CN102008493A - Application of panaxadiol saponin in preparing medicines combined with glucocorticoid receptor - Google Patents
Application of panaxadiol saponin in preparing medicines combined with glucocorticoid receptor Download PDFInfo
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Abstract
The invention relates to application of panaxadiol saponin (PDS) in preparing medicines combined with a glucocorticoid receptor, and belongs to the field of medicines. The panaxadiol saponin is applied to preparing the medicines combined with the glucocorticoid receptor, and the panaxadiol saponin and cis-platinum are jointly applied to resisting tumors; and in the study that the PDS and the cis-platinum are combined to resist prostatic cancer transplanting tumors, the PDS has antiallergic and attenuation effects like Dexa, but the PDS group has the survival time, spleen index, fur brightness degree and the like superior to those of the Dexa group. Mice in a Dexa and cis-platinum combined administration group all die before the 13th days in the course of treatment particularly. Therefore, the PDS has the same molecular mechanism as the Dexa, and has important significance for developing new medicines in which the PDS replaces the Dexa.
Description
Technical field
The invention belongs to field of medicaments.
Background technology
Dexamethasone (Dexamethasone, glucocorticoid effect and the record of side effect on pharmacology's textbook such as Dexa): (1) pharmacotoxicological effect is that Dexa has antiinflammatory, antiendotoxin, inhibition immunity, shock and strengthens pharmacological action such as stress, so being widely used in each section treats multiple disease, as autoimmune disease, irritated, inflammation, asthma and department of dermatologry, ophthalmic diseases etc.In recent years, dexamethasone clinical application amount increases year by year, and China has become maximum in the world dexamethasone market so far.(2) description of side effect summary is as follows: 1. long-range or use Dexa that iatrogenic cushing's syndrome face and figure, hypokalemia syndrome, bleeding tendency can take place in a large number; 2. bad, the osteoporosis of wound healing so that ischemic necrosis of femoral head takes place, digestive tract irritability is hemorrhage etc.; 3. the recurrence of accompanying infection or old tuberculose focus also is the untoward reaction of adrenocortical hormone.
Dexamethasone (Dexa) has write the medicine description as the adjuvant of paclitaxel antitumor action: write in paclitaxel injection " usage and dosage " column: " in order to prevent to take place anaphylaxis, at preceding 12 hours oral dexamethasone 10mg of paclitaxel treatment, treat preceding 6 hours oral dexamethasone 10mg again, treat and gave diphenhydramine intramuscular injection 20mg in preceding 30～60 minutes ".Dexa has been widely used in the clinical oncotherapy as the treatment adjuvant of paclitaxel and cisplatin etc., plays an important role at the allergy and the toxic reaction of prevention sickened body to chemotherapeutics.Thereby the chemotherapy of tumor can be carried out smoothly.But the acute stress due to the glucocorticoid is hemorrhage to be caused danger disease death and becomes stealthy killer.Exploring a kind ofly has the effect of glucocorticoid sample, and it is significant not have the medicine of its side effect.
Summary of the invention
The invention provides the application of a kind of Panaxadiol saponin in preparation and glucocorticoid receptor (GR) bound drug.Purpose is to disclose the molecular mechanism that Panaxadiol saponin PDS has the effect of Dexa sample, provides theoretical foundation for Panaxadiol saponin is developed to the medicine that substitutes glucocorticoids such as dexamethasone.
The application of Panaxadiol saponin in preparation and glucocorticoid receptor (GR) bound drug.
Panaxadiol saponin (Panaxadiol Saponins, PDS) effect of dexamethasone sample is arranged but do not have the experimentation of hormonelike side effect: (1) finds that PDS has the anti-hemorrhagic shock effect with the Dexa sample, do not cause the side effect that gastrointestinal stress is hemorrhage and have: for disclosing material base and the mechanism of motherland's medical science about " Ginseng Decoction has the effect of coming back to life ", and done serial research, carried out the anti-hemorrhagic shock experimental study of effect of PDS with the positive contrast medicine of Dexa.Found that PDS has the similar anti-hemorrhagic shock effect with Dexa, and does not have the hormonelike side effect.Be surprised to find that in experiment Dexa group dog has the mean blood pressure of 2 dogs (2/15) to reduce to 0 rapidly in blood-letting after mean arterial pressure drops to 40mmHg, blood pressure still sharply descends and death after feeding back whole blood, postmortem finds that gastrointestinal tract is full of blood, and it is hemorrhage to be diagnosed as the gastrointestinal stress ulcer.Clinically, in the rescue of danger diseases such as various shocks, also have similar unexpected the generation when widely applying Dexa, but can't differentiate, be difficult to cause the attention of clinical experts with the death that danger disease causes.So, be referred to as " stealthy killer ".For further this significant discovery certainly, we have finished PDS antiendotoxin shock, research that anti-losing blood/the endotoxin doubles are hit again in succession, its result proves that Panaxadiol saponin in the research of anti-various shocks, all shows the effect that is better than the dexamethasone class.
In with PDS and the research of cisplatin combined anti-carcinoma of prostate transplanted tumor, find that PDS has Dexa sample antiallergic and Attenuation, but the bright degrees of PDS group time-to-live, index and spleen index and fur etc. all are better than the Dexa group.Particularly Dexa and cisplatin combined medication group mice are all dead before the 13rd day course of treatment.Therefore, the application of Panaxadiol saponin in preparation and glucocorticoid receptor (GR) bound drug, illustrate the molecular mechanism that PDS has the Dexa same purpose, the new drug that substitutes Dexa for exploitation PDS is significant.
Description of drawings
Fig. 1 is luciferase reporter gene detection figure.Among the figure:
1.GRE-Iuciferase combine the fluorescence intensity that produces with Glucocorticoid Receptor.
2. fluorescence intensity increases by 36 times after adding Dexa.
3. add PDS 25 μ g/ml fluorescence intensities and do not have influence.
4. adding PDS 50 μ g/ml fluorescence intensities increases by 500.
5. add PDS100 μ g/ml fluorescence intensity and increase by 12 times.
The specific embodiment
Experimental example: various dose PDS is to the influence of glucocorticoid receptor (GR) transcriptional activity.
Use in the reporter gene (GRE-Luciferase) and glucocorticoid receptor (GR) (Glucocorticoid Receptor) cotransfection 293 cells of glucocorticoid receptor response element startup.Observation after adding dexamethasone to the influence of glucocorticoid receptor (GR) transcriptional activity; Reuse PDS replaces dexamethasone, and whether to glucocorticoid receptor (GR) transcriptional activity influential, whether be dose dependent and carry out overall merit if observing various dose PDS.
Materials and methods:
1.293 cell line is available from U.S. ATCC.Put and contain in 5% (V/V) calf serum and antibiotic RPMI 1640 (the GIBCO BRL company) culture fluid, in 37 ℃, 5%CO
2Cultivate in the incubator.
2. medicine Stem and leaf of Radix Ginseng glycol saponins (PDS) is provided by Jilin University natural drug research department, is patent (ZL 98100070.3) product, dissolves with normal saline or 5% glucose during use; Cisplatin and dexamethasone all are that clinical hospital pharmacy provides.
3. luciferase reporter gene detection method
3.1 in 12 well culture plates, cell density is about 1 * 10 with 293 cell inoculations
5/ hole is cultivated and was treated that cell carried out plasmid transfection when growing into 90% fusion in 24 hours.The plasmid total amount that is used for transfection is the 300ng/ hole, comprises 100ng GRE-Luciferase/ hole and 200ng/ hole glucocorticoid receptor expression plasmid.Used transfection reagent is Lipofectamine 2000 (an Invitrogen company), and transfection 24～48 hours adds Dexamethasone or PDS respectively and handles cell.
3.2Dexa it is 100 μ mol/L that group adds the concentration of Dexamethasone, after the drug treating after 12 hours, collecting cell, and carry out the Luciferase activity analysis according to the requirement of the Luciferase Assay Kit Instruction manual of STRATAGENE company.
3.3PDS group adds 1 μ l, 2 μ l, 4 μ l (25 μ g/ml, 50 μ g/ml, 100 μ g/ml) respectively.After the drug treating 12 hours, collecting cell, and carry out the Luciferase activity analysis according to the requirement of the Luciferase Assay Kit Instruction manual of STRATAGENE company.
The result
The result as shown in Figure 1, at reporter gene (GRE-Luciferase) and glucocorticoid receptor (GR) (Glucocorticoid Receptor) cotransfection 293 cells that glucocorticoid receptor response element starts, fluorescence intensity is very low.When adding dexamethasone (Dexamethasone100 μ mol/L), the transcriptional activity of glucocorticoid receptor (GR) increases by 36.5 times.As follows to the influence of glucocorticoid receptor (GR) transcriptional activity when replacing dexamethasone: when 1. adding 25 μ g/ml PDS, to the not influence of transcriptional activity of glucocorticoid receptor (GR) with various dose PDS; The transcriptional activity of glucocorticoid increases by 5.5 times when 2. adding PDS and being 50 μ g/ml; 3. the transcriptional activity of glucocorticoid increases by 12 times when adding PDS is 100 μ g/ml.As seen, Panaxadiol saponin combines with glucocorticoid receptor (GR), has and the similar biological effect of dexamethasone, and has shown as tangible dose-dependence.
Experimental example:
PDS, Dexa and cisplatin combined medication are to the influence of mice-transplanted tumor growth with lymphocyte function
Before using antineoplastons such as cisplatin, purple triol, oral dexamethasone is in order to the anaphylaxis of prevention body routinely clinically.Yet the side effect of glucocorticoids such as dexamethasone particularly can obviously weaken the resistance function of tumor of body to immune inhibitory action.PDS has the effect with the receptors bind of glucocorticoids such as dexamethasone, but does not have the side effect of dexamethasone sample, this experiment paired observation PDS and Dexa respectively as effect and the side effect of adjuvant in anti-mice carcinoma of prostate transplanted tumor of cisplatin.
1 materials and methods
1.1 laboratory animal: 60 of male C57 mices, body weight 18-20 gram is purchased Beijing Experimental Animal Center in the Chinese Academy of Sciences, by breeding of the Experimental Animal Center SPF of Jilin University level animal feeding room and raising.
1.2 prepare before the Mus source property prostate gland cancer cell strain RM-1 cell transplantation: cultivate in containing the IMDM culture fluid of 10% calf serum, put 37 ℃, 5%CO
2In the incubator of humidifying, when treating that the RM-1 cell grows to 95% culture bottle with PBS flushing culture bottle 2 times, add 0.125% trypsinization, the cell that digestion is good sucks in the centrifuge tube, the centrifugal 5min of 950rpm/min, abandon supernatant, add the IMDM culture fluid that does not contain calf serum, suction pipe piping and druming mixing (cell counting), centrifugal again, abandon supernatant, the RM-1 cell density is transferred to 1 * 10
7/ ml is standby.
1.3 mice-transplanted tumor replication of Model and laboratory animal grouping:
1.3.1 mouse subcutaneous transplanting tumor replication of Model: get RM-1 cell 1 * 10
7/ ml, nearby the limb side is subcutaneous to be inoculated in mouse back, each inoculation point injection RM-1 cell 0.1ml (1 * 10
6/ point).Observe the transplantation tumor success rate, surveyed each radial line of tumor every 2 days, the general state of record animal.Grow at about 2.5 * 3.0 o'clock at the tumor cell volume, carry out the animal grouping.
1.3.2 animal grouping and administration: up to 100%, gross tumor volume grows to 11.7 (2.5 * 3 at the 7th day C57 mice tumor formation rate of inoculation RM-1 cell
2* 0.52) mm
3Shi Suiji is divided into 6 groups, 10 every group.1. model group: only give normal saline 100ul, 2. cisplatin group: give cisplatin (1.5mg/kg) with the normal saline dilution, 3. Dexa organizes: only give Dexa 2.0mg/kg, 4. cisplatin+Dexa organizes: (1.5mg/kg+2.0mg/kg), 5. PDS group (12.5mg/kg) 6. cisplatin+PDS organize (1.5mg/kg+12.5mg/kg), more than each the group, intraperitoneal injection is 1 time every other day, be 14d the course of treatment.24h after the last administration, anesthesia is put to death animal, the weighing heart, liver, spleen, lung, kidney and tumor weight, and design factor through the eyeball blood sampling down.
1.3.3 the calculating of gross tumor volume and tumour inhibiting rate: the 1. measurement of gross tumor volume and calculating:, press general formula [V (mm with the vernier caliper measurement subcutaneous transplantation tumor line of apsides
3)=L * W
2* 0.52] calculates gross tumor volume V.2. calculate tumour inhibiting rate, tumour inhibiting rate %=[1-(the average tumor of the average tumor weight/matched group of experimental group is heavy)] * 100%.3. organ coefficient, organ coefficient %=organ weights/body weight * 100%.
1.4MTT method detects the inductive splenocyte breeder reaction of ConA:
1.4.1 the pre-treatment of splenocyte preparation and cell: 1. get under the sterilising conditions and respectively organize mouse spleen, place the plate-Lve that adds 5ml Hank ' s liquid in advance to tilt to place) with tweezers spleen is smashed to pieces, and remove piece of tissue with filtered through gauze cell suspension, filtrate moves in the centrifuge tube, wash 1 time (1000r/min) 5 minutes.2. with the IMDM that contains 10% calf serum suspension cell again, and get 100 μ l cell suspension and add in the cell counting liquid of 900 μ l, microscopically carries out cell counting.3. with the IMDM that contains 10% calf serum each group splenocyte cell concentration is transferred to 5 * 10
6/ ml adds 96 well culture plates, 100 μ l cell suspension/holes.4. the preparation of ConA storing solution: be mixed with the storing solution that ConA content is 200 μ l/ml with 1 * PBS; 5. get 96 well culture plates that are loaded with 100 μ l cell suspension/holes, every hole adds ConA 100 μ l/ holes, every group 3 multiple hole, and other establishes 3 multiple holes and only adds culture fluid as negative control group.6. 96 orifice plates of handling through ConA place 37 ℃, cultivate 40-48h in the 5%CO2 incubator.
1.4.2MTT colorimetry detects and calculates lymhocyte transformation rate: 1. take out culture plate from incubator, every hole adds MTT10 μ l, continues to cultivate 4h; 2. 1000r/min is centrifugal 5 minutes, and careful the suction abandoned supernatant, adds the inferior maple 100 μ l/well of dimethyl again, slowly acts on 10min on micro-oscillator.3. on microplate reader in 570nm place photometry absorption value (A).4. shown in lymhocyte transformation rate is calculated as follows
1.5 statistical analysis measurement data: X ± SD represents, adopts SPSS Windows for 11 softwares, one factor analysis of variance, and carry out between the group of sample average relatively.
2 results
2.1 medicine is to the influence of tumor-bearing mice survival rate with general situation:
2.1.1 survival rate: each medication group there is no death administration the 14th day, and survival rate is 100%.
2.1.2 cisplatin+PDS group general state obviously is better than cisplatin+Dexa group: cisplatin group during the medication, cisplatin+Dexa group, activity obviously reduces, and food ration significantly descends, and drinking-water reduces, performance such as become thin and fur is unglazed.But the activity of cisplatin+PDS group, diet and amount of drinking water, the bright degree of fur all are better than being better than cisplatin group and cisplatin+Dexa group.
2.2 compare between the group of mice body weight change percent and tamor index:
2.2.1 use Dexa and PDS separately body weight is not had obvious influence: 1. under the situation of using Dexa and PDS every other day, the body weight when experiment finishes all is positive growth, sees Table 1.2. tumor weight and the model group of using Dexa and PDS group merely relatively do not have significant difference, no matter prove Dexa, and still the independent application of PDS does not all have therapeutical effect to transplanted tumor, sees Table 2.
2.2.2PDS the adjuvant as cisplatin is better than dexamethasone: 1. for the influence (table 1) of body weight change percent: PDS cisplatin group, animal average weight percent change shows and alleviates 27% before and after the experiment.And single with cisplatin group and Dexa cisplatin group, the demonstration of animal average weight percent change alleviates 36% and 39% respectively before and after the experiment.Relatively PDS cisplatin group and Dexa cisplatin group can find out clearly that PDS can increase the body weight of cisplatin group, improve the functional status of body, and Dexa does not then have this effect.2. to the influence (shown in the table 2) of gross tumor volume and weight: as seen in Table 2, PDS cisplatin coupling group gross tumor volume is significantly less than cisplatin group and Dexa cisplatin group.
Table 1PDS and Dexa and cisplatin combined medication are to the influence of transplanted tumor mice body weight
*: compare P＜0.05 with model group; △: compare P＜0.05 with the cisplatin group
The influence that table 2PDS and DEXA and cisplatin combined medication are heavy to prostate cancer tumor
*: compare P＜0.05 with model group; △: compare P＜0.05 with the cisplatin group
2.3.PDS the spleen weight of cisplatin group obviously overweights Dexa cisplatin group:
2.3.1 cisplatin group body weight when spleen heavily alleviates also obviously descends, it is not really remarkable that index and spleen index descends, yet, Dexa cisplatin group is when losing weight, it is particularly evident that spleen weight alleviates, and reduces the most obvious, as shown in table 3 at each group spleen coefficient with Dexa cisplatin group.
2.3.2PDS the tumor-bearing mice spleen is had protective effect, and PDS cisplatin group is in weight increase, spleen is heavy also to be increased than cisplatin group and Dexa cisplatin group, and this shows that fully PDS is better than dexamethasone as the adjuvant of cisplatin.Demonstration has the effect (seeing table 3 for details) of weight increase and index and spleen index to mice with tumor.
Table 3PDS and Dexa and cisplatin combined application heavily reach the influence of spleen index to the carcinoma of prostate mice spleen
*: compare P＜0.05 with model group; △: compare P＜0.05 with the cisplatin group; #: compare P＜0.05 with Dexa cisplatin group
2.4PDS and Dexa and cisplatin combined application are to the influence of transplanted tumor mouse lymphocyte conversion ratio:
2.4.1 cisplatin obviously suppresses ConA induction of lymphocyte conversion ratio.Dexa cisplatin group, the inductive lymhocyte transformation rate of ConA is lower than the cisplatin group.As seen, Dexa has suppressed the lymphocyte transformation function significantly, has weakened the antitumor action of body, and is as shown in table 4.
2.4.2PDS the inductive lymhocyte transformation rate 3.29% of cisplatin group ConA, apparently higher than Dexa cisplatin group (18.61%), prompting PDS has the effect of protection lymphocyte function.See table 4 for details.
Table 4PDS and Dexa and cisplatin combined medication are to the influence of ConA induction of lymphocyte conversion ratio
| Group | Control wells A value | Stimulate back A | 48h conversion ratio (%) |
| Model group (normal saline) | 0.578±0.084 | 0.585±0.103 | 1.565 |
| Cisplatin | 0.358±0.066 | 0.310±0.024 | -13.24* |
| Dexamethasone group | 0.423±0.083 | 0.421±0.103 | -1.05△ |
| Dexa cisplatin group | 0.324±0.062 | 0.259±0.043 | -18.61*△ |
| PDS cisplatin group | 0.378±0.073 | 0.389±0.097 | 3.29*△# |
| The PDS group | 0.685±0.156 | 0.879±0.201 | 27.83*△ |
*: compare P＜0.05 with model group; △: compare P＜0.05 with the cisplatin group; #: compare P＜0.05 with Dexa cisplatin group
3. brief summary:
Cisplatin is the metal complex of platinum, and action target spot is DNA, and cisplatin forms DNA and CDDP complex in tumor cell DNA interchain and chain, thereby, disturb dna replication dna and albumen synthetic, promote apoptosis.Originally experiment showed, that cisplatin group body weight when tumor weight obviously alleviates also significantly descends.
Use dexamethasone and Panaxadiol saponin separately and do not have antitumor action.But PDS can obviously improve the inductive lymhocyte transformation rate of ConA, and dexamethasone has then reduced the inductive lymphocytic conversion ratio of ConA.
Dexa cisplatin group and PDS cisplatin group compare: 1. the tumor coefficient (2.17 ± 1.35) of PDS cisplatin group shows that less than Dexa cisplatin group (3.12 ± 1.23) PDS and cisplatin combined application have the effect of synergistic antitumor; 2. PDS cisplatin treated animal general state is better than Dexa cisplatin group; PDS and cisplatin combinedly be applied in weight increase and the spleen weight aspect obviously is better than Dexa cisplatin group; 3. PDS cisplatin group has the effect that improves cisplatin group lymhocyte transformation rate, and Dexa cisplatin group has then reduced the inductive lymhocyte transformation rate of ConA significantly.
In sum, Panaxadiol saponin is better than glucocorticoids such as dexamethasone after substituting the adjuvant of dexamethasone as chemotherapy of tumors.
Claims (3)
1. the application of Panaxadiol saponin in preparation and glucocorticoid receptor (GR) bound drug.
2. Panaxadiol saponin and the cisplatin combined application that is applied in the antitumor.
3. Panaxadiol saponin as claimed in claim 2 and the cisplatin combined application that is applied in the antitumor, it is characterized in that: the mass ratio of Panaxadiol saponin and cisplatin is 25: 3.
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Cited By (2)
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| CN102437239A (en) * | 2011-11-30 | 2012-05-02 | 中山大学 | Full back electrode aluminium back junction solar cell manufacture technology |
| CN110664821A (en) * | 2019-11-08 | 2020-01-10 | 延边大学 | Application of panaxadiol in preparing medicine for inhibiting expression of PD-L1 and tumor cell proliferation protein |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1939311A (en) * | 2006-10-01 | 2007-04-04 | 沈阳药科大学 | Ginseng sapogenin aglycone derivative biological preparation and its usage |
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| Publication number | Priority date | Publication date | Assignee | Title |
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Open date: 20110413 |