| Objective: To observe the effects of formula of regulating qi, resolving phlegm,activating blood and its three separate components (formula of regulating qi, formulaof resolving phlegm, formula of activating blood) and formula of tonifying qi,nourishing yin, clearing heat and its three separate components (formula of tonifyingqi, formula of nourishing yin, formula of clearing heat) on the proliferationinhibition and EGFR—PLC-γ1—P-tyr—PKC-α与TSHR—PLC-β3—PI3K—PKCαsignaling transduction pathway of human papillary thyroid carcinoma cell TPC-1;study the mechanism of treatment of regulating qi, resolving phlegm, activatingblood and treatment of tonifying qi, nourishing yin, clearing heat on thyroidcarcinoma, explore the relationship of traditional Chinese medicine pathogenisis ofqi stagnation, phlegm coagulating, blood stasis, heat toxin, etc. and thyroidcancerization and EGFR—PLC-γ1—P-tyr—PKC-α与TSHR—PLC-β3—PI3K—PKCα signal transduction.Methods: Mathematical statistics methods such as Frequency distribution analysis,principal component analysis and clustering analysis were used to make syndrome,formula, traditional Chinese medicine analysis and rule exploration of thyroid tumorand carcinoma treated by traditional Chinese medicine; human papillary thyroidcarcinoma cell TPC-1was selected asthe experimental object, cells were observedby inverted microscope, and calculated the inhibition rate with MTT method forscreening effective herbs; orthogonal design was used to select the best curativeeffect prescription; after establishing the control group and using the formula ofregulating qi,resolving phlegm, activating blood and its three separate components,formula of tonifying qi,nourishing yin,clearing heat and its three separatecomponents to effect cells， cell growth state was observed under inverted microscope; flow cytometry method was used to neasure the cell cycle of TPC-1cells after effected by the formula of regulating qi,resolving phlegm, activatingblood and its three separate components, formula of tonifying qi,nourishingyin,clearing heat and its three separate components; Western blot method was usedto check the protein expression in EGFR—PLC-γ1—P-tyr—PKC-α与TSHR—PLC-β3—PI3K—PKCα signal transduction pathway, so that we canobserved the effect of all treatmet groups on TPC-1cells from the proteinexpression.Results:①Through literature statistical analysis on traditional Chinese medicinedifferentiating and treating thyroid tumor and carcinoma,32syndromes wereinvolved, among which, syndrome of qi stagnation and phlegm coagulating,syndrome of blood stasis, syndrome of liver depression and qi stagnation,syndrome of deficiency of both qi and yin, syndrome of multual stagnation ofphlegm and damp, syndrome of multual congulating of phlegm and heat, syndromeof liver depression and spleen deficiency were the main syndromes;13pathogenesis elements were achieved, among which,9were excess pathogenesis,mainly were qi stagnation, coagulation of phlegm, blood stasis, fire and heat,4were deficiency pathogenesis, mainly were qi-deficiency and yin-deficiency;disease location mainly in liver, spleen, kidney, heart.,6clustered syndromes wereachieved through hierarchical cluster analysis on156symptoms and signs, theywere syndrome of stagnation of liver qi, spleen deficiency and phlegm congulating,mutual stagnation of phlegm and heat, deficiency of both qi and yin, qi stagnationand blood stasis, pathogenesis heat generated by liver depression;396formulaewere attained, among which,56were set prescriptions,340were experimentalprescriptions, the set prescriptions mainly were types of harmonizing prescription,tonifying prescription and phlegm-removing prescription;289kinds of Chinesemedicine were involved in, could be classified into19types, among which,phlegm-resolving, antitussive and antiasthmatic Chinese medicine, heat-clearingmedicine, tonifying medicine, activating blood stasis medicine, qi-regulatingmedicine, stabilizing live and removing wind were used in higher frequencyactivating blood regulation and removing stasis. Classified meridians of Chinesemedicine of treating hyperthyroidism mainly were liver, lung, spleen, stomach,kidney and heart.②Formula of regulating qi, resolving phlegm, activating bloodand its three separate components and formula of tonifying qi, nourishing yin, clearing heat and its three separate components could obviously inhibit TPC-1cellproliferation, their effects on cellular morphology were obviously different with theblank control group. Cell in the group of formula of regulating qi presented longfusiform; cell in the group of formula of resolving phlegm became shrinking,presented fusiform, rough surface, and the intracellular was full of a large number ofgranulate or vacuolus-liking object; cell amount in the group of formula of activatingblood evidently reduced, cellular morphology obviously shrank and diminished, thetransmittance of light reduced, anomaly morphology, cellular space increased withbad adherence; cell in the group of formula of regulating qi, resolving phlegm,activating blood shrank with anomaly morphology, rough surface, most cellspresented vacuous-liking construction, cell debris increased (saw the attacheddrawing from1to15). Cell in the group of formula of tonifying qi became shrinkingand small, roundness of fusiform, rough surface, much of cell debris; cell in thegroup of formula of nourishing yin obviously became round, some cells becameloose and swell, cell space increased, and the intracellular was full of a large numberof granulate or vacuolus-liking object; cellular morphology in the group of clearingheat jobviously became round, loosed and swell, rough surface, cellular fringepresented vacuolus-liking construction, the intracellular was full of a large numberof granulate object; cell in the group of formula of tonifying qi, nourishing yin,clearing heat presented anomaly fusiform or round, rough surface, most cellspresented vacuolus-liking construction, the intracellular was full of a large numberof granulate object and cell debris (saw the attached drawing from16to30).③Formula of regulating qi, resolving phlegm, activating blood and its three separatecomponents and formula of tonifying qi, nourishing yin, clearing heat and its threeseparate components could all inhibit TPC-1cell proliferation, and effects of the twowhole formulae were the strongest, and presented dosage-effects and time-effectsdependent relationship for the inhibition of TPC-1cell proliferation with theincreasing of medicine concentration and time.④Formula of regulating qi,resolvingphlegm, activating blood and its three separate components and formula of tonifyingqi, nourishing yin, clearing heat and its three separate components had obviouslyeffects on TPC-1cell cycle, cellular percent of G1stage reduced in all treatmentgroup of formula of regulating qi, resolving phlegm, activating blood and its threeseparate components, and the whole formula group reduced most obviously, cellularpercent of G2stage increased in all treatment group of formula of regulating qi, resolving phlegm, activating blood and its three separate components, and the wholeformula group increased most evidently. Cellular percent of G1stage reduced and ofG2stage increased in all treatment group of formula of tonifying qi, nourishing yin,clearing heat and its three separate components, and the group of formula ofnourishing yin reduced or increased the most; cellular percent of S stage in the groupof nourishing yin increase. Those rusults indicated that formula of regulating qi,resolving phlegm, activating blood and its three separate components and formula oftonifying qi, nourishing yin, clearing heat and its three separate componentsinhibited cell proliferation in the G2stage.⑤EGFR, PLC-γ1, P-tyr, PKCα, TSHR,PLC-β3, PI3K were overexpressed in human papillary thyroid carcinoma cell lineTPC-1.⑥When the cells were treated with IC50dosage, the protein expression levelof EGFR, PLC-γ1, P-tyr, PKCα, TSHR, PLC-β3, PI3K in the cells decreased.Conclusions:①Formula of regulating qi, resolving phlegm, activating blood and itsthree separate components and formula of tonifying qi, nourishing yin, clearing heatand its three separate components significantly inhibit the growth of TPC-1cell, Inwhich the two whole formula groups are the strongest that shows regulating qi,resolving phlegm and activating blood have synergistic effects, or tonifying qi,nourishing yin and clearing heat have synergistic effects.②Formula of regulating qi,resolving phlegm, activating blood and its three separate components and formula oftonifying qi, nourishing yin,clearing heat and its three separate components inhibitedcell proliferation in the G2stage.③There is close relationship between theoccurrence of thyroid carcinoma and EGFR—PLC-γ1—P-tyr—PKCα andTSHR—PLC-β3—PI3K—PKC-α signal transduction in TTPC-1cell.④Formula ofregulating qi, resolving phlegm, activating blood and its three separate componentsand formula of tonifying qi, nourishing yin, clearing heat and its three separatecomponents down-regulate the protein expression level ofEGFR—PLC-γ1—P-tyr—PKCα and TSHR—PLC-β3—PI3K—PKC-α signaltransduction. They may inhibit the growth of TPC-1cells through inhibiting theabove two signal transduction pathways.④Qi stagnation, phlegm congulation,blood stasis, heat toxin, both deficiency of qi and yin have close relationship withthe marked increment of EGFR—PLC-γ1—P-tyr—PKCα andTSHR—PLC-β3—PI3K—PKC-α signal molecular expression and transduction inthe phathogenesis of human thyroid carcinoma. |
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