CN106727500A - The application of dihydroartemisinine, suppression medicine and its application - Google Patents

Abstract

The application of dihydroartemisinine, suppression medicine and its application.The present invention relates to a kind of application of antimalarial agent-dihydroartemisinine (DHA) in signal transduction and activating transcription factor (STAT3) abnormal activation medicine is suppressed.Found by experiment in vivo and in vitro, DHA can Selective depression STAT3 phosphorylation activation, this inflammatory factor IL 6 that acts on stimulates, can also play under low-oxygen environment.Suppression STAT3 abnormal activation medicines containing DHA, can be used to treat the diseases related to STAT3 abnormal activations such as tumour, autoimmune disease and diseases associated with inflammation.

Description

The application of dihydroartemisinine, suppression medicine and its application

Technical field

The invention belongs to field of medicaments, and in particular to antimalarial agent-dihydroartemisinine (DHA) is suppressing STAT3 abnormal activations Application in medicine.Meanwhile, the invention further relates to a kind of suppression medicine containing the dihydroartemisinine, and the suppression medicine Using.

Background technology

DHA is the important derivatives and main active of antimalarial Chinese medicine qinghaosu, and its drug effect is that other artemisines spread out Biological 3~5 times.DHA and qinghaosu are slaughtered cry of a deer professor and find and thereby is achieved the Nuo Bei of 2015 by famous pharmacy man The prize of your physiological medical science, both have been used for treating various malaria and especially pernicious malaria, resistance malaria and encephalic malaria and obtain Good result, it is considered to be the first-line drug for the treatment of malaria.In vivo, after qinghaosu and its derivative rapidly transform into DHA Competence exertion antimalarial active, the peroxide bond in molecular structure is the key point that DHA eliminates plasmodium.In liver and gall tubule Interior, then DHA enters small enteral by UDP-glucoronosyl/transferase glucuronic acid through choledochus, is followed by multiple liver sausage Ring is excreted eventually through urine and excrement.Its characteristic distributions is concentration highest in spleen, is secondly kidney, adrenal gland and liver It is dirty.Research finds, except antimalarial and anti-schistosomiasis, DHA also has regulation inflammatory reaction, improves lupus erythematosus, antibacterial and disease-resistant The multiple drug effect such as poison.

Numerous researchs have confirmed, STAT3 (Signal transducers and activators of Transcription 3, STAT3) to tumour generation development have important facilitation.Under physiological status, the STAT3 of monomer It is in cytoplasm and inactive, it is necessary to phosphorylation activation play effect.STAT3 molecules have Tyr 705 and Ser 727 two Phosphorylation activation site, Tyr 705 is STAT3 dimerizations, nuclear translocation and the critical sites for combining DNA；Ser 727 is then ERK1/2 and JNK etc. activates the site of STAT3.Jak2, EGFR and SRC etc. pass through acceptor/non-receptor activation pathway activation monomer STAT3, dimerization forms homologous or heterodimer, into karyon in specific target gene combination activated transcription, regulate and control downstream The expression of albumen.In normal structure, the activation of STAT3 is rapid and of short duration, and in tumour cell, STAT3 is then persistently activated.Swash STAT3 living not only promotes normal cell canceration, Tumor Angiongesis and cancer cell migration, and maintains breast cancer and incidence " stem cell " characteristic of the tumor stem cells such as tumour cell (HNSCC).Lung cancer, liver cancer, head and neck scale carcinoma, breast cancer, prostate cancer Expression high, activation STAT3 is found that with leukaemia etc., its incidence is more than 50% in liver cancer, lung cancer and breast cancer, And then it is up to more than 95% in HNSCC cells.Numerous result of study promptings, the activation of STAT3 is the required of normal cell canceration Condition, is the requirement for maintaining tumour cell and its stem cell properties, is the requirement for maintaining tumor cell proliferation growth, It is the requirement of some other oncogene activation.Additionally, STAT3 produces radiation and chemotherapy to resist tumour cell also important Facilitation, can reverse this chemicotherapy to resist if it being blocked with inhibitor or Decoy oligonucleotides and is activated.

Many factors can activate STAT3, such as SRC, EGFR, IL-6, IL-10, CXCL12 and COX-2 albumen and the factor STAT3 can be directly or indirectly activated, and in environment such as hypoxemia, can equally activate STAT3.Because STAT3 is many signals The joint of path and shared signal protein, therefore, except tumour, it is also closely related with various diseases.The inflammation such as IL-6 because Son activation STAT3 release pyrogens influence immunocyte and then cause acute inflammation.STAT3 can Targeted-control blood vessel endothelium The expression of growth factor (VEGF), therefore its generation development to heart and vascular diseases has a major impact, and be dilatancy cardiac muscle The necessary condition that disease progression is aggravated.Research it has also been found that, STAT3 activation after can with the clinical symptoms of heavy system lupus erythematosus, And aggravate rheumatoid arthritis inflammation performance, cause the exacerbation of the state of an illness.Additionally, the oneself state of STAT3 also with Crow grace Disease is relevant with the generation of ulcerative colitis, and promotes ulcerative colitis to change to malignant tumour.

STAT3 is by sustained activation in tumour cell and inflammatory tissue, blocked can reverse disease generation development and Lapse to.At present, the activation that various directly or indirectly inhibitor block STAT3 is had been developed for.Direct inhibitor include Stattic, S3I-1757 and BP-1-102 etc., such inhibitor is combined with STAT3 molecules, is allowed to that activation function can not be played；Suppress indirectly Agent then suppresses STAT3 activation, AG490, AZD1480, Cucurbitacin B and turmeric indirectly by regulating and controlling the albumen such as SRC, EGFR and Jak2 Element etc. belongs to indirect inhibitor.Although the above-mentioned significant anticancer effect of inhibitor, due to stability, water-soluble and toxic and side effect Etc. reason, only only a few inhibitor is used for zoopery and clinical experimental study.

The content of the invention

It is an object of the present invention to provide a kind of antimalarial common drug-dihydroartemisinine (Dihydroartemisinin, DHA) Application in STAT3 abnormal activations are suppressed.

Dihydroartemisinine is used to suppress STAT3 abnormal activations, and the molecular formula of the dihydroartemisinine is as follows：

Other STAT3 inhibitor are compared, the DHA from plant can synthesize on a large scale, and medicine cost is relatively It is low, it is very helpful to reducing medical expense and medical burden, the economy of raising drug therapy.

DHA clinically using for a long time, is clearly slapped to its medicine pharmacokinetics in itself and pharmacodynamics Hold, these clinical experiences can help select optimal drug dosage, medication cycle and administrated method etc., can greatly shorten clinical examination The time is tested, is that other any STAT3 inhibitor can not be when.

The inhibitory action that DHA is activated to STAT3 is notable and has specificity higher, only to abnormal cell and tissue STAT3 activation has important inhibitory action, and normal cell and tissue are affected by it very little, is conducive to the clinical toxic and side effect that reduces to carry The tolerance level of patient high.

Further, dihydroartemisinine is used to suppress the STAT3 activation when hypoxemia or/and IL-6 protein factors stimulate.

Hypoxemia is intracellular common microenvironment, and not only in normal oxygen, Selective depression STAT3 is activated dihydroartemisinine, this Effect can also be played under low oxygen conditions.IL-6 protein factors can directly or indirectly activate STAT3, and in environment such as hypoxemia, STAT3 can be equally activated, dihydroartemisinine is to the inhibitory action of STAT3 abnormal activations in hypoxia condition or/and IL-6 albumen The factor can also be played when stimulating.

One kind suppresses medicine, and the suppression medicine contains dihydroartemisinine.

Further, the suppression medicine contains drug excipient or carrier.

Further, the drug excipient or carrier include glycerine, ethanol, BS, dimethyl sulfoxide (DMSO), physiology salt One or more in water.

Further, double blue and green artemisin concentration are 40-200 μm of ol/L in the suppression medicine.

Further, the described medicine that suppresses is used in the Disease Intervention with STAT3 abnormal activation characteristics.

Further, it is described suppress medicine be used for the Disease Intervention time with STAT3 abnormal activation characteristics >= 12h。

Further, the disease of the STAT3 abnormal activations characteristic includes tumour, autoimmune disease, inflammatory disease Disease.

The autoimmune pathologies and diseases associated with inflammation, including ulcerative colitis, systemic loupus erythematosus, class wind Wet arthritis, ankylosing spondylitis, idiopathic thrombocytopenic purpure, psoriasis, multiple sclerosis, chronic nephritis, The diseases such as gastritis, pancreatitis, inflammatory lung conditions, bronchial astehma, obstructive nephropathy, the retinitis.

The tumour include liver cancer, breast cancer, cancer of pancreas, Huppert's disease, leukaemia, lymthoma, lung cancer, stomach cancer, Cancer of the esophagus, the carcinoma of the rectum, colon cancer, cutaneum carcinoma, melanoma, prostate cancer, brain tumor and cervical carcinoma etc..

Further, the suppression medicine is used in combination with cancer therapy drug or anti-inflammatory drug.

The present invention carries out experiment in vitro first, from three kinds of representative head and neck scale carcinoma cells, using Western blotting The technology such as (Western blotting) and plasmid transfection, it was demonstrated that DHA can suppress the activation of Jak2/STAT3 signal paths, this Kind can also be played under acting on inflammatory factor IL-6 stimulations, low-oxygen environment.Then by itself and the most frequently used STAT3 inhibitor AG490 and AZD1480 compare, it is found that three kinds of medicines can suppress STAT3 activation with drug concentration increase, show that DHA is one Plant STAT3 activation inhibitors.Transplantable tumor animal model is finally set up, further proves that DHA can significantly inhibit the growth of transplantable tumor, And the activator protein of Jak2 and STAT3 is lowered, experimental animal is without obvious toxic and side effect.The experimental result of in vitro and in vivo shows Show, suppress Jak2/STAT3 signal activations by specificity, DHA can significantly inhibit the growth of HNSCC cells.

Brief description of the drawings

Fig. 1 is the activation of DHA suppression HNSCC cells STAT3 under normal oxygen condition.

Fig. 2 is the activation of DHA suppression HNSCC cells STAT3 under hypoxia.

Fig. 3 is that DHA suppresses HNSCC cells p-STAT3 expression with specificity.

Fig. 4 is that DHA suppresses STAT3 signal paths upstream p-Jak2 protein expressions.

Fig. 5 is Jak2 and the STAT3 activation that DHA suppresses that IL-6 causes.

Fig. 6 is DHA as a kind of new Jak2/STAT3 signal pathway inhibitors.

Fig. 7 is the activation that DHA suppresses Jak2/STAT3 signal paths in zoografting tumor tissue.

Specific embodiment

Embodiment one

DHA suppresses the activation of HNSCC cells STAT3 under normal oxygen condition.

Prepare DHA solution.DHA pulvis 1g are weighed, dimethyl sulfoxide (DMSO) (DMSO) solution 17.584ml is added, under gnotobasis With 1ml pipettors, pressure-vaccum, by complete drug dissolution, is prepared into the DHA solution of 200mM/L repeatedly, is distributed into sterile vials, be placed in- 80 DEG C freeze standby, and the used time now matches somebody with somebody.

Western blotting detect DHA to HNSCC cellular associated proteins expressions.The extraction of cell protein：Choosing Take the logarithm growth period Fadu, Cal-27 and Hep-2 head and neck scale carcinoma cell, digestion count after with 4 × 10⁵/ 2ml/well cells are close Degree is inoculated into 6 well culture plates, after existing prepare, the culture medium 2ml containing various concentrations DHA of addition after cell attachment.Medicine is done The μ l of RAPI lysates 100 are added to extract cell protein after terminating in advance.Tumor tissue protein extraction：Tumor tissue is taken out from liquid nitrogen, will It inserts homogenizer homogenate after being cut into tiny fragment, adds RIPA lysates of the 500 μ l containing PMSF, and 30min is placed on ice, cracks During repeatedly homogenate grinding thoroughly cracking histocyte.Then by Tissue Lysates move into centrifuge tube in, under the conditions of 4 DEG C, 12000rpm is centrifuged 15min, and Aspirate supernatant is placed in liquid nitrogen and saves backup.Using BCA standard measure protein concentrations, calculate and carry The protein concentration of each sample for taking, is distributed into the centrifuge tubes of 200 μ 1, and often pipe contains 40 μ g albumen, then carries out albuminous degeneration.Match somebody with somebody The separation gel of system 8~12%, room temperature places 30min.Treat that gelling knot, in g., jelly-like, prepares 4% and concentrates glue, insert comb.Treat glue Comb is extracted after condensation, electrophoresis liquid is poured into, injector adds protein sample and standard items albumen Marker as control, connects electricity Stream, carries out SDS-PAGE electrophoresis, time about 2h.Filter paper, pvdf membrane, gel are sequentially placed into after end, both positive and negative polarity is noted, electricity is poured into Turn liquid, by the albumen transferring film on separation gel to pvdf membrane, the time is about 4h.Then 1h is closed and with containing primary antibody with skimmed milk power Skimmed milk power and 4 DEG C of overnight incubations of pvdf membrane.Next day washes film and is incubated secondary antibody 1h, is developed using ECL methods.

DHA suppresses the activation of HNSCC cells STAT3 under normal oxygen condition.Three kinds of tumour cells Fadu, Hep-2, Cal-27 are used DHA intervenes 24h, Fadu DHA concentration be 0,20,40,80,160 μm of ol/L, Hep-2 and Cal-27 DHA concentration be 0,10, 20、40、80μmol/L；Or the DHA of doses acts on three kinds of cancer cells, and Fadu DHA concentration is 160 μm of ol/L, Hep-2 With Cal-27 with DHA concentration be 80 μm of ol/L, with Western blotting detect 0,6h, 12h, 24h and 48h when p-STAT3 Expression.Result is as shown in Figure 1：P-STAT3 in tumour cell subtracts with the increase of DHA concentration and intervention time Few, control group is without significant change.This explanation, DHA substantially suppresses the activation of STAT3, and is closed in dependence with dosage and time is intervened System.

Embodiment two

DHA suppresses the activation of HNSCC cells STAT3 under hypoxia.

Hypoxemia is microenvironment common in malignant cell, and tumor cell proliferation, invasion and attack, transfer and generation can be promoted to put Chemoresistance, therefore the Synergistic action of research DHA is significant under low oxygen conditions.

Prepare DHA solution.DHA pulvis 1g are weighed, dimethyl sulfoxide (DMSO) DMSO solution 10.284ml, ethanol solution is added 7.3ml, with 1ml pipettors, pressure-vaccum, by complete drug dissolution, is prepared into the DHA solution of 200mM/L repeatedly under gnotobasis, packing Enter sterile vials, be placed in -80 DEG C and freeze standby, the used time now matches somebody with somebody.

Hypoxemia device and hypoxia condition.Hypoxemia equipment in microbiological incubator is home-made contrivance, and hypoxemia device is by six pieces The poly (methyl methacrylate) plate bonding of thick 8mm is formed, and is divided into room and lid two parts, has the pipe of control gas turnover respectively in the both sides of room Road, air intake duct covers and pads last layer soft rubber pad, with anti-gas-leak, tighten fixation near bottom, gas off-take near top in closing Screw, it is ensured that anoxic case is completely closed.The hypoxemia Related Experimental Study that my laboratory is carried out in the past can confirm that the device is used for Study the feasibility of hypoxemia.This experiment hypoxia condition is 37 DEG C, 5%CO₂, 1%O₂, 94%N₂.Institute in charge flow rate and container Zirconia oxygen analyzer is determined aerobic partial pressure reaching standard time again.Gaseous mixture (95%N is passed through by air intake duct₂, 5%CO₂), while beating Feed channel is outputed, draft speed is 3L/min, after ventilation 8min, then to being passed through (1%O in hypoxemia device₂, 94%N₂, 5%CO₂) Gaseous mixture, draft speed is 3L/min, after ventilation 5min, in case oxygen concentration change by zirconia oxygen analyzer monitored over time, most The whole low oxygen concentration for needed for experiment.When partial pressure of oxygen is up to standard in hypoxemia device, draft speed is changed to low-flow ventilation 0.8L/min, To maintain oxygen concentration in low-oxygen box, to ensure the stabilization of oxygen concentration in low-oxygen box.Obtained by the gaseous mixture for preparing different oxygen Corresponding low oxygen concentration, this model error it is minimum (<0.02) low oxygen concentration of stabilization, can especially be obtained.This model is convenient It is easy, the need for various different hypoxics researchs can be met.In addition, this modelling economy, simple to operate, horizontal high voltage can be entered Sterilizing etc. is disinfected, easy to utilize.

Western blotting detect DHA to HNSCC cellular associated proteins expressions.The extraction of cell protein：Choosing Take the logarithm growth period Fadu, Cal-27 and Hep-2 head and neck scale carcinoma cell, digestion count after with 4 × 10⁵/ 2ml/well cells are close Degree is inoculated into 6 well culture plates, after existing prepare, the culture medium 2ml containing various concentrations DHA of addition after cell attachment.Medicine is done The μ l of RAPI lysates 100 are added to extract cell protein after terminating in advance.Tumor tissue protein extraction：Tumor tissue is taken out from liquid nitrogen, will It inserts homogenizer homogenate after being cut into tiny fragment, adds RIPA lysates of the 500 μ l containing PMSF, and 30min is placed on ice, cracks During repeatedly homogenate grinding thoroughly cracking histocyte.Then by Tissue Lysates move into centrifuge tube in, under the conditions of 4 DEG C, 12000rpm is centrifuged 15min, and Aspirate supernatant is placed in liquid nitrogen and saves backup.Using BCA standard measure protein concentrations, calculate and carry The protein concentration of each sample for taking, is distributed into the centrifuge tubes of 200 μ 1, and often pipe contains 40 μ g albumen, then carries out albuminous degeneration.Match somebody with somebody The separation gel of system 8~12%, room temperature places 30min.Treat that gelling knot, in g., jelly-like, prepares 4% and concentrates glue, insert comb.Treat glue Comb is extracted after condensation, electrophoresis liquid is poured into, injector adds protein sample and standard items albumen Marker as control, connects electricity Stream, carries out SDS-PAGE electrophoresis, time about 2h.Filter paper, pvdf membrane, gel are sequentially placed into after end, both positive and negative polarity is noted, electricity is poured into Turn liquid, by the albumen transferring film on separation gel to pvdf membrane, the time is about 4h.Then 1h is closed and with containing primary antibody with skimmed milk power Skimmed milk power and 4 DEG C of overnight incubations of pvdf membrane.Next day washes film and is incubated secondary antibody 1h, is developed using ECL methods.

The condition of this experimental selection hypoxemia is 1%O₂, DHA intervention tri- kinds of HNSCC cell 24h of Fadu, Hep-2, Cal-27, It is that 160 μm of ol/L, Cal-27 and Hep-2 DHA intervene concentration for 80 μm of ol/L that Fadu DHA intervene concentration.Result such as Fig. 2 institutes Show, p-STAT3 protein expressions are still subject to substantially suppression, and (to add relative medicine or hypoxemia treatment, "-" is addition solvent pair to "+" According to Actin is albumen internal reference).Therefore, under low-oxygen environment, DHA can also suppress the activation of STAT3 albumen.

Embodiment three

DHA suppresses HNSCC cells p-STAT3 expression has specificity.

Prepare DHA solution.DHA pulvis 1g are weighed, glycerine 10.584ml, BS 7ml is added, used under gnotobasis Pressure-vaccum, by complete drug dissolution, is prepared into the DHA solution of 200mM/L to 1ml pipettors repeatedly, is distributed into sterile vials, is placed in -80 DEG C freeze standby, the used time now matches somebody with somebody.

Western blotting detect DHA to HNSCC cellular associated proteins expressions.The extraction of cell protein：Choosing Take the logarithm growth period Fadu, Cal-27 and Hep-2 head and neck scale carcinoma cell, digestion count after with 4 × 10⁵/ 2ml/well cells are close Degree is inoculated into 6 well culture plates, after existing prepare, the culture medium 2ml containing various concentrations DHA of addition after cell attachment.Medicine is done The μ l of RAPI lysates 100 are added to extract cell protein after terminating in advance.Tumor tissue protein extraction：Tumor tissue is taken out from liquid nitrogen, will It inserts homogenizer homogenate after being cut into tiny fragment, adds RIPA lysates of the 500 μ l containing PMSF, and 30min is placed on ice, cracks During repeatedly homogenate grinding thoroughly cracking histocyte.Then by Tissue Lysates move into centrifuge tube in, under the conditions of 4 DEG C, 12000rpm is centrifuged 15min, and Aspirate supernatant is placed in liquid nitrogen and saves backup.Using BCA standard measure protein concentrations, calculate and carry The protein concentration of each sample for taking, is distributed into the centrifuge tubes of 200 μ 1, and often pipe contains 40 μ g albumen, then carries out albuminous degeneration.Match somebody with somebody The separation gel of system 8~12%, room temperature places 30min.Treat that gelling knot, in g., jelly-like, prepares 4% and concentrates glue, insert comb.Treat glue Comb is extracted after condensation, electrophoresis liquid is poured into, injector adds protein sample and standard items albumen Marker as control, connects electricity Stream, carries out SDS-PAGE electrophoresis, time about 2h.Filter paper, pvdf membrane, gel are sequentially placed into after end, both positive and negative polarity is noted, electricity is poured into Turn liquid, by the albumen transferring film on separation gel to pvdf membrane, the time is about 4h.Then 1h is closed and with containing primary antibody with skimmed milk power Skimmed milk power and 4 DEG C of overnight incubations of pvdf membrane.Next day washes film and is incubated secondary antibody 1h, is developed using ECL methods.

Three kinds of tumour cells Fadu, Hep-2, Cal-27 with DHA intervene 24h, Fadu with DHA concentration be 0,20,40,80, 160 μm of ol/L, Hep-2 and Cal-27 are 0,10,20,40,80 μm of ol/L with DHA concentration, as a result as shown in figure 3, p-STAT3 is bright It is aobvious to be suppressed, and p-Erk1/2 and p-Akt albumen is without significant change.

Example IV

DHA suppresses STAT3 signal paths upstream p-Jak2 protein expressions.

Prepare DHA solution.DHA pulvis 1g are weighed, glycerine 7.584ml, physiological saline 10ml is added, used under gnotobasis Pressure-vaccum, by complete drug dissolution, is prepared into the DHA solution of 200mM/L to 1ml pipettors repeatedly, is distributed into sterile vials, is placed in -80 DEG C freeze standby, the used time now matches somebody with somebody.

Western blotting detect DHA to HNSCC cellular associated proteins expressions.The extraction of cell protein：Choosing Take the logarithm growth period Fadu, Cal-27 and Hep-2 head and neck scale carcinoma cell, digestion count after with 4 × 10⁵/ 2ml/well cells are close Degree is inoculated into 6 well culture plates, after existing prepare, the culture medium 2ml containing various concentrations DHA of addition after cell attachment.Medicine is done The μ l of RAPI lysates 100 are added to extract cell protein after terminating in advance.Tumor tissue protein extraction：Tumor tissue is taken out from liquid nitrogen, will It inserts homogenizer homogenate after being cut into tiny fragment, adds RIPA lysates of the 500 μ l containing PMSF, and 30min is placed on ice, cracks During repeatedly homogenate grinding thoroughly cracking histocyte.Then by Tissue Lysates move into centrifuge tube in, under the conditions of 4 DEG C, 12000rpm is centrifuged 15min, and Aspirate supernatant is placed in liquid nitrogen and saves backup.Using BCA standard measure protein concentrations, calculate and carry The protein concentration of each sample for taking, is distributed into the centrifuge tubes of 200 μ 1, and often pipe contains 40 μ g albumen, then carries out albuminous degeneration.Match somebody with somebody The separation gel of system 8~12%, room temperature places 30min.Treat that gelling knot, in g., jelly-like, prepares 4% and concentrates glue, insert comb.Treat glue Comb is extracted after condensation, electrophoresis liquid is poured into, injector adds protein sample and standard items albumen Marker as control, connects electricity Stream, carries out SDS-PAGE electrophoresis, time about 2h.Filter paper, pvdf membrane, gel are sequentially placed into after end, both positive and negative polarity is noted, electricity is poured into Turn liquid, by the albumen transferring film on separation gel to pvdf membrane, the time is about 4h.Then 1h is closed and with containing primary antibody with skimmed milk power Skimmed milk power and 4 DEG C of overnight incubations of pvdf membrane.Next day washes film and is incubated secondary antibody 1h, is developed using ECL methods.

Three kinds of tumour cells Fadu, Hep-2, Cal-27 with DHA intervene 24h, Fadu with DHA concentration be 0,20,40,80, 160 μm of ol/L, Hep-2 and Cal-27 DHA concentration are 0,10,20,40,80 μm of ol/L, observe p-EGFR, p-Jak2, p-SRC And p-STAT3 protein expression situations.Result such as Fig. 4 point out, three kinds of tumour cell p-Jak2 and p-STAT3 protein expressions with The increase of DHA dosage and decline, and two kinds of albumen of p-EGFR and p-SRC then without substantially change.

Embodiment five

DHA suppresses Jak2 and the STAT3 activation that IL-6 causes.

Prepare DHA solution.Weigh DHA pulvis 1g, add dimethyl sulfoxide (DMSO) DMSO solution 6.584ml, glycerine 11ml is aseptic With 1ml pipettors, pressure-vaccum, by complete drug dissolution, is prepared into the DHA solution of 200mM/L repeatedly under environment, is distributed into aseptic small Bottle, is placed in -80 DEG C and freezes standby, and the used time now matches somebody with somebody.

Western blotting detect DHA to HNSCC cellular associated proteins expressions.The extraction of cell protein：Choosing Take the logarithm growth period Fadu, Cal-27 and Hep-2 head and neck scale carcinoma cell, digestion count after with 4 × 10⁵/ 2ml/well cells are close Degree is inoculated into 6 well culture plates, after existing prepare, the culture medium 2ml containing various concentrations DHA of addition after cell attachment.Medicine is done The μ l of RAPI lysates 100 are added to extract cell protein after terminating in advance.Tumor tissue protein extraction：Tumor tissue is taken out from liquid nitrogen, will It inserts homogenizer homogenate after being cut into tiny fragment, adds RIPA lysates of the 500 μ l containing PMSF, and 30min is placed on ice, cracks During repeatedly homogenate grinding thoroughly cracking histocyte.Then by Tissue Lysates move into centrifuge tube in, under the conditions of 4 DEG C, 12000rpm is centrifuged 15min, and Aspirate supernatant is placed in liquid nitrogen and saves backup.Using BCA standard measure protein concentrations, calculate and carry The protein concentration of each sample for taking, is distributed into the centrifuge tubes of 200 μ 1, and often pipe contains 40 μ g albumen, then carries out albuminous degeneration.Match somebody with somebody The separation gel of system 8~12%, room temperature places 30min.Treat that gelling knot, in g., jelly-like, prepares 4% and concentrates glue, insert comb.Treat glue Comb is extracted after condensation, electrophoresis liquid is poured into, injector adds protein sample and standard items albumen Marker as control, connects electricity Stream, carries out SDS-PAGE electrophoresis, time about 2h.Filter paper, pvdf membrane, gel are sequentially placed into after end, both positive and negative polarity is noted, electricity is poured into Turn liquid, by the albumen transferring film on separation gel to pvdf membrane, the time is about 4h.Then 1h is closed and with containing primary antibody with skimmed milk power Skimmed milk power and 4 DEG C of overnight incubations of pvdf membrane.Next day washes film and is incubated secondary antibody 1h, is developed using ECL methods.

Fadu DHA concentration is that 160 μm of ol/L, Hep-2 and Cal-27 DHA concentration are 80 μm of ol/L, and DHA intervenes 24h Afterwards, (to add relative medicine treatment, "-" is addition solvent control to "+", and Actin is to add people to recombinate IL-6 (20ng) interventions 1h Albumen internal reference).Result such as Fig. 5 is pointed out, and IL-6 can be such that p-Jak2 the and p-STAT3 protein expressions in three kinds of cancer cells substantially increase Plus, and DHA then significantly reduces two kinds of albumen.

Embodiment six

DHA can be used as a kind of new Jak2/STAT3 signal pathway inhibitors.

Prepare DHA solution.DHA pulvis 1g are weighed, ethanol 17.584ml is added, under gnotobasis with 1ml pipettors repeatedly Complete drug dissolution is prepared into the DHA solution of 200mM/L by pressure-vaccum, is distributed into sterile vials, and AZD1480 and AG490 are The dimethyl sulfoxide (DMSO) DMSO solution of 100mM/L concentration, three kinds of medicines are placed in -80 DEG C and freeze standby, and the used time now matches somebody with somebody.

Western blotting detect DHA to HNSCC cellular associated proteins expressions.The extraction of cell protein：Choosing Take the logarithm growth period Fadu, Cal-27 and Hep-2 head and neck scale carcinoma cell, digestion count after with 4 × 10⁵/ 2ml/well cells are close Degree is inoculated into 6 well culture plates, and intervention experiment is distinguished after DHA, AZD1480 and AG490 of various concentrations is used after cell attachment Three kinds of squamous cell carcinomas.Pharmaceutical intervention adds the μ l of RAPI lysates 100 to extract cell protein after terminating.Tumor tissue protein extraction：From Tumor tissue is taken out in liquid nitrogen, homogenizer homogenate is inserted after being cut into tiny fragment, add RIPAs of the 500 μ l containing PMSF to crack Liquid, places 30min on ice, and homogenate grinding thoroughly cracks histocyte repeatedly in cracking process.Then Tissue Lysates are moved into In centrifuge tube, under the conditions of 4 DEG C, 12000rpm centrifugation 15min, Aspirate supernatant is placed in liquid nitrogen and saves backup.It is legal using BCA Amount protein concentration, calculates the protein concentration of each sample of extraction, is distributed into the centrifuge tubes of 200 μ 1, and often pipe contains 40 μ g albumen, Then carry out albuminous degeneration.8~12% separation gel is prepared, room temperature places 30min.Treat that gelling knot, in g., jelly-like, prepares 4% dense Contracting glue, inserts comb.Wait comb is extracted after the knot that is gelled, electrophoresis liquid is poured into, injector adds protein sample and standard items albumen Marker carries out SDS-PAGE electrophoresis, time about 2h as control, turn-on current.Be sequentially placed into after end filter paper, pvdf membrane, Gel, notes both positive and negative polarity, pours into electricity and turns liquid, and by the albumen transferring film on separation gel to pvdf membrane, the time is about 4h.Then degreasing is used Milk powder closing 1h and with contain primary antibody skimmed milk power and 4 DEG C of overnight incubations of pvdf membrane.Next day washes film and is incubated secondary antibody 1h, uses ECL methods are developed.

AZD1480 and AG490 are two kinds of STAT3 signal path specific inhibitors, and this part is studied first by both medicines Fadu, Cal-27 and Hep-2 cell 24h are intervened with different dosage, it is suppressed then to observe three kinds of tumor cell activities with mtt assay Situation processed, the corresponding IC50 values of AZD1480 and AG490 are calculated according to its result.Determine two kinds of inhibitor further according to IC50 values Intervention concentration (with reference to the selection of DHA concentration, i.e. maximum dose more than IC50 values, and its residual value is less than IC50 values, and concentration gradient is again Than setting).After inhibitor concentration setting, three kinds of squamous carcinomas of intervention experiment are distinguished with DHA, AZD1480 and AG490 of various concentrations Cell 24h.The expression of p-Jak2 and p-STAT3 albumen declines with the concentration of Experimental agents.This result as shown in fig. 6, It is similar to specific STAT3 inhibitor AZD1480 and AG490 effect, DHA equally can substantially suppress head and neck scale carcinoma cell Jak2 and The activation of STAT3 albumen.And, in same concentration, DHA presses down compared with AG490 to Hep-2 Carbazole alkaloids to two kinds of albumen of activation System is more notable.

Embodiment seven

DHA suppresses the activation of Jak2/STAT3 signal paths in zoografting tumor tissue.

Prepare DHA solution.DHA pulvis 1g are weighed, ethanol 9.584ml, physiological saline 8ml is added, 1ml is used under gnotobasis Pressure-vaccum, by complete drug dissolution, is prepared into the DHA solution of 200mM/L to pipettor repeatedly, is distributed into sterile vials, is placed in -80 DEG C Freeze standby, the used time now matches somebody with somebody.

The raising of BALb/c male nude mouses and the foundation of Transplanted tumor model.14 BALb/c male nude mouses (4~6 weeks, 18~ 20g weights), to raise in Bethune International Peace Hospital's Experimental Animal Center (SPF grades), receptacle is at 24 DEG C of constant temperature and sound insulation Reason, the daily light and shade time is 12/12h, and lamina air flow filtering, aseptic feed and aqua sterilisa freely eat for animal, and animal is raised Support in cage, most 5 animals of each cage, regularly replace bedding and padding.Nude mice reaches Experimental Animal Center and raises 1 week, treats that it is adapted to After new environment and observation animal ordinary circumstance is good, the Cal-27 cells of exponential phase are collected in digestion, after counting that its is dense It is condensed to 5 × 10⁷The unicellular solution of/ml Hanks, by every 0.2ml be inoculated in left side groin on the outside of it is subcutaneous, be successfully established shifting Plant knurl animal model.Zoopery is whole to be carried out under the supervision and accreditation of Ethics Committee of Bethune International Peace Hospital. Animal packet and DHA intervene：Transplanted tumor in nude mice diameter it is long to 5mm or so when be grouped with random table, i.e. DHA groups and control group (n=7).DHA groups give 50mg/kg, and control group then gives DMSO, and two groups of animals are administered using intraperitoneal injection, continuous note Penetrate 2/3 day and rest 1 day, inject 5 times weekly, intervention time amounts to 28 days.The measurement of transplantable tumor volume and the weight of animals：See daily Examine the ordinary circumstances such as the animal state of mind, diet and defecation, pay special attention to whether there is bite, tumor by local skin whether there is ulceration etc.. With gross tumor volume of electronics vernier caliper measurement, while electronic scale claims the weight of animals, register and statistical measurement number within every 4 days According to noticing control group and experimental group body weight whether there is significant difference.Gross tumor volume is calculated with below equation：Volume (cm³)= It is wide²(cm²) × long (cm)/2.After last time DHA intraperitoneal injections 24h, CO is first used₂Anesthetized animal, then uses at disconnected neck method Dead experiment nude mice.The transplanting tumor tissue for stripping is divided into two parts, and a part is placed in paraformaldehyde solution for immunohistochemistry Dyeing；Another part is placed in liquid nitrogen is used for Western blot analysis Jak2/STAT3 signal paths GAP-associated protein GAP changes.

Cell immunohistochemical staining method.Animal tumor tissue sample presses 4 μ m thick serial section, is then attached to rely through poly On the slide of propylhomoserin treatment, 60 DEG C overnight.Will dewaxing 20min 2 times in section dimethylbenzene；It is dipped in 100% ethanol 2min 2 It is secondary；95% ethanol 2min 2 times；90% ethanol 2min 3 times；85% ethanol 2min 2 times；75% ethanol 2min 2 times.Will section It is placed in citric acid reparation liquid, in heating in pressure cooker；Pressure cooker stops heating after boiling 2~3min, is then down to room temperature, uses PBS washing slice 5min, totally 3 times.The activity that 3% hydrogen peroxide closes peroxidase is added dropwise, then 5min3 is washed with PBS It is secondary, lowlenthal serum room temperature closing 20min is added dropwise.P-Jak2 (Tyr1007/1008) and p-STAT3 (Tyr705) primary antibody is added dropwise, presses 1：100 dilution proportions, overnight incubation in 4 DEG C of refrigerators.With PBS washing slices 5min 3 times, goat antirabbit secondary antibody is added dropwise, room temperature is incubated 20min is educated, then 5min is washed with PBS 3 times.In horseradish enzyme mark strepto- avidin working solution is added dropwise in section, 37 DEG C are incubated 20min, PBS washing 5min 3 times.Colour developing is controlled with DAB under microscope, is cut into slices with running water cleaning down.Haematoxylin redyeing 3min, 1% hydrochloride alcohol differentiation, ammoniacal liquor returns indigo plant, gradient alcohol dehydration, each 2min, xylene soak 5min2 times, neutral tree Glue mounting.The positive and negative tissue of DHA experimental groups and DMSO control groups mutually using 400 × microphotograph preservation.

Western blotting detect DHA to HNSCC cellular associated proteins expressions.The extraction of cell protein：Choosing Take the logarithm growth period Fadu, Cal-27 and Hep-2 head and neck scale carcinoma cell, digestion count after with 4 × 10⁵/ 2ml/well cells are close Degree is inoculated into 6 well culture plates, after existing prepare, the culture medium 2ml containing various concentrations DHA of addition after cell attachment.Medicine is done The μ l of RAPI lysates 100 are added to extract cell protein after terminating in advance.Tumor tissue protein extraction：Tumor tissue is taken out from liquid nitrogen, will It inserts homogenizer homogenate after being cut into tiny fragment, adds RIPA lysates of the 500 μ l containing PMSF, and 30min is placed on ice, cracks During repeatedly homogenate grinding thoroughly cracking histocyte.Then by Tissue Lysates move into centrifuge tube in, under the conditions of 4 DEG C, 12000rpm is centrifuged 15min, and Aspirate supernatant is placed in liquid nitrogen and saves backup.Using BCA standard measure protein concentrations, calculate and carry The protein concentration of each sample for taking, is distributed into the centrifuge tubes of 200 μ 1, and often pipe contains 40 μ g albumen, then carries out albuminous degeneration.Match somebody with somebody The separation gel of system 8~12%, room temperature places 30min.Treat that gelling knot, in g., jelly-like, prepares 4% and concentrates glue, insert comb.Treat glue Comb is extracted after condensation, electrophoresis liquid is poured into, injector adds protein sample and standard items albumen Marker as control, connects electricity Stream, carries out SDS-PAGE electrophoresis, time about 2h.Filter paper, pvdf membrane, gel are sequentially placed into after end, both positive and negative polarity is noted, electricity is poured into Turn liquid, by the albumen transferring film on separation gel to pvdf membrane, the time is about 4h.Then 1h is closed and with containing primary antibody with skimmed milk power Skimmed milk power and 4 DEG C of overnight incubations of pvdf membrane.Next day washes film and is incubated secondary antibody 1h, is developed using ECL methods.

Fig. 7 is the activation that DHA suppresses Jak2/STAT3 signal paths in zoografting tumor tissue, is as a result shown, control group Jak2 and STAT3 activation is expression high, and the activation of two kinds of albumen of DHA groups is significantly inhibited.

Presently preferred embodiments of the present invention is the foregoing is only, is not intended to limit the invention, it is all in essence of the invention Within god and principle, any modification, equivalent substitution and improvements made etc. should be included within the scope of the present invention.

Claims (10)

1. a kind of application of dihydroartemisinine, it is characterised in that：The dihydroartemisinine is used to suppress STAT3 abnormal activations, institute The molecular formula for stating dihydroartemisinine is as follows：

2. the application of dihydroartemisinine according to claim 1, it is characterised in that：

The dihydroartemisinine is used to suppress the STAT3 activation when hypoxemia or/and IL-6 protein factors stimulate.

It is 3. a kind of to suppress medicine, it is characterised in that：The suppression medicine contains dihydroartemisinine as claimed in claim 1.

4. suppression medicine according to claim 3, it is characterised in that：The suppression medicine contains drug excipient or load Body.

5. suppression medicine according to claim 4, it is characterised in that：The drug excipient or carrier include glycerine, second One or more in alcohol, BS, dimethyl sulfoxide (DMSO), physiological saline.

6. suppression medicine according to claim 5, it is characterised in that：Double blue and green artemisin concentration are 40-200 μm of ol/ L。

7. the application of a kind of suppression medicine as any one of claim 3 to 6, it is characterised in that：The suppression medicine For in the Disease Intervention with STAT3 abnormal activation characteristics.

8. the application for suppressing medicine according to claim 7, it is characterised in that：The intervention time of the suppression medicine >= 12h。

9. the application for suppressing medicine according to claim 8, it is characterised in that：The disease includes tumour, autoimmunity Property disease, diseases associated with inflammation.

10. the application for suppressing medicine according to claim 9, it is characterised in that：Suppression medicine and the cancer therapy drug or Anti-inflammatory drug is used in combination.