| Objective:To study the effects of improving proliferation and ischemia-like injury protection of CNKC on PC12cells, neuroprotective effects of CNKC on SH-SY5Y cell apoptosis induced by Aβ peptide, and the effect of CNKC liquid extract on the apoptosis cascade reaction of hippocampus neurons in aging model rats induced by D-galactose. Moreover, to illustrate elementarily pharmacological mechanism which can provide scientific basis for further development of CNKC.Methods:(1) The effects of improving proliferation and ischemia-like injury protection of CNKC on PC12cells.①PC12cells were used as experimental models.0.05mg/mL,0.20mg/mL,0.80mg/mL and2.40mg/mL CNKC extracts were prepared with DMEM and added to the PC12cells as experimental group. Equal volume of DMEM were added to PC12cells as control group. Cell morphous were observed and A value was assayed by MTT at540nm to investigate cell proliferation.②Model group:Ischemia-like injury cell model was induced by Earle’s solution and NaCN solution(final concentration was20mmol/L). Drug group:0.05mg/mL,0.20mg/mL,0.80mg/mL and2.40mg/mL CNKC extracts were prepared with DMEM and added to the PC12cells, then cultivated for4hours. The liquid was blotted and NaCN solution(final concentration was20mmol/L) was added to the cells, cultivated for15mins. Suck the culture solution, wash with D-Hank’s solution twice, add0.5ml DMEM solution and cultivate for24hours. Cell morphous were observed, A value was assayed by MTT at540nm, and LDH in supernatant was assayed. Inhibition ratio of PC12cells was calculated.(2) The effects of improving proliferation and ischemia-like injury protection of serum contain CNKC on PC12cells.112Kunming mice were randomly divided into14groups:control group, Nimodipine group and CNKC groups(96mice,8mice for every group).Equal volume of solvent was given to control group. And16mg/kg of Nimodipine was given to Nimodipine group, bid for6days. Blood samples were collected one hour after the last dose. CNKC group were divided into three groups:(i) single dose,(ii) the second dose after4hours,(iii)bid for3days, eight dose totally. Blood samples were collected0.5h,lh,2h,3h after last dose, and separated serum. Ischemia-like injury PC12cells induced by NaCN were as experimental model cells. Effect of Serum containing CNKC exacts collected in different administration times and different blood collection time on PC12model cells were investigated.1%,2%,4%,8%,16%serum containing CNKC, Nimodipine, blank blood serum were added to culture media. PC12model cells were cultured by using these culture media. A values were tested by MTT assay. Inhibition ratio of PC12cells was calculated.(3) Neuroprotective effects of CNKC on SH-SY5Y cell apoptosis induced by Aβ peptide.①SH-SY5Y cell was cultivated. Different concentration of CNKC exacts were added to the cells to optimize the drug concentration for experiment by MTT assay. SH-SY5Y cells were divided into three groups:control group, model group(Aβ25-3525μmol/L) and drug group(0.725g/L CNKC exacts+AP25-3525μmol/L). After cultivated for24hrs, cellular morphous was observed by inverted microscope. LDH was assayed at440nm. A values were determined at540nm by MTT assay and cellular survival rate was calculated.②mRNA levels of AIF,Cyt C,Bax,Bcl-2assayed by Real Time-PCR. SH-SY5Y cells were divided into three groups:control group, model group(Aβ25-3525μmol·L-1) and drug group(0.725g/L CNKC extracts+Aβ25-3525μmol/L). After cultivated for24hrs, the cells were collected and cellular total RNA was extracted to assay the mRNA levels. The steps of mRNA assay were sample collecting, total RNA extracting, cDNA synthesis by reverse transcription and real time PCR amplification. Beta actin was as reference, and Ct was calculated by the intersection point of amplification curve and valve curve. Relative quantitation was analysed by ΔCt. The expression of destination gene was2ΔCt=2Ctm-Ctn.③AIF, Cyt C, Bax, and Bcl-2protein levels were assayed by Western Blotting. SH-SY5Y cells were divided into three groups:control group, model group(Aβ25-3525μmol/L) and drug group(0.725g/L CNKC exacts+Aβ25-3525μmol/L). After cultivated for24hr, the cells were collected and cellular total protein was extracted to assay the protein levels.④Apoptosis assay. SH-SY5Y cells were divided into three groups:control group, model group(A(325-3525μmol/L) and drug group(0.725g/L CNKC extracts+Aβ25-3525μmol/L). After cultivated for24hr, the cells were collected and assayed by flow cytometry. Excitation wave Ex=488nm, green fluorescence of Annexin V-FITC were detected by FITC(FLl) path and red fluorescence(FL2) was detected by PI (FL2) path.(4) Effect of CNKC extracts on the apoptosis cascade reaction of hippocampus neurons in aging model rats induced by D-galactose.0100valid healthy mice were randomly divided into five groups:normal control group(E), D-galactose model group(D), high dose of CNKC group(A), middle dose of CNKC group(B), and low dose of CNKC group(C).20mice were given to each group. D-galactose was given to the mice of A,B,C,D groups, subcutaneous injection,60mg/kg per day.0.65g/kg, 0.35g/kg and0.15g/kg of CNKC extracts were intragastric administrated to the mice of A,B,C groups respectively. Equal dose of sodium chloride was given to the mice of E group. Continuous rear for three months to obtain AD model. Every group performed the place navigation test and spatial probe test.②Pathology survey. The mice were executed by decapitation. The left hippocampus was taken and carried out the procedure of fixation, dehydration, paraffin imbedding, slice, deparaffinage, hydration, and staining. Morphous of hippocampus tissue was observed under light microscope and neuron amount at CA2-CA3zone of hippocampus was counted. Morphological changes of brain tissue were surveyed after HE staining.③Immunohistochemistry staining. Brain tissue of experimental mice were taken and fixed, midpiece was performed paraffin section and immunohistochemistry staining. Cellular morphous was surveyed and masculine cells were counted under light microscope.④The mice were executed by decapitation. Hippocampus on both sides were separated, weighted and conserved in liquid nitrogen. mRNA levels of AIF, Cyt C, Bax, and Bcl-2were tested by Real Time-PCR.⑤Protein levels of AIF, Cyt C, Bax, and Bcl-2tested by Western Blotting. The mice were executed by decapitation. Hippocampus on both sides were separated and weighted. Lysate was added to tissue homogenate and centrifuge. Total protein quantitation of supernatant was tested by BCA method.(5)SPSS13.0statisties software was used to analyze the data.The measurement data indicated by the mean value and standard deviation. The results comparison of proliferation effect on PC12cells, mice learning train scores were by repeated-measure variance analysis and multiple comparisons with LSD. The result of PC12cells ischemic injury effected by different concentrations of drug serum was by factorial design univariate and LSD. All of other data were analyzed by One-Way ANOVA and multiple comparisons with LSD(Homogeneity of variance) or Dunnett T3(Heterogeneity of variance). Significance level was a=0.05(bilateral), and P<0.05expressed the difference with statistics significance.Result:(1)①Cellular Morphous:Observed with inverted microscope. Control group:After cultured for24hrs, the cells grew rapidly. Most of them were fusiform and minority were triangle. After cultured for48hrs, the cells were in logarithmic phase. After cultured for72hrs, the cells grew retardation and there were some floating cells in some holes. Drug group:cellular morphous had no changes and no injury. After cultured for48hrs, cells grew well. After cultured for72hrs, the cells grew slowly. The results of MTT showed that, compared with control group, Absoption values of0.05mg/ml group at24hr,48hr24hr had no significant difference and no cellular proliferation (F=20.043, P=0.476; F=28.729, P=0.957; F=15.977,P=0.597). A values of0.20mg/mL,0.80mg/mL,2.40mg/mL groups at24hr,48hr24hr had significant difference and cellular proliferation (P<0.05).All of them could improve proliferation.②In model group, the cellular apophysis disappeared, cellular swelled, diopter descended and some cells were splited to fragment. Pretreated by CNKC extracts, the morphology of ischemia-like injury cells could change obviously. Fewer cellular apophysis disappeared and cell debris could be observed. The results of MTT showed that, compared with model group(1.52±0.15), A value of normal control group was1.98±0.02(Welch F=561.380, P=0.001) and cellular vigor of model group declined significantly. A values of0.05mg/ml group had no statistical significance (P=0.976). A values of0.20mg/mL (P=0.007),0.80mg/mL (P=0.001),2.40mg/mL (P=0.000) groups were higher than those of model group. Cell injury inhibition ratio were13.0%,60.9%,80.4%, and93.5%. And it showed that CNKC extracts could improve cellular vigor obviously. The results of LDH assay showed that, compared with model group(657.2±47.2), LDH value of normal control group was270.8±16.0(Welch F=273.717, P=0.000) and lower than the value of model group. LDH values of0.05mg/ml group had no statistical significance (P=0.978). LDH values of0.20mg/ml,0.80mg/ml, and2.40mg/ml groups were lower than those of model group. The cell injury inhibition ratio were6.0%,28.6%,66.8%, and91.8%. LDH would increased if cell injuried. LDH of model group was higher than that of normal control group. CNKC extracts could lower the release of LDH and improve cellular damage obviously.(2)①Effect of serum containing CNKC extracts collected in different administration times and different blood collection time on PC12cells was investigated. Compared with model group(A value was1.43±0.12), control group was significantly different (F=142.627, P=0.000).A values of single dose serum containing CNKC extracts collected0.5hr,1hr,2hrs,3hrs were1.37±0.09,1.41±0.09,1.45±0.10,1.45±0.07(P=1.000), and showed no cytoprotection. A values of twice doses serum containing CNKC extracts collected1hr,2hrs were1.77±0.13, and1.78±0.12(P<0.05), much higher than those of model group and showed obvious cytoprotection. A values of eight doses serum containing CNKC extracts collected0.5hr,1hr,2hrs,3hrs were1.86±0.06,1.90±0.08,1.810±0.09, and1.79±0.11. All of them showed obvious cytoprotection, especially for0.5hr and lhr(P<0.01).②Effect of Serum containing different concentration of CNKC extracts on PC12cells was investigated. Compared with model group(A value was1.41±0.08), A values of serum containing2%CNKC extracts was1.75±0.09and showed cytoprotection. Both serum containing4%CNKC extracts and Nimodipine showed cytoprotection.(3) The result of cellular morphous showed that after effected by Aβ25-35for72hrs, cellular diopter of the model group dropped, cell rounded and gathered, axon length and cell body were shorter than that of normal control group. However, compared with model group, cellular morphous of drug group was greatly improved. Their cellular synapse and diopter increased and the cells were well-distributed. The results of MTT assay showed that A value of model group(0.590±0.006) was much lower than that of control group(0.631±0.005)(F=147.104, P=0.000), and A value of drug group(0.622±0.004) was higher than that of model group(P=0.000). Cytoactive of SH-SY5Y could be degraded by Aβ25-35, and CNKC extracts could lessen cellular injury. The results of LDH assay showed that LDH of model group(1345.7±98.7) was higher than that of control group(789.1±63.5)(F=181.790, P=0.000), and LDH of drug group(675.7±56.6) was lower than that of model group (P=0.007). This result also showed that Cytoactive of SH-SY5Y could be degraded by Aβ25-35, and CNKC extracts could lessen cellular injury.②Result of mRNA levels. A260nm/A280nm of RNA was between1.8and2.0. Electrophoresis results showed that the straps of28S and18S RNA were very clear. All these illustrated that RNA was with high purity and had no degradation. RNA levels of control group, model group and drug group were1140ng/μl,1044ng/μl, and1020ng/μl respectively. According to the result of real-time PCR, AIF/actin mRNA relative amount of control group, model group and drug group were0.0102+0.0027,0.1023±0.0138,0.0252±0.0043; Cyt C/actin were0.0256±0.0065,0.0788±0.0062,0.0331±0.0013; Bax/actin were0.0203±0.0012,0.1707±0.0096,0.0380±0.0010; Bcl-2/actin were0.0409±0.0052,0.0090±0.0015,0.0252±0.0013。 Compared with control group, AIF,Cyt C,Bax mRNA amounts of model group were increased (F=60.846, P=0.013; F=89.670, P=0.000; F=420.101, P=0.002),and Bcl-2mRNA amount was reduced (F=104.859, P=0.012). Compared with model group, AIF,Cyt C,Bax mRNA amounts of drug group were reduced (P=0.014; P=0.000; P=0.002), and Bcl-2mRNA amount was increased (P=0.000).The result showed that CNKC exacts could protect injury SH-SY5Y cells induced by AP25-35.③ Protein concentration of control group, model group and drug group were16.170μg/μl,16.146μg/μl, and16.335μg/μl, respectively. According to the grey values, AIF levels of control group, model group and drug group were AIF levels were0.0145±0.0003,0.0502±0.0003,0.0261±0.0010, Bax levels were0.0252±0.0003,0.0871±0.0008,0.0519±0.0011, Cyt C levels were0.0240±0.0008,0.1045±0.0006,0.0653±0.0007, Bcl-2levels were0.0910±0.0057,0.0144±0.0003,0.0697±0.0010. Compared with control group, AIF/beta actin,Bax/beta actin.Cyt C/beta actin of model group were increased (Welch F=20950.876, P=0.000; Welch F=13197.318, P=0.000; Welch F=6704.384, P=0.000),and Bcl-2/beta actin was reduced(F=14872.548, P=0.000). Compared with model group, AIF/beta actin,Bax/beta actin,Cyt C/beta actin of drug group were reduced (P=0.000; P=0.000; P=0.000), and Bcl-2/beta actin was increased(P=0.000).The result showed that CNKC exacts could protect injury SH-SY5Y cells induced by AP25-35-.④Apoptosis result showed apoptosis rate of control group, model group and drug group were3.71±1.74,17.12±3.52and7.20±1.15. Compared with control group, apoptosis rate of model group were increased (F=26.038, P=0.000). Compared with model group, apoptosis rate of drug group were reduced (P=0.002).The result showed that CNKC exacts could protect injury SH-SY5Y cells induced by Aβ25-35.(4)①ompared with model group, in the first quadrant(NE), there were no differences of the time and distance to safe platform of group C,D,E(P>0.05). However, from the second quadrant, the data was fewer than that of model group(P<0.05). The result showed that CNKC extracts could improve the skill of learning of AD mice.②The result of HE staining showed that there was no obvious impairment in neurons of hippocampal formation (E group), and the cells were normal with clear structure. Morphous of neuron cells were integrity, arranged orderly, with large nucleus and clear nucleoli. Hippocampal formation of AD model group had integrated tissue. However, less cellular layer, incompleted neuron morphous, raritas and disordered cellular arrangement, smaller nucleus, anachromasis, obscure structure and derangement nerve fiber could be seen. Result of pathological section (group A,B,C) showed that there was light impairment in hippocampal formation, and the cellular layer was acceptable with clear structure.③Result of mRNA levels. A260nm/A280nm of RNA was between1.8and2.0. Electrophoresis results showed that the straps of28S and18S RNA were very clear. All these illustrated that RNA was with high purity and had no degradation. RNA levels of group E,D,A,B,C were1248ng/μl,1328ng/μl,1156ng/μl,1220ng/μl, and1304ng/μl. According to the result of real-time PCR, AIF/actin mRNA relative amount of group E,D,A,B,C were0.0036±0.0007,0.0092±0.0008,0.0045±0.0005,0.0051±0.0004,0.0075±0.0012; Cyt C/actin were0.0030±0.0005,0.0128±0.0023,0.0059±0.0015,0.0085±0.0022,0.0102±0.0008; Bax/actin were0.0028±0.0003,0.0127±0.0024,0.0050±0.0009,0.0060±0.0014,0.0094±0.0014; Bcl-2/actin were0.0281±0.0012,0.0027±0.0002,0.0152±0.0021,0.0103±0.0014,0.0072±0.0015. Compared with control group, AIF/beta actin,Bax/beta actin,Cyt C/beta actin of model group were increased (P<0.05),and Bcl-2/beta actin was reduced (P<0.05). Compared with model group, AIF/beta actin,Bax/beta actin,Cyt C/beta actin of drug groups were reduced (P<0.05), and Bcl-2/beta actin was increased(P<0.05).The result showed that CNKC exacts could protect apoptosis cascade reaction of hippocampus neurons in aging model rats induced by D-galactose.④Protein concentration of group E,D,A,B,C were17.995μg/μl,18.082μg/μl,18.388μg/μl,17.901μg/μl,18.239μg/μl, respectively. According to the analysis of grey value, AIF levels of group E,D,A,B,C were0.2978±0.0195,0.7464±0.0389,0.3312±0.0297,0.6133±0.0426,0.5559±0.0393, Bax were01989±0.0120,0.7294±0.0512,0.2617±0.0144,0.3309±0.0235, 0.4755±0.0326, Cyt C were0.2292±0.0133,0.8553±0.0501,0.4185±0.0300,0.4928±0.0381,0.5802±0.0394, Bcl-2were0.5673±0.0294,0.1994±0.0115,0.4456±0.0318,0.4304±0.0313,0.1987±0.0119.⑤Results of immunohistochemistry of Cyt C showed that masculine cells of group E were widespread distributed in hippocampus and cell population was significant deviation from group D. Masculine cell population of model group was3.7times, than that of normal control group. Abnormality of these proteins express could be recovered by CNKC extracts in some degree.Conclusions:(1)CNKC extracts could improve proliferation and protection on ischemia-like injury PC12cells induced by NaCN.(2) Serum containing CNKC extracts could improve proliferation and protection on ischemia-like injury PC12cells induced by NaCN and increase survival cells.(3) CNKC extracts had neuroprotective effects on SH-SY5Y cell apoptosis induced by Aβ25-35.(4) CNKC extracts had the effect on the apoptosis cascade reaction of hippocampus neurons in aging model rats induced by D-galactose. In addition, the pharmacological mechanism of CNKC could be illustrated elementarily so as to provide scientific basis for further AD clinical treat. |
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