| ObjectiveFirst, to study the inhibitory effect of Benefiting Qi for Removing Phlegm formula on growth of human lung cancer A549cells xenografts in nude mice.Second, to study the effect of Benefiting Qi for Removing Phlegm formula induce tumor cells apoptosis in nude mice with human lung cancer A549cells xenografts and A549cells apoptosis in vitro.Third, to study the effect of Benefiting Qi for Removing Phlegm formula on the activation of PERK-eIF2α signaling pathway.MethodsFirst, Building human lung cancer A549cells xenografts models in nude mice and grouping to give medicine:By adopting the method of cell trans-plantation to inoculate A549cell suspension in BalB/CA-nu nude mice right axillary subcutaneous. When the right axilla of each nude mouse could be palpable to the subcutaneous tumor diameter reached5mm, the nude mice were randomly divided into4groups according to the matching design,8nude mice in each group, groups:Model group, Traditional Chinese Medicine group, Chemo-therapy group and Combined group. Administration:In Model group, each nude mouse was given physiological saline0.4mL/day to fill the stomach, once a day, for2weeks; in Traditional Chinese Medicine group, each nude mouse was given Benefiting Qi for Removing Phlegm formula about0.4mL/day dose (27.3g/kg/day) to fill the stomach, once a day, for2weeks; in Chemotherapy group, each nude mouse was given0. lg/mL of cisplatin saline solution0.4mL/day (2mg/kg/day) to intraperitoneal injection, once a day, in the first five days; in Combined group, each nude mouse was given Benefiting Qi for Removing Phlegm formula about0.4mL/day dose (27.3g/kg/day) to fill the stomach, once a day, for2weeks; Each nude mouse in Combined group were given0. lg/mL of cisplatin saline solution0.4mL/day (2mg/kg/day) to intraperitoneal injection, once a day, in the first five daysSecond, weighing the weight of nude mice and measuring tumor diameter: Weighed the weight of nude mice with human lung cancer A549cells xenografts by a precision electronic balance two times every week (Dayl, Day5, Day9, Day14)during the period of delivery. At the same time, measured long diameter (a) and short diameter (b)of tumor of nude mouse by micrometer and the tumor volume was calculated by V=0.5ab2. According to the statistical analysis method for volume difference of tumors in nude mice between Dayl4and Dayl, evaluated the effect of Ben-efiting Qi for Removing Phlegm formula inhibit solid tumor growth in nude mice with human lung cancer A549cells xenografts.Third, weighing the weight of tumor:After stopping to give medicine1days,3nude mice with human lung cancer A549cells xenografts of each group were randomly selected to observe the survival time and the remaining5were sacrificed. Then stripped the subcutaneous tumor of nude mice with human lung cancer A549cells xenografts, weighed the weight of tumor and calculated the tumor rowth inhibition rate and tumor weight index.Fourth, detection of tumor cell apoptosis rate by flow cytometry:Excised tumor tissue blocks of about0.1g and ground on100steel screen and rinsed with precooling PBS buffer.Collected cell suspension and filtered through300mesh nylon net to flow cytometry tubes (in ice-water bath). After the centrifugal machine centrifuge1200rpm for5min, discarded supernatant,then added500uL Binding Buffer to mix cells in the model group, and added5uL Annexin V-FITC to a flow tube in the model group, at the same time, added5uL Propidium Iodide to mix in the other flow tube. To added5uL Annexin V-FITC and5uL Propidium Iodide to mix in all other flow tube at room temperature, away from light for10min, all flow tubes are added lmL PBS for testing on the machine.Fifth,pathological observation of organs:Conducted autopsy on the nude mice after stripping tumors, removed the heart, liver, spleen, lung, kidney and fixed these organs in4%formaldehyde solution for24h, rinsed with water for24h, then preserved in75%ethanol. After dehydration, embedding, slicing processing and HE staining, observed the morphological structure of organ tissues in nude mice with human lung cancer A549cells xenografts under bi-ological microscope.Sixth, Immunohistochemical assay:Excised tumor tissue blocks of about0.3g and rinsed with normal saline. Fixed tumor tissue blocks in4%formal-dehyde solution5mL for24h, and then rinsed with water for several times. After dehydration, embedding, sectioning and other processing, Detected expression of PERK, eIF2α, p-eIF2α in tumor tissue by immunohistochemical SP method.Seventh, preparation of blank serum and medicated serum:the SPF SD rats were randomly divided into2groups,15rats in each group, groups:Negative control group, Traitional Chinese Medicine group. Began to give medicine when body weight of rats in each group reached about200g. In Negative Control group: Each rat was given0.5%sodium carboxymethyl cellulose2mL/day once daily for7days to fill the stomach; in Traitional Chinese Medicine:Each rat was given Benefiting Qi for Removing Phlegm formula with0.5%carboxymethyl methyl cellulose2mL/day (63g/kg/day) to fill the stomach once daily for7days. After administration of1～2h in the first seven days, each rat was intraperitoneal injected10%chloral hydrate anesthesia to narcotize. Under sterile conditions, blooded from abdominal aortic with10mL syringe tip. Injected blood slowly into sterile test tubes, discarded the end of the blood to prevent excessive extrusion hemolysis. Let blood stand for2h, the centrifugal machine cen-trifuge3000rpm for5min, packed in1.5mL EP tube, stored at-20℃refri-gerator spare. Preparation of30%blank rat serum culture medium:In15mL centrifuge tube, added thawed early blank rat serum into containing10%FBS RPMI1640medium with a Pasteur pipette of3mL, mixed, and then sealed plastic sealed tube, placed at4℃refrigerator spare. Preparation of30%medicated rat serum culture medium:In15mL centrifuge tube, added thawed early3mL medicated rat serum into containing10%FBS RPMI1640medium with a Pasteur pipette of3mL, mixed, and then sealed plastic sealed tube, placed at4℃refrigerator spare.Eighth, A549cells were observed by inverted microscope:A549cells in Control group were treated with30%blank rat serum medium for24h; A549cells in Experimental group were treated with30%medicated rat serum culture medium for24h. Then A549cells were observed under inverted microscope.Ninth, A549cells were observed by transmission electron microscope: Poured the culture medium’out of culture flasks in Control group and Experi-mental group, digested with trypsin, then added the right amount of culture fluid, centrifuged1000rpm for10min. Poured culture fluid after centrifug-ation. Added slowly the right amount of3%glutaraldehyde solution into the tube to fix at4℃for the night. After soaking double fixation (using3%glutaraldehyde,1%osmium tetroxide), dehydration, infiltration, embedded system, embedded block processing, slicing and uranyl acetate and lead ci-trate double staining process, A549cells were observed under Tecnai G2Spirit Twin type transmission electron microscope.Tenth, Western blot assay:Extracted the total protein in A549cells in Control group and Experimental group, determined protein sample concentration by BCA method, conducted SDS-PAGE electrophoresis, transferred to a membrane, conducted immunoblotting and other steps to detect the expression of PERK, p-PERK, eIF2α, p-eIF2a.Eleventh, Statistical analysis:Run the SPSS17.0software package for statistical analysis, the experimental data presented was analysed as mean±standard deviation and used the single factor analysis of variance (One-Way ANOVA), used the LSD test for comparison between groups, P<0.05was considered statistically significant.ResuLtsFirst, Survival time of nude mice with human lung cancer A549cells xenografts in Model group, Traditional Chinese Medince group, Chemotherapy group and Combined group were more than three months.Second, the body weight of nude mice with human lung cancer A549cells xenografts in Combined group increased basically smoothly; the body weight of nude mice with human lung cancer A549cells xenografts in Traditional Chinese Medince group increased gradually in the early and middle phase of drug delivery, but decreased compared with the previous phase in the later period of drug delivery; the body weight of nude mice with human lung cancer A549cells xenografts in Chemotherapy group decreased gradually in the early phase of drug delivery, but increased in the middle and later period of drug delivery; the body weight of nude mice with human lung cancer A549cells xenografts in model group decreased gradually in the early phase of drug delivery, but increased in middle phase of drug delivery,then decreased in the later period of drug delivery. Third, tumor volume of nude mice with human lung cancer A549cells xenografts in Traditional Chinese medicine group, Chemotherapy group, and Combined group were saller than Model group. The tumor volume difference of nude mice between Day14and Dayl in the three groups were also saller than Model group. There was no statistical significance among Traditional Chinese medicine group, Chemotherapy group, Combined group and Model group(P>0.05)Fourth, tumor weight of nude mice with human lung cancer A549cells xenografts in Model group, Traditional Chinese Medicine group, Chemotherapy group and Combined group were0.532±0.052g,0.492±0.087g,0.394±0.090g,0.250±0.094g. The tumor weight of nude mice with human lung cancer A549cells xenografts in Traditional Chinese Medicine group, Chemotherapy group, and Combined group were saller than Model group. There was no statistical significance between Traditional Chinese Medicine group and Model group(P>0.05); there was statistical significancebetween Chemotherapy group and Model group (P<0.05); there was statistical significancebetween Combined group and Model group (P<0.001). The tumor weight of nude mice with human lung cancer A549cells xenografts in Chemotherapy group was smaller than Traditional Chinese Medicine group, but there was no statistical significance (P>0.05); the tumor weight of nude mice with human lung cancer A549cells xenografts in Combined group was smaller than Traditional Chinese Medicine group with statistical significance (P<0.001). The tumor weight of nude mice with human lung cancer A549cells xenografts in Combined group was smaller than Chemotherapy group with statistical significance (P<0.05).The tumor inhibition rate of Chemotherapy group was25.9%, significantly higher than7.5%of Traditional Chinese Medicine group; the tumor inhibition rate of Combined group was53%, significantly higher than Chemotherapy group, which indicated that Benefiting Qi for Removing Phlegm formula combined with cisplatin has synergistic antitumor effects. The tumor weight index of nude mice with human lung cancer A549cells xenografts in Model group, Traditional Chinese Medicine group, Chemotherapy group and Combined group were2.256±0.237%,1.862±0.301%,1.628±0.171%,1.034±0.371%. The tumor weight index of nude mice with human lung cancer A549cells xenografts in Traditional Chinese Medicine group, Chemotherapy group, Combined group were smaller than Model group. There was statistical significancebetween Traditional Chinese Medicine group and Model group (P<0.05);there was statistical significance between Chemotherapy group and Model group (P<0.01); there was statistical signif icancebetween Combined group and Model group (P<0.001). The tumor weight index of Chemotherapy group was smaller than Traditional Chinese Medicine group, but there was no statistical significance(P>0.05);the tumor weight index of Combined group was smaller than Traditional Chinese Medicine group with statistical significance (P<0.001).The tumor weight index of Combined group was smaller than Chemotherapy group with statistical significance (P<0.01)Fifth, tumor cell apoptosis rate in Model group, Traditional Chinese Medicine group, Chemotherapy group, Combined group were4.60+2.21%、12.33±4.30%、18.17±3.95%、25.60±4.68%. There was statistical significance (P <0.05) between Traditional Chinese Medicine group and Model group; there was statistical significance (P<0.01) between Chemotherapy group and Model group; there was statistical significance (P<0.001)between Combined group and Model group. The tumor cell apoptosis rate in Chemotherapy group was higher than Traditional Chinese Medicine group but there was no statistical significance (P>0.05);the tumor cell apoptosis rate in Combined group was higher than Traditional Chinese Medicine group with statistical significance(P<0.01) The tumor cell apoptosis rate in Combined group was higher than Chemotherapy group with statistical significance(P<0.05)Sixth, Under Biological microscope, central hepatic vein blood stasis were found in liver tissue of nude mice with human lung cancer A549cells xenografts in Model group, Traditional Chinese Medicine group, Chemotherapy group, Combined group. But congestion between groups did not differ significantly, suggesting possible reason could be that bleeding was not completely when put to death animals, nothing to do with drug toxicity. Each group of nude mice with human lung cancer A549cells xenografts remaining organs showed no abnormalities.Seventh, By immunohistochemical method, detection of PERK in tumor tissue of nude mice with human lung cancer A549cells xenografts showed cytoplasmic staining. Strong positive expression rate of PERK in Model group was80%, weak positive expression rate was20%; PERK showed weak positive expression in Traditional Chinese Medicine group, with expression rate of100%. eIF2a showed cytoplasmic staining in tumor tissue of nude mice with human lung cancer A549cells xenografts. Strong positive expression of eIF2a showed completely in Model group, with the expression rate of100%; eIF2a showed completely weak positive expression in Traditional Chinese Medicine group, with expression rate of100%. p-eIF2α showed cytoplasmic staining in tumor tissue of nude mice with human lung cancer A549cells xenografts. The weak positive expression rate of p-eIF2a was20%in Model group, the negative expression rate was80%; p-eIF2a showed weak positive expression in Traditional Chinese Medicine group, with expression rate of100%.Eighth, under the inverted microscope, A549cells in Control group grew well, showed tight connections between cells, fusiform or more bumps, cytoplasm full uniform and nucleus was located in the center of cell tended. Part of A549cells in Experimental group increased cell volume, thin envelope bubble; some cells appear protuberance shortening, cytoplasmic reduction, smaller in size, rounded, and even some cell shed and suspended in culture medium.Ninth, under transmission electron microscopy, Control group showed the characteristics of malignant tumor cells,the nucleus enlarged, deformed, imbalance between the nucleus and cytoplasm, the nucleus membrane integrity, clarity, cell membrane integrity, mitochondria swelling, rough endoplasmic reticulum normal. Experimental group showed the characteristics of early apoptosis. Cell volume was significantly reduced, the structure of cell membrane was complete, nuclear shape is irregular, nuclear fractured, gathered to the edge, condensed into lumps or blocky, distributed in the inner side of the nuclear membrane, mitochondria swelled, rough endoplasmic reticulum expanded.Tenth, Western blot results was that gray value of PERK, eIF2a in Control group were significantly higher than Experimental group; p-PERK, p-eIF2α in Experimental group were significantly higher than Control group.ConclusionFirst, Benefiting Qi for Removing Phlegm formula can inhibit growth of human lung cancer A549cells xenografts in nude mice.Second, Benefiting Qi for Removing Phlegm formula can induce the tumor cells apoptosis in nude mice with human lung cancer A549cells xenografts and A549cells apoptosis in vitro, indicating that inducing tumor apoptosis may be one of the mechanisms that Benefiting Qi for Removing Phlegm formula inhibit growth of human lung cancer A549cells xenografts in nude mice.Third, Benefiting Qi for Removing Phlegm formula combined with cisplatin in inhibiting growth of human lung cancer A549cells xenografts in nude mice and promoting A549cells apoptosis had synergistic effect.Fourth, Benefiting Qi for Removing Phlegm formula can activate PERK-eIF2a signaling pathways, and its effect on apoptosis of A549cells may be related to the activation of PERK-eIF2α signaling pathway. |
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