| ObjectiveTo evaluate effect of WKKL alleviates CRF which caused by the tumor and the chemotherapy of paclitaxel (PTX) in breast cancer mice CRF model. Reveal the mechanism of WKKL alleviates CRF and chemotherapy related fatigue.Methods1. The metabolism mechanism of WKKL alleviates CRF.Before the experiment, all female BALB/c mice were trained in the swimming tank (25℃±1℃) for2days (5min/day). Then, exclude the mice which the swimming time is too long or too short and too excited or too quiet. Randomly selected10mice as normal controls (NS, po,20ml/kg, qd) mice, and the rest mice were prepared for breast cancer cell inoculation.4TI breast cancer cells were inoculated in subcutaneous of the fourth pair breast fat pad region of mouse right side of the abdominal wall to prepare the mouse model of CRF. Each mouse was injected0.1ml4T1breast cancer cells (1×106cells/ml). When the tumor grows to50mm3and100mm3(about day7after inoculation), the mice were divided in to four groups (10mice/group) according to the tumor volume. The four groups are tumor control (NS, po,20ml/kg, qd) group, PTX (PTX, ip,10mg/kg, q2d) group, WKKL (WKKL, po,1.5g/kg, qd) group and WKKL+PTX (WKKL, po,1.5g/kg qd; PTX, ip,10mg/kg, q2d) group. Then the following aspects of the experiment were carried out on the5groups of mice (NC group, TC group, PTX group, WKKL group and WKKL+ PTX):(1) The effect of WKKL on the general situations of the tumor-bearing mice:The Weight, activity, mental state, fur appearance changes, and diet of mice in each group were observed during the experiment. The general situations of the mice was evaluated for each week.(2) The effect of WKKL on the behavior of the tumor-bearing mice:All mice were conducted open field test at the12th day after administration. The total movement distance, the distance of peripheral lattice, the distance of central lattice, frequency of grooming, climbing frequency and stool amount of mice in the experimental tank were observed.30min after the last administration, observe the immobility time of all mice by the TS-200tail suspension test instrument.(3) The effect of WKKL on the survival time of tumor-bearing mice:Observe the survival time of mice of the behavior experiment without any interventions. When all mice were died, calculate the mean survival time, median survival time and the life extension rate of mice in each group.(4) The effect of WKKL on the anti-fatigue effects of the tumor-bearing mice: Take another of the5groups of mice. Then observe the exhaustive swimming time of all mice in the weight-loaded swimming test. A tin wire (7%of body weight) was loaded on the tail root of each mouse. The mice were tested three times (0d,10d and20d after administration).(5) The effect of WKKL on the tumor growth inhibition of the tumor-bearing mice:Take another of the5groups of mice. Two-dimensional measurements were taken with calipers every three days and calculate the tumor volume. After the last administration, all mice were sacrificed, collected the blood and separate the plasma, stored at-80℃for metabolomics research. Then peel the tumor tissue and weighted. And the tumor inhibitory rate was calculated.(6) The effect of WKKL on the organs index of the tumor-bearing mice: weighted the spleen, thymus and adrenal gland take from the previous experiment, and calculate the organs index.(7) The effect of WKKL on the plasma metabolomics of CRF mice model: Metabolomic finger printing was acquired by1H-NMR. Multivariate data statistic (PCA,PLS-DA,OPLS-DA) was used to pattern recognition. Then we got the5groups of mice plasma metabolic profiles. Then we compare the data group between group. Then chosen the VIP and normalized integral value filter out the differences metabolites (potential molecular markers) in plasma between groups. The endogenous metabolites were assayed by HMBD data base and other document. Then, analyze the changed metabolic pathways. At last, reveal the mechanism of CRF and how WKKL alleviates the CRF.2. The gene mechanism of WKKL alleviates chemotherapy related fatigue:Before the experiment,25female BALB/c mice were trained in the swimming tank (25℃±1℃) for2days (5min/day). Then, exclude the mice which the swimming time is too long or too short and too excited or too quiet. The mice were divided in to three groups as normal controls (NS, po,20ml/kg, qd) group, PTX (PTX, ip,10mg/kg, q2d) group, and WKKL+PTX (WKKL, po,1.5g/kg qd; PTX, ip,10mg/kg, q2d) group. Then the following aspects of the experiment were carried out on the3groups of mice:(1) The effect of WKKL on the general situation of the chemotherapy-bearing mice:The Weight, activity, mental state, diet, and fur appearance changes of mice in each group were observed during the experiment. The general situation of the mice was evaluated for each week.(2) The effect of WKKL on the anti-fatigue effects of the chemotherapy-bearing mice:Observe the exhaustive swimming time of all mice in the weight-loaded swimming test. A tin wire (7%of body weight) was loaded on the tail root of each mouse. The mice were tested three times (0d,7d and14d after administration).(3) The effect of WKKL on the skeletal muscle genomics of chemotherapy related fatigue mice model:Gene chip technology was acquired to obtain the original genetic information in the skeletal muscles of mice in the experiments, Affmetrix GCOSvl.4(gene chip operating software versionl.4) was used to of obtain the fluorescence signal intensity and ratio calculation and statistical analysis to get single analysis results of each chip to chip quality control process. Compare the difference of gene expression between the three groups. Analyze the related pathways, reveal the correlation of fatigue mechanism of chemotherapy, and the mechanism of WKKL alleviate chemotherapy related fatigue.Results1. The metabolism mechanism of WKKL alleviates CRF.(1) The effect of WKKL on the general situation of the tumor-bearing mice: Before the administration, the general mental state of mice between the5groups had no significant difference. Mice in each group were lively. All mice were sensitive to food and escape quickly. The situation of mice in the NC group keeps the same during the experiment. Compared with NC group mice, the other4groups mice’s hair were gradually sparse, and they gradually inactive, move slowly and eating less. At the end of the experiment, the general situation of mice in this4groups from the poor to relatively good order were TC group, PTX group, WKKL+PTX group and WKKL group. Before administration the body weight of mice in each group had no significant difference (F=0.100,P=0.982). However, after1week administration, body weight in NC group were significantly increased compared to the previous (P<0.01), and significantly heavier than PTX(P=0.000). After2weeks administration, body weight in NC group were significantly increased compared to the beginning (P<0.01), but there had no significant increase compared with intervention1st week (P>0.05). The body weight of mice in NC group were significantly heavier than other4groups (P<0.01). The body weight of mice in TC group, WKKL group and WKKL+PTX group had no significant changes to the beginning (P>0.05), but significant changes to the1st week (P<0.05). The body weight of mice in PTX group had significant changes to the beginning and1st week (P<0.01). Compared with TC group mice, the body weight of mice in PTX group had significantly changes (P<0.01). Compared with PTX group mice, the body weight of mice in WKKL group and WKKL+PTX group had significant changes (P<0.01). After3weeks administration, the body weight of mice in each group maintain at the level of the2week. (2) The effect of WKKL on the behavior of the tumor-bearing mice:(a) open-field experimental results:The farthest total movement distances were the mice in NC group, and PTX group were the least (PTC=0.014、PPTX=0.003). Compared with mice in NC group, the total movement distance of mice in TC group and PTX group were significantly differences (PTC=0.000)、PPTX=0.000). The farthest distance of peripheral lattice was mice in NC group, and TC group (P=0.006) were the least. Compared with mice in NC group, the distance of peripheral lattice of TC group, PTX group and WKKL group of mice were significantly reduced, and had no significant difference in WKKL+PTX group(P>0.05). Compared with mice in TC group, the stool amount in NC group, PTX group and WKKL+PTX group were significantly decreased (P<0.05～0.01). Compared with mice in PTX group, the stool amount in NC group, TC group, WKKL Group and WKKL+PTX group were significantly increased (P<0.05～0.01). The frequencies of climbing of mice in NC group were the most, and least in WKKL group. Compared with mice in NC group, the frequencies of climbing of mice in WKKL group and WKKL+PTX group were significantly decreased (P<0.05～0.01). The mice in PTX group and WKKL+PTX group had higher frequencies of grooming than other groups, the mice in TC group had the least. Compared with mice in NC group, the frequency of grooming of mice in TC group and WKKL group were significantly decreased (P<0.05～0.01). Compared with mice in TC group, the frequency of grooming of mice in NC group, PTX group and WKKL+PTX group were significantly increased (P<0.01). Compared with mice in PTX group, the frequency of grooming of mice in TC group and WKKL group were significantly decreased (P<0.05～0.01).(b) Tail suspension test results: The immobility time of mice in TC group were longer than NC group, but there was no significant difference between the two groups (P=0.308). Compared with mice in TC group, the immobility time of mice in WKKL group (P=0.004) and WKKL+PTX group (P=0.001) were significantly shorter(3) The effect of WKKL on the survival time of tumor-bearing mice:Compared with mice in TC group, the median survival time and the average survival time in other3groups were significantly prolonged (P<0.05-0.01). Compared with mice in PTX group, the median survival time and the average survival time in WKKL+PTX group were significantly prolonged (P=0.039), and significantly decreased in TC group. Compared with mice in TC group, the survival time of WKKL+PTX group, WKKL group and PTX group were prolonged21.61%,13.07%and8.54%, respectively.(4) The effect of WKKL on the anti-fatigue effects of the tumor-bearing mice:(a) Swimming time between the groups at same time points:There were no significant differences between the5groups (P=0.992) at the beginning. At10th day after administration, the swimming ability of mice in NC group were significantly longer than TC group, PTX group and WKKL+PTX group (P=0.01). Compared with mice in TC group, the swimming time in PTX group, WKKL group and WKKL+PTX group had no significant differences (P=0.05). Compared with mice in PTX group, the swimming ability of mice in NC group and WKKL group were significantly prolonged (P=0.05～0.01). At20th day after administration, the swimming ability of mice in NC group were significantly longer than other4groups (P=0.000) Compared with mice in TC group, the swimming time of mice in PTX group, WKKL group and WKKL+PTX group had no significant differences (P>0.05). Compared with mice in PTX group, the swimming ability of mice in WKKL+PTX group and WKKL group were significantly prolonged (P<0.01).(b) Swimming time at different time points of each group mice:With the intervention time increased, the swimming ability of mice in NC group showed a gradual increase trend, and a gradual decrease trend in TC group, PTX group and WKKL+PTX group. Compared with the mice in NC group at0day, the swimming ability was significantly increased at10th day and20lh day (P=0.05～0.01). Compared with the mice in TC group at0day, the swimming ability had no significantly changes at10th day (P>0.05), but was significantly decreased at20th day (P<0.01). Compared with the mice in PTX group at0day, the swimming ability was significantly decreased at10th day and20th day (P<0.01). Compared with the mice in WKKL group at0day, the swimming ability had no significantly changes at10th day (P>0.05), but was significantly decreased at20th day (P<0.05). Compared with the mice in WKKL+PTX group at0day, the swimming ability was significantly decreased at10th day and20th day (P=0.05～0.01).(5) The effect of WKKL on the tumor growth inhibition of the tumor-bearing mice:Compared with mice in TC group, the tumor weight in WKKL+PTX group (P=0.007) and PTX group (P=0.037) were significantly lighter. Compared with mice in TC group, the tumor volume in WKKL+PTX group were significantly smaller (P<0.05). The tumor inhibition rates of WKKL+PTX group, WKKL group and PTX group were22.24%,15.36%and16.89%, respectively.(6) The effect of WKKL on the organs index of the tumor-bearing mice:(a) Spleen index:Compared with mice in NC group, the spleen index in tumor bearing mice (TC group, PTX group, WKKL group and WKKL+PTX group) were significantly increased (P<0.01). Compared with mice in TC group, the spleen index in PTX group, WKKL group and WKKL+PTX group have no difference (P>0.05). Compared with mice in PTX group, the spleen index in WKKL group and WKKL+PTX group have no difference (P>0.05).(b) Thymus index:The thymus index of mice in NC group was higher than other groups, and the least was in PTX group. Compared with mice in NC group, the thymus index of mice in tumor bearing mice (TC group, PTX group, WKKL group and WKKL+PTX group) were significantly decreased (P<0.01). Compared with mice in TC group, the thymus index of mice of PTX group were significantly decreased (P=0.001). Compared with mice in PTX group, the thymus index of mice in WKKL group (P=0.010) and WKKL+PTX group (P=0.006) were significantly increased,(c) Adrenal index: Compared with mice in NC group, the adrenal index of mice in TC group and PTX group were significantly increased (P<0.01). Compared with mice in TC group, the adrenal index of mice in WKKL+PTX group were significantly decreased (P=0.037). Compared with mice in PTX group, the adrenal index of mice in WKKL group and WKKL+PTX group had no difference (P>0.05).(7) The effect of WKKL on the plasma metabolomics of CRF mice model:(a) The5groups1H-NMR-based plasma analysis of CRF mice:The samples of5groups were divided by the PLS-DA model, and calculated2principal components,(b) Compare between NC group and TC group:The PCA and PLS-DA model can divided the samples of NC group and TC group. And the OPLS-DA model was divided better. Then, found16significant metabolites by the load scatter plot, S-plot, VIP value and normalized integral value (P<0.05-0.01). Compared with mice in NC group, the levels of Lactic acid, glycerol phosphate, spermine, leucine, alanine, glucose-6-phosphoric acid,3-hydroxy butyric acid were increased in TC group, and the levels of Choline, inositol phosphate, coenzyme A, octanoyl glycine,3-hydroxy isovaleric acid, acid, methyl malonic acid, aspartic acid were decreased in TC group,(c) Compare between TC group and PTX group:The PCA and PLS-DA model were divided the samples of TC group and PTX group. And the OPLS-DA model was divided better. Then, found13significant metabolites by the load scatter plot, S-plot, VIP value and normalized integral value (P<0.05-0.01). Compared with mice in TC group, the levels of citrulline were increased in PTX group, and the levels of Choline, inositol phosphate, citric acid, glycerol phosphate, linoleic acid, glutamine, coenzyme A, aspartic acid, serine, cysteine, erythrose were decreased in PTX group,(d) Compare between TC group and WKKL group:The PCA and PLS-DA model can divided the samples of TC group and WKKL group. And the OPLS-DA model was divided better. Then, found1significant metabolites by the load scatter plot, S-plot, VIP value and normalized integral value (P<0.05-0.01). Compared with mice in TC group, the level of glycerol3-phosphate was increased in WKKL group,(e) Compare between TC group and WKKL+PTX group:The PCA and PLS-DA model were divided the samples of TC group and WKKL+PTX group. And the OPLS-DA model was divided better. Then, found17significant metabolites by the load scatter plot, S-plot, VIP value and normalized integral value (P<0.05-0.01). Compared with mice in TC group, the levels of Propionyl glycine, citrulline, isoleucine, octanoyl glycine,2-hydroxy butyric acid were increased in WKKL+PTX group, and the levels of Inositol, dihydroxy acetone, phosphoric acid, choline, spermine,3-hydroxy butyric acid and malonic acid, aspartic acid, serine, L-valine, pyruvic acid, succinic acid and aldehyde, amino caproic acid, beta alanine were decreased in WKKL+PTX group.(f) Compare between PTX group and WKKL+PTX group:The PCA and PLS-DA model were divided the samples of PTX group and WKKL+PTX group. And the OPLS-DA model was divided better. Then, found5significant metabolites by the load scatter plot, S-plot, VIP value and normalized integral value (P<0.05-0.01). Compared with mice in PTX group, the levels of Nervonic acid, coenzyme A, phosphoenolpyruvate, inositol and erythrose were increased in WKKL+PTX group,(g) Compare between WKKL group and WKKL+PTX group:The PCA and PLS-DA model were divided the samples of PTX group and WKKL+PTX group, but here are still some overlap between the two groups. And the OPLS-DA model was divided better. Then, found12significant metabolites by the load scatter plot, S-plot, VIP value and normalized integral value (P<0.05-0.01). Compared with mice in WKKL group, the levels of Propionyl glycine, citrulline, glycerophosphate,2-hydroxy butyric acid, octanoyl glycine, isoleucine, corticosterone, folic acid, ferulic acid, hydrogen, phenylalanine, alanine were increased the levels of Choline phosphatein was decreased in WKKL+PTX group.2. The gene mechanism of WKKL alleviates chemotherapy related fatigue:(1) The effect of WKKL on the general situation of the chemotherapy-bearing mice:Before the administration, the general mental state, activity, diet, and fur appearance of mice between the3groups had no significant difference. Mice in each group were lively. Their fur was bright, clean and tidy. Their eyes were bright. All mice were sensitive to food and escape quickly. The situation of mice in the NC group keeps the same during the experiment. Compared with NC group mice, the other2groups mice’s hair were gradually sparse, and they gradually inactive, move slowly and eating less. At the end of the experiment, the general situation of mice in this2groups from the poor to relatively good order were PTX group, WKKL+PTX group. Before administration the body weight of mice in each group had no significant difference (F=0.066, P=0.936). However, after1week administration, body weight in NC group were significantly increased compared to the previous (P=0.002) After2weeks administration, body weight in NC (P=0.001) and WKKL+PTX group (P=0.002) were significantly increased compared to the beginning, but there had no significant increase compared with intervention1st week (P>0.05). Compared with PTX group mice, the body weight of mice in NC group (P=0.000) and WKKL+PTX group (P=0.000) had significant changes.(2) The effect of WKKL on the anti-fatigue effects of the chemotherapy-bearing mice:(a) Swimming time between the groups at same time points:There were no significant differences between the3groups (P=0.992) at the beginning. At7th day after administration, the swimming ability of mice in NC group were significantly longer than PTX group(P=0.000) and WKKL+PTX group(P=0.001). Compared with mice in PTX group, the swimming ability of mice in NC group (P=0.000) and WKKL+PTX group (P=0.007) were significantly prolonged. At14th day after administration, the swimming ability of mice in NC group were significantly longer than other2groups (P=0.01). Compared with mice in PTX group, the swimming ability of mice in WKKL+PTX group and NC group were significantly prolonged (P <0.01).(b) Swimming time at different time points of each group mice:With the intervention time increased, the swimming ability of mice in NC group showed a gradual increase trend, and a gradual decrease trend in PTX group and a volatility trend in WKKL+PTX group. Compared with the mice in NC group at0day, the swimming ability was significantly increased at14th day (P=0.026). Compared with the mice in PTX group at0day, the swimming ability was significantly decreased at14th day (P=0.002)(3)The effect of WKKL on gene mechanism:There were47different genes between PTX and WKKL+PTX group.39were up-regulated, and8were down-regulated. Atp4a, Pla2g1b, Pikfyve, Lpin2, Pld4, Ppm1h, Acot10, etc. participated in the mitochondrial energy metabolism. S100a9, Igh-6, Ghrl, etc. participated in in the inflammatory response, humoral immune response, cell adhesion, defense response. There were involved in multiple signal transduction pathways, including VEGF signaling pathway, Fc epsilon RI signaling pathwaysignaling pathway and MAPK signaling pathway GnRH signaling pathway and Phosphatidylinositol signaling system, etc.Conclusion(1) The mouse inoculation with4TI breast cancer cells at the breast fat pad region of the right side of the abdominal wall subcutaneous exist CRF. The degrees of CRF were related to the tumor growth. The occurrence of CRF might related to the disturbance of the metabolic pathways of tricarboxylic acid cycle, fatty acid metabolism, β-alanyl acid metabolism, amino acid metabolism, gluconeogenesis, pyruvate metabolism,glucose alanine cycle, mitochondrial electron transport chain and malate-aspartate shuttle.(2) PTX can aggravated CRF, and the degrees of CRF were aggravated according to the chemotherapy period prolonged. The mechanism of PTX aggravated CRF might related to the disturbance of the metabolic pathways of Pyruvate metabolism, tricarboxylic acid cycle, metabolism of amino acid metabolism, and spermine and spermine, metabolism of fatty acid metabolism, Lipid metabolism, glycerol phosphate shuttle, synthesis of phospholipids, mitochondrial electron transport chain.(3) WKKL can improve the general situation of the tumor-bearing mice, prolong the survival time. WKKL can’t alleviate CRF. Thoes effect of WKKL might related to the function of regulated the metabolic pathways of inositol metabolism, glycerol lipid metabolism, glycerol phosphate shuttle and mitochondrial electron transport chain.(4) The combination therapy of WKKL and PTX can significantly alleviate CRF which caused by the breast cancer and the chemotherapy of PTX. The effect of alleviate CRF of the combination therapy of WKKL and PTX was better than only use WKKL. The mechanism of combination therapy of WKKL and PTX alleviate CRF might related to the function of regulated the metabolic pathways of The Krebs cycle, metabolism of fatty acid metabolism, propionic acid, pyruvate metabolism, spermine and spermine metabolism, amino acid metabolism, sugar, glucose and alanine cycle dysplasia, pyruvate metabolism, muscle metabolism of alcohol, glycerol phosphate shuttle, mitochondrial electron transport chain, malic acid-aspartate shuttle, the synthesis of phospholipids.(5) WKKL can improve the fatigue of the chemotherapy-bearing mice, The mechanism of WKKL alleviate chemotherapy related fatigue might related to the genes expression of energy metabolism, immune function, signal transduction pathways. |
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