose of New Bitongling has obvious protective effect on cardiac muscle.? TNF-a,IL-6 and IL-17 serum cytokines detected based on ELISA:compared with EAM group,the contents of TNF-a,IL-6 and IL-17 are reduced(P<0.05)in New Bitongling high-dose and medial-dose groups and Methotrexate group,and the content of TNF-a is reduced(P<0.05)in New Bitongling low-dose group.It's indicated that the Nexw Bitongling can inhibit the secretion of infla/cmmatory cytokines.3.The influence of New Bitongling on joint inflammation and general state of rats(D General state:the feeding state,redness and swelling of joints and movement situation of rats in all New Bitongling groups are improved to different extents after the drug administration of 2 weeks.?Arthritis index:compared with the rats in EAM group,the arthritis indexes of rats in New Bitongling low-dose and medial-dose groups are reduced obviously((P<0.05),the arthritis index of rats in New Bitongling high-dose group is reduced significantly(P<0.01),while that of rats in Methotrexate group is reduced slightly without any statistical difference after the drug administration of 28 days.? Thickness of toes:compared with the rats in EAM group,the thickness of right rear toes for rats in New Bitongling low-dose group and Methotrexate group is reduced obviously(P<0.05)after 25 days;that in New Bitongling medial-dose group is reduced obviously(P<0.05,0.01)after 19 days;and that in New Bitongling high-dose group is reduced obviously(P<0.05,0.01)after 7 days.? HE staining:compared with the rats in EAM group,the inflammatory cell infiltration of rats in all groups is alleviated to different extents.? Masson trichrome staining:compared with the rats in EAM group,the proliferation of collagenous fibers for rats in all groups is alleviated to different extents.4.The influence of New Bitongling on the expression of TLR4,p38MAPK,NF-kB,TNF-a and IL-6 mRNA in cardiac muscles for rats in EAM group:compared with the rats in norrnmal group,the expression of TLR4,TNF-a and IL-6 mRNA in myocardial tissues of rats in EAM group is increased(P<0.01)and the expression of p38MAPK and NF-?B m RNA has no statistical significance.Compared with the EAM group,the expression of TLR4,TNF-a and IL-6 mRNA can be reduced(P<0.01)in New Bitonglin high-dose group and Methotrexate group.5.The influence of New Bitongling on the protein expression of TLR4,My D88,p-p38MAPK(phosphorylation p38MAPK)and NF-?B/p-p65(phosphorylation p-p65)in total proteins of cardiac muscle for rats in EAM group:compared with the rats in normal group,the protein expression of TLR4,My D88,NF-KB/p-p65 and p-p38MAPK in myocardial tissues for rats in EAM group is up-regulated obviously(P<0.05),and compared with the rats in EAM group,the above-mentioned indexes are reduced to different extents(P<0.05)for rats in all New Bitongling groups and Methotrexate group.It's indicated that the New Bitongling can inhibit the abnormal activation of TLR4/MAPK/NF-?B signal pathway,which is one of the main mechanisms for New Bitongling to exert therapeutic effect.6.The influence of New Bitongling on the protein expression of CaMK ?,PLB,p-PLB and SERCA2 in calcium pumps of cardiac muscle for rats in EAM group:compared with the rats in blank group,the protein expression of SERCA2 is down-regulated obviously(P<0.05)and that of CaMK?,PLB and p-PLB is up-regulated(P<0.05)for rats in EAM group.Compared with the rats in EAM group,the protein expression of SERCA2 is up-regulated obviously(P<0.05)and that of CAMKII,p-PLB is down-regulated obviously(P<0.05)for rats in all New Bitongling groups and Methotrexate group.The protein expression of PLB has no statistical significance in all New Bitongling groups.It's indicated that all doses of New Bitongling and Methotrexate can significantly inhibit the down-regulated protein expression of SERCA2 and up-regulated protein expression of CAMKII and p-PLB,which are all induced by EAM.7.The influence of New Bitongling on the protein expression of p-p38MAPK,NF-KB/p-p65 and IRF3 in nucleoproteins of cardiac muscle for rats in EAM group:compared with the rats in normal group,the protein expression of p-p38MAPK and NF-?B/p-p65 in nucleoproteins of cardiac muscle is up-regulated obviously(P<0.05)for rats in EAM group.Compared wit the rats in EAM group,the protein expression of p-p38MAPK and NF-?B/p-p65 is obviously down-regulated(P<0.05)for rats in all New Bitongling groups and Methotrexate group.The protein expression of IRF3 has no statistical significance in EAM group.Compared with the EAM group,the protein expression of IRF3 has no statistical significance in all New Bitongling groups and Methotrexate group.It's indicated that the New Bitongling may protect the cardiac muscle by inhibiting the activation of NF-kB signal pathway.8.The influence of New Bitongling on the protein expression of TLR4,NF-KB/p-p65(phosphorylation p-p65)and p-p38MAPK(phosphorylation p38MAPK)in synovial tissues for rats in EAM group:compared with the rats in normal group,the protein expression of TLR4,p-p38MAPK and NF-?B/p-p65 in synovial tissues is strengthened obviously((P<0.05)for rats in EAM group.Compared with the rats in EAM group,the protein expression of TLR4 is reduced obviously(P<0.05)for rats in New Bitongling medial-dose group;the protein expression of NF-KB/p-p65 is reduced obviously(P<0.05)for rats in all New Bitongling groups and Methotrexate group;and the protein expression of p-p38MAPK is reduced obviously(P<0.05)for rats in New Bitongling high-dose and low-dose groups and Methotrexate group.It's indicated that the New Bitongling may alleviate the joint synovitis by inhibiting the activation of TLR4/MAPK/NF-?B signal pathwayConclusion1.The myocardial inflammation and damage of rats in New Bitongling groups are alleviated and the heart function is improved.Functional mechanism I:reduce the generation of IL-6,IL-17 and TNF-a by inhibiting TLR4/MAPK/NF-?B signal pathway and thus achieve the objectives of anti-inflammation and immunoregulation;functional mechanism II:inhibit the myocardial apoptosis by down-regulating the expression of Caspase-3 and Bax and up-regulating the expression of Bcl-2;and functional mechanism III:correct the abnormal protein expression of CaMK ?,SERCA2,PLB and p-PLB,improve the Ca2+distribution and dysfunction in myocardial cells and restore the process of Ca2+ mediate excitation-excitation-contraction coupling.From the above,New Bitongling can improve the heart function,alleviate the myocardial inflammatory response,inhibit the myocardial damage and eventually achieve the therapeutic effect of EAM.2.According to the comparison on the therapeutic effects of New Bitongling low-dose,medial-dose and high-dose groups,the inhibiting effect and therapeutic effect on TLR4/MAPK/NF-kB and CaMK II signal pathways are enhanced,which indicates that there is a certain correlation between the therapeutic effect and the dose of New Bitongling.3.The good effect for New Bitongling to alleviate the joint synovitis may be achieved by inhibiting the abnormal activation of TLR4/MAPK/NF-?B signal pathway. |
PDF Full Text Request