| Colorectal carcinoma is one of the most common malignant tumor in the digestive system, its indicidence situates the third place of all the malignant tumors and continues to show a rising trend. So far, the pathogenesis of colorectal cancer has not yet entirely clear, the treatment means of colorectal cancer include operation, chemotherapy, radiotherapy and biological therapy, etc. Because of its occult onset, when the diagnosis was confirmed, most patients has already been at the intermediate and advanced stage, the tumor grows rapidly, metastasizes distance in the early stage, the major causes which lead to the failure of therapy are the tumor recurrence and its distant metastasis. Thus, seeking the antineoplastic medicine which is little toxicity, safe and effective becomes research focus. By inhibiting colorectal cancer SW480 metastasis activity and its cell proliferation with extracted sample of Ramulus cinnamomi, then the further study of the induction in vivo for cell apoptosis of tumor-bearing mice's tumor and the inhibition of its metastasized tumor. This study can explain the mechanisms that the ramulus cinnamomi resist the colorectal carcinoma, also provide the theory basis for its therapy. The research methods: Making a chemical analysis of the traditional Chinese medicine-ramulus cinnamomi by using HPLC fingerprint analysis methods. Detecting the inhibitory effect of various ramulus cinnamomi extract to the MMPs by using the enzyme method; detecting the inhibitory effect of three ramulus cinnamomi extracts which have inhibition to MMP-9 by using the zymography analysis method; utilizing the ramulus cinnamomi primary water extract to experiment SW480 cell and oberving the change of SW480 cell morphocytology after taking medicine; testing the situation that the ramulus cinnamomi extract inhibits adhesion, metastasis, invasion of colorectal cancer SW480 cell and cell proliferation in vitro; detecting the apoptosis by using flow cytometry; finally, carrying on the zoopery that the ramulus cinnamomi extract inhibits colorectal cancer and detecting the inhibion of ramulus cinnamomi extract to the tumor-bearing mice orthotopic transplantation tumor and the metastasis of colorectal carcinoma of mouse, comparing and analyzing the volume, weight and the amount of hepatic metastases of the control group and experimental group; after the paraffin section of tumor tissues, carrying on HE stains and immunohistochemical stains, comparing the tumor cell structural changes and the expression of MMP-2,9 of experimental group and control group; using the method of Western Blot to test the MMP-2 and MMP-9 protein expressions of tumor tissues.The results indicate: The results of ramulus cinnamomi HPLC fingerprint analysis reveal: the chemical constituents of ramulus cinnamomi are cinnamic acid 2-methoxy cinnamic acid, cinnamic aldehyde and ethyl cinnamate, etc. The inhibitory effects of ramulus cinnamomi final water extract acted on the MMP-2, MMP-9 and MMP-16 are MMP-2:IC50=8μg/ml,MMP-9:IC50=7.5μg/ml,MMP-16 : IC50=10μg/ml respectively. Ramulus cinnamomi n-butyl alcohol extract are MMP-2 : IC50=87μg/ml , MMP-9 : IC50=32μg/ml , MMP-16 :IC50=151μg/ml respectively; final water extract are MMP-2:IC50=23μg/ml,MMP-9 : IC50=24μg/ml , MMP-16 : IC50=21μg/ml. With the increased concentration of ramulus cinnamomi extractive, the colour of the precursor activation MMP-9 striated membranes is lighter and lighter until fade. Observing it under inverted microscope: the untreated SW480 cell grows well, it has irregular shape which is spindle and polygon, it can fuse into colony formation and has nested phenomenon in growing, its nuclei are large and atypia, nuclear-cytoplasmic ratio is high, it has abnormal nuclear division; The experimental group: after the different concentrations ramulus cinnamomi extract acting on the SW480 cell, the suspension cell has become increase, cytolymph is cloudy, the cell body dwindle, round, shrivel, it begins karyopyknosis and karyorrhexis, cell refraction ratio weaks, granular substances appear in the cell and cell fragments appear in the nutrient fluid. Observing it under the electron microscope, the morphology of normal SW480 cell is larger, the shape is polygon, the cell membrane is intact, there is a lot of floss ecptoma on the surface, intercellular is interconnection. The nuclei are large and irregular, have 1～3 nucleolus, nuclear-cytoplasmic ratio is high, endo-chromatin are abundant. Chromatin margination is not obvious, the organelles in the cytoplasm are abundant, there is little swelling in the organelles such as endoplasmic reticulum, mitochondria and lysoome; the experimental group ramulus cinnamomi extract, the tumor cells show the retrogression in varying degrees, the typical apoptotic cell can be seen: cell size dwindle, epicyte is intact, cell body shrink, the cytoplasmic processes of cell border are large vesiculiform, endochylema condense, electron density deep in endochylema, there is no destroy in cellular organ, nucleolus disappear, karyopyknosis heterochromatin aggregation distribute into gobbet on the nuclear membrane after karyorrhexis, and form the apoptotic body. Karyorrhexis disintegrates into several fragments and form the apoptotic body. After 72 hours, the tumescent necrosis can be shown. The adhere inhibition ratio is 20.92% when the ramulus cinnamomi treated group is 100μg/ml, the adhere inhibition ratio is 26.76% when the ramulus cinnamomi treated group is 200μg/ml. Compared with the number(116±6) of SW480 cell through the microporous membrane in the untreated group (control group), the number(76±6) in the ramulus cinnamomi extractive treated group of migration experiment decreased(P<0.05) obviously, the adhere inhibition ratio is 34.48%; the number(52±7) of SW480 cell which invaded Matrigel adhesive through microporous membraneramulus in the cinnamomi extractive treated group of invasion experiment decrease (P<0.05) than the number(88±9) that of untreated group(control group), the invaded inhibition ratio is 40.91%;the result of MTT experiment reveals that when the medicine concentration changes within 0～200 μg/ml the cytotoxicity is low, the inhibitory effect on the cell proliferation is not clear, when the medicine concentration changes within 200～700μg/ml, the inhititory effect which has positive correlation with concentration is strong. The flow cytometry shows that SW480 cell cycle is regulated by ramulus cinnamomi extract treatment, cells in G2/M period are relative increase, the tumor cell begin apoptosis. The volumn and weight of the mouse orthotopic transplantation tumor have been obviously inhibited and the inhibitory rate grew up with dosage concentration increasing . The inhibitory rate of 20mg/d for each one in the treated group is 32.93% and 40mg/d for each one in the treated group is 45.80%. Compared with the number 11±4 of liver metastatic tumors in the control group, the number of liver metastatic tumors 20mg/d for each one in the treated group is 7±2, P<0.05. Compared with control group,the number of liver metastatic tumors is 5±2 40 mg /d for each one in the treated group, P<0.01. The observation under the HE stained light-microscopy indicates that the tumor cell disseminate distribution in mouse tumor tissues of the both groups, a lot of tumor cell lines into glandular shape. Compared with control group, the tumor cell density of tumor-bearing mice tumor cell in the experimental group is sparse, the stroma is more, the inflammatory cell infiltration and necrosis of even stained red structurelessness also can be revealed, the number of vessel which around the tumor reduced clearly. The result of immunohistochemistry of colorectal transplantation tumor manifested that number of MMP-2 and MMP-9 in the experimental group is less than the control group apparently. The method of Western blot tested that MMP-2 and MMP-9 are significant differences in that of between experimented group and control group, P<0.01.Conclusion: 1.Ramulus cinnamomi can inhibit the activity and expression of colorectal carcinoma transplantation tumor MMPs in mice. 2.Ramulus cinnamomi contains an matrix metalloproteinase inhibitor that is an natural matrix metalloproteinase inhibitor. 3.Ramulus cinnamomi extract can inhibit adhesion, infiltration and metastasis of colorectal carcinoma. 4.Ramulus cinnamomi can inhibit colorectal carcinoma cell proliferation, induce the apoptosis of colorectal carcinoma. 5.Ramulus cinnamomi extract can inhibit SW480 cell course from G1 to S, and then slow down cells proliferation. 6. Because ramulus cinnamomi extract has more than four active ingredients, it can satisfy the combined therapy of polypragmasy and multi-targets. 7. Ramulus cinnamomi in the body can inhibit proliferation and metastasis of colorectal carcinoma transplantation tumor, inhibit the neovascularization and then inhibit recurrence and spread of colorectal cancer, we suppose that its mechanism is related to the activity of matrix metalloproteinase inhibitor. 8. Because ramulus cinnamomi extract has the traits of low toxicity and strong inhibition, it will provide a new therapy for the colorectal carcinoma.
|
PDF Full Text Request