CN107281213B - Application of kohlrabi polysaccharide in preparation of radioactive xerostomia medicines - Google Patents

Abstract

The invention relates to the technical field of medicines, in particular to application of bolete polysaccharide in preparation of a medicine for preventing and treating radioactive xerostomia, and animal experiments prove that the bolete polysaccharide is suitable for preventing and treating the radioactive xerostomia, has definite curative effect and has good medicinal value in preparation of the medicine or health-care products for preventing and treating the radioactive xerostomia; the method for preparing the dogliver vegetable essence polysaccharide has the advantages of simple process, easily obtained raw materials, high yield, high content, no toxic or side effect, good taste and good development value, and further reduces the preparation cost.

Description

Application of kohlrabi polysaccharide in preparation of radioactive xerostomia medicines

[ technical field ] A method for producing a semiconductor device

The invention relates to the technical field of medicines, and in particular relates to application of rabdosia koenigii polysaccharide in preparation of a medicine for treating radioactive xerostomia.

[ background of the invention ]

At present, radiotherapy is still one of the treatment methods for head and neck malignant tumors, but due to the position of salivary glands and the high sensitivity of salivary glands to radioactive rays, malignant tumor cells are killed, and meanwhile, adjacent salivary gland tissues are also damaged, so that various complications of the salivary glands are caused, wherein the radioactive xerostomia seriously affects the life quality of patients, and therefore, the research on the prevention and treatment measures for the salivary gland radioactive xerostomia is always a hot point concerned by scholars at home and abroad.

Herba Cibotii whole plant (Dicliptera chinensis L. Ness) is mainly produced in southern China such as Guangdong and Guangxi, has the functions of clearing heat and detoxicating, cooling blood and arresting bleeding, promoting fluid production and diuresis, and is commonly used for common cold and fever, summer heat and polydipsia, dysuria and other symptoms. It is originally recorded in Lingnan's herbal collection recorded in Xiaopedodan of China, "pedunculate, phyllanthus and almond, cold in nature and heat-dissipating. It is called local antelope. Modern researches show that the polysaccharide of the herba et Gemma Agrimoniae is a main effective component in the whole herb of the herba et Gemma Agrimoniae, and has physiological activities of resisting oxidation, protecting liver, regulating immunity, etc. The polysaccharide of the kohlrabi has not only very wide biological activity, but also extremely low toxic and side effects in vivo, and has become one of the research hotspots in the field of medicine.

The aquaporin family is a water-specific aquaporin on cell membranes, is ubiquitous on animal and plant and microbial cell membranes, and has increasingly prominent functions and effects particularly in animals. The prior research on the molecular mechanism of salivary gland radioactive damage suggests that radiation can inhibit the function of aquaporin family, so that the expression of aquaporin family is reduced, thereby possibly influencing the aquaporin-mediated transport process of water across acinar cell membranes, aquaporin 5(AQP5) as one of aquaporin family members is one of a few members which are found to be positioned in rat salivary glands so far, and the reduction or deletion of aquaporin can influence the water transport pathway, so that osmotic pressure imbalance is caused, and then xerostomia is caused, and in addition, Ma confirms that AQP5 plays an important role in the salivary gland secretion function through knocking out rat AQP5 gene.

So far, only the heparan setariae polysaccharide (DCP1A) is prepared in the prior art, and the monosaccharide components and the structures of the heparan setariae polysaccharide are not analyzed.

[ summary of the invention ]

The invention aims to: provides an application of the dog liver vegetable essence polysaccharide in the treatment of radioactive xerostomia and a method for preparing the dog liver vegetable essence polysaccharide. The invention firstly separates, extracts and purifies the whole herb of the kohlrabi to prepare the kohlrabi extract polysaccharide (DCP1A) which acts on a radioactive injury rat model, and determines whether the kohlrabi polysaccharide has a certain protective effect on salivary gland radioactive xerostomia by dynamically observing the salivary gland morphology and function with the function of saliva secretion; through observing the expression change condition of AQP5 in rat submandibular gland tissue from two aspects of protein level and molecular level, the expression condition of AQP5 capable of influencing salivary gland secretion function is deeply observed, thereby providing scientific basis for the treatment of radioactive xerostomia. The dog liver vegetable essence polysaccharide can restore the function and the form of salivary gland tissues with salivary secretion function in animal experiments, and the AQP5 in salivary line tissues is detected through protein and molecular level, so that the dog liver vegetable essence polysaccharide can improve the expression quantity, and can be used for treating and preventing the radioactive xerostomia.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a composite of herba Sedi Aizoon polysaccharide for preventing and treating radiation xerostomia is prepared by adding adjuvants allowable by national and industrial standards and regulations, and making into tablet, capsule, pill, granule, powder and injection, and its preparation method is common to that of existing corresponding dosage forms.

Molecular weight of 2.40-2.48X 10⁴The composition is added with auxiliary materials allowed by national and industrial standards and regulations to be added, and is used for preparing tablets, capsules, pills, granules, powder and injections.

The invention discloses a herba cynanchi wallichii polysaccharide which is prepared by extracting crude polysaccharide in plants from herba cynanchi wallichii whole plants by adopting an alcohol precipitation water extraction method, removing crude polysaccharide free protein by a papain hydrolysis method, removing pigment by a hydrogen peroxide oxidation method, removing micromolecular substances by using dialysis and membrane separation technologies, separating uniform polysaccharide components by respectively adopting an anion exchange column chromatography method and a gel column chromatography method to obtain herba cynanchi wallichii extract polysaccharide (DCP1A) with relatively uniform components, and determining the molecular weight and the purity of the extract polysaccharide by a high performance liquid permeation chromatography;

the alcohol precipitation water extraction method comprises the steps of carrying out water extraction on the cibotium barometz to obtain a water extract, carrying out alcohol precipitation on the water extract, freeze-drying the water extract by using a vacuum freeze-drying machine, and collecting crude cibotium barometz polysaccharide;

the method for removing free protein of crude polysaccharide by papain hydrolysis comprises dissolving dried crude polysaccharide of herba Selaginellae Doederleinii, adding papain to water bath, inactivating enzyme at high temperature, centrifuging, collecting supernatant, and freeze drying to obtain deproteinized crude polysaccharide of herba Selaginellae Doederleinii;

the anion exchange column chromatography method comprises the steps of sequentially adjusting DEAE-cellulose filler to be neutral through an alkaline substance and then through an acidic substance, adjusting the DEAE-cellulose filler to be neutral through an alkaline substance and then through distilled water, then enabling the DEAE-cellulose filler to enter a chromatographic column, adding crude polysaccharide after the DEAE-cellulose filler is adjusted to be neutral, dissolving the DEAE-cellulose filler with distilled water, unscrewing a spiral clamp from the chromatographic column, sequentially adding distilled water, a NaCl solution and a NaOH solution for elution, collecting more than 10mL of eluent at intervals of 60min to perform absorbance detection on the eluent, combining the eluents with the same peak position of 490nm, performing evaporation concentration, dialyzing and freeze-drying to obtain a primarily purified polysaccharide sample (DCP 1);

the gel column chromatography is to pretreat Sephacryl S-300HR filler, then pack the filler, adjust the inner gel of the chromatographic column until the volume is maintained unchanged, close the bottom spiral clamp, precisely absorb the freeze-dried NaCl fraction to prepare aqueous solution, slowly drop-add the aqueous solution onto the upper surface of the gel, open the bottom spiral clamp for balancing, close the spiral clamp after the sample completely enters the gel bed, drop eluent, start the column elution, concentrate the elution fraction under reduced pressure, then carry out alcohol precipitation, centrifuge, freeze-dry, and further purify the herba Setariae viridis seminiferous polysaccharide (DCP1A) at the maximum peak position in the elution diagram. The content of polysaccharide in DCP1A fraction was 95.22% by HPGPC.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

the invention overcomes the existing research thinking inertia and limitation in the technical field, and through a large amount of experimental summarization and analysis for a long time, the adopted dogliver vegetable medicinal material resources are rich, the preparation process of the method for preparing the dogliver vegetable essence polysaccharide is simple, the raw materials are easy to obtain, the preparation cost is further reduced, and the yield is high, the content is high, no toxic or side effect is caused, the taste is good, and the development value is good. In addition, the invention discovers that the bolete polysaccharide is suitable for preventing and treating the radioactive xerostomia, has definite curative effect, has good medicinal value in preparing medicines or health-care products for preventing and treating the radioactive xerostomia, and provides a research basis for further and deeply developing the bolete polysaccharide.

[ description of the drawings ]

FIG. 1 is an elution profile of a DEAE-52 cellulose chromatography column of the present invention;

FIG. 2 is an elution curve of Sephacryl S-300HR chromatography column of the present invention;

FIG. 3 is a high performance coacervation permeation chromatogram of DCP1A of the present invention;

FIG. 4 is a graph of the effect (g) of the heparan Canitis extract polysaccharide of the invention on the mean value of weight gain in radioactively injured rats;

FIG. 5 shows the change of staining of the mandibular gland HE (HE, x 200) after 1 week and 12 weeks of application of DCP1A of the present invention to a radioactively injured rat (notes A-D are respectively a 0Gy blank group, a 0Gy low dose group, a 0Gy medium dose group, and a 0Gy high dose group at 1 week after irradiation; E-H are respectively an 18Gy irradiation group, an 18Gy low dose group, an 18Gy medium dose group, and an 18Gy high dose group at 1 week after irradiation; I-L are respectively a 0Gy blank group, a 0Gy low dose group, a 0Gy medium dose group, and a Gy 0 high dose group at 12 weeks after irradiation; M-P are respectively a 18Gy blank group, an 18 low dose Gy blank group, an 18Gy medium dose group, and an 18Gy high dose group at 12 weeks after irradiation);

FIG. 6 shows the PAS staining pattern (PAS, x 200) of the submandibular gland of a DCP1A of the present invention after the radioactive injury of rats for 1 week and 12 weeks (A-D are a 0Gy blank group, a 0Gy low dose group, a 0Gy medium dose group, and a 0Gy high dose group after the irradiation of rats for 1 week, respectively, E-H are a 18Gy irradiation group, an 18Gy low dose group, a 18Gy medium dose group, and a 18Gy high dose group after the irradiation of rats, respectively, I-L are a 0Gy blank group, a 0Gy low dose group, a 0Gy medium dose group, and a 0Gy high dose group after the irradiation of rats for 12 weeks, and M-P are a 18Gy blank group, a 18Gy low dose group, a 18Gy medium dose group, and a 18Gy high;

FIG. 7 shows the change of AQP5 immunohistochemical staining (immunohistochemistry, x 200) of DCP1A of the present invention in the submaxillary gland after 1 week and 12 weeks of radiation injury in rats (A-D are 0Gy blank group, 18Gy low dose group, 18Gy medium dose group, 18Gy high dose group, respectively, 1 week after irradiation, E-H are 18Gy irradiation group, 18Gy low dose group, 18Gy medium dose group, 18Gy high dose group, respectively, I-L are 0Gy blank group, 0Gy low dose group, Gy 0 medium dose group, 0Gy high dose group, respectively, 12 weeks after irradiation, M-P are 18Gy blank group, 18Gy low dose group, 18Gy medium dose group, 18Gy high dose group, respectively, after irradiation);

FIG. 8 shows the relative expression level of AQP5mRNA of DCP1A acting on the submaxillary gland of a rat with radioactive damage

Note that ① represents the 0Gy dosing of each group vs0Gy blank groups,

②

represents 18Gy4 week middle, high dose group vs18Gy8 week middle, low dose group vs18Gy12 week middle, high dose group vs18Gy12 week middle, high dose group,

figure 9 internal reference GAPDH and AQP5 amplification and dissolution curves of interest.

[ detailed description ] embodiments

In order to make the technical means, the creation features, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the embodiment. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1:

1 separation and extraction of polysaccharide from herba Canitis

Weighing 2.00kg of dried whole dogliver herb, cutting into pieces with the length of about 2cm, adding 8L of distilled water, extracting at 90 ℃ for 4h, repeating for 3 times, combining extracting solutions, concentrating at 65 ℃ in a rotary evaporator to 200ml, slowly pouring into absolute ethyl alcohol (V concentrated solution: V absolute ethyl alcohol is 1:4), shaking fully, standing overnight, collecting alcohol precipitates the next day, freeze-drying in a vacuum freeze-drying machine, and collecting crude dogliver herb polysaccharide for later use.

1.2 separation and extraction of crude polysaccharide from Cibotium Sativum

1.2.1 Deproteinization of crude polysaccharide from herba Selaginellae Doederleinii

Taking the freeze-dried crude polysaccharide of the dogliver dish, adding distilled water to enable the final concentration to be 5.00% (g/mL), adding papain to enable the final concentration to be 1.00% (g/mL), fully stirring, putting in a water bath at 50 ℃ for 3h, inactivating enzymes at high temperature, centrifuging at 3000r/min for 10min, collecting supernatant, and concentrating and freeze-drying by a vacuum freeze-drying machine to obtain the deproteinized crude polysaccharide of the dogliver dish.

1.2.2 decolouring of crude polysaccharide from herba Selaginellae Doederleinii

Adding distilled water to obtain deproteinized herba Setariae viridis crude polysaccharide with final concentration of 5.00% (g/mL), adding 30% hydrogen peroxide to obtain final concentration of 1.00% (g/mL), reacting in 45 deg.C water bath for 4h, detecting pH once every 30min, adjusting pH to 8.0 with 0.50 mol/L-1 NaOH solution to maintain pH of the system at 8.0, cooling to room temperature, and reacting with 0.50 mol/L^-1Adjusting HCl to be neutral, adding NaCl to enable the final concentration to be 5.00% (g/mL), after NaCl is completely dissolved, slowly adding equal volume of absolute ethyl alcohol into the system, shaking up fully, standing overnight at 4 ℃, centrifuging after 24h, collecting alcohol precipitate, and freeze-drying by a vacuum freeze-dryer to obtain the depigmented crude polysaccharide of the green bolete.

1.2.3 removing small molecular impurities from herba Selaginellae Doederleinii crude polysaccharide

Taking the crude polysaccharide of the cabbages, which is subjected to protein removal and pigment removal, adding a proper amount of distilled water, completely dissolving the crude polysaccharide, putting the crude polysaccharide into a dialysis bag with the molecular weight cutoff of 3500, dialyzing for 48 hours, concentrating the dialyzate, and freeze-drying by a vacuum freeze-drying machine to obtain the crude polysaccharide of the cabbages, which is subjected to small molecular impurity removal.

1.3 separation and extraction of Boletus edulis extract polysaccharide

1.3.1DEAE-52 cellulose column chromatography purification of crude polysaccharide of herba Selaginellae Doederleinii

Weighing 150.00g of DEAE-cellulose filler, then carrying out DEAE-cellulose filler pretreatment, soaking the filler in 600mL of distilled water, stirring, adding 400mL of distilled water after 24 hours, standing for 30min, carrying out suction filtration to remove suspended particles, adding 0.50 mol.L^-1Soaking 500mL of NaOH solution for 30min, repeatedly washing with distilled water to neutrality after suction filtration, and then using 0.50 mol.L^-1Soaking 500mL of HCl solution for 30min, repeatedly washing the HCl solution to be neutral by using distilled water after suction filtration, and placing the filler in a solution of 0.50 mol.L^-1Soaking in NaOH solution for 30min, vacuum filtering, washing with distilled water to neutrality, loading into chromatography column with specification of 2.5cm × 80cm, fixing on tripod, adding distilled water into chromatography column to make the distilled water level reach half of the column height, slowly adding pretreated DEAE-cellulose suspension, opening spiral clamp at column bottom, and maintainingKeeping the flow rate at 1 drop/6 s, slowly pouring the pretreated DEAE-cellulose suspension again when the liquid level of the distilled water is close to the liquid level of the cellulose, repeating the above operation until the DEAE-cellulose liquid level in the column is kept unchanged to a position about 5cm below the opening of the column, closing the spiral clamp, checking that the column surface is flat and free of bubbles, taking the distilled water as a mobile phase at the flow rate of 1 drop/10 s, balancing for 12h, accurately weighing 2.00g of the crude polysaccharide sample, adding 20ml of distilled water for dissolving, slowly dropwise adding the sample solution, unscrewing the spiral clamp, sequentially using the distilled water and 0.125 mol.L^-1And 0.30 mol. L of NaCl solution^-1Eluting with NaOH solution at flow rate of 1 drop/6 s, collecting one tube (about 10mL) every 60min, measuring absorbance of eluate at 490nm, mixing eluates with the same peak position, performing rotary evaporation and concentration at 50 deg.C, dialyzing for 48 hr, and lyophilizing to obtain primarily purified polysaccharide sample (DCP 1).

The polysaccharide of different components can be separated by eluting through DEAE-52 cellulose column by using different mobile phases, the experiment uses distilled water, 0.125mol/LNaCl solution and 0.30mol/LNaOH as the mobile phases for elution in turn, the elution curve is shown in figure 1, and the three components obtained by fractionation according to the elution curve are named DCP1, DCP2 and DCP3 in turn according to the sequence of the peak time (figure 1).

1.3.2Sephacryl S-300HR column chromatography purification of crude polysaccharide of herba Selaginellae Doederleinii

Weighing 5.00g of Sephacryl S-300HR filler for pretreating the Sephacryl S-300HR filler, adding 50mL of deionized water for soaking for 24 hours, pouring out the upper layer liquid, adding NaOH solution with the same volume, standing for 1 hour, pouring out the liquid, repeatedly washing the deionized water until the deionized water is neutral, then loading the column, fixing the column on a tripod, injecting distilled water into the column at 1/4 column height in advance, pouring the gel solution into the column along the tube wall after stirring uniformly until the gel solution is close to the column height, opening a bottom spiral clamp, supplementing the gel in the column until the volume is unchanged, keeping the position of which the upper plane is about 5cm below the column height, balancing the mobile phase with the flow rate of 1 drop/10S until the column surface of the gel does not drop any more, closing the bottom spiral clamp, precisely sucking the freeze-dried NaCl fraction to prepare an aqueous solution, slowly dropping on the upper surface of the gel, opening the bottom spiral clamp for balancing, and (3) closing the spiral clamp after the sample completely enters the gel bed, dropwise adding an eluent, starting column elution, eluting with distilled water in the experiment at the flow rate of 1 drop/4 s, carrying out alcohol precipitation on the eluted fraction after reduced pressure concentration, centrifuging, freeze-drying, and further purifying the dicliptera chinensis semina polysaccharide (DCP1A) at the maximum peak position in the elution diagram.

Purifying DCP1A by Sephacryl S-300HR gel filtration chromatography, eluting with NaCl, collecting normal elution peak, concentrating under reduced pressure, precipitating with ethanol, and lyophilizing to obtain DCP1A and DCP1B, as shown in FIG. 2.

1.3.3 determination of molecular weight of DCP1A by High Performance Gel Permeation Chromatography (HPGPC)

The relative molecular weight of DCP1A was determined by High Performance Gel Permeation Chromatography (HPGPC), and 1.00g of lyophilized DCP1A polysaccharide sample was precisely taken and dissolved in ultra-water to give a final concentration of 1mg/mL, and standard dextran40 (molecular weights 5900, 11800, 22800, 47300, 112000, 21200 and 404000, respectively). The HPLC conditions were as follows: a chromatographic column: TSKgelG3000PWXL (Φ 7.8 mm. times.300 mm); a detector: ELSD-LH II low temperature type evaporation light scattering detector (Sedex 85), gain 1, drift tube temperature 50 ℃, carrier gas pressure 50 psi; a separation unit: ultrapure water, flow rate 0.8mL/min, column temperature 40 ℃.

As shown in fig. 3, it can be seen that the elution peak of High Performance Gel Permeation Chromatography (HPGPC) of DCP1A is single and has good symmetry, indicating that DCP1A is a homogeneous component, which can be used for the post-experimental study, and the standard regression equation of molecular weight is: y-0.5493 x +9.1967, R2-0.9936. The molecular weight of DCP1A was calculated to be 2.42X 10 by standard equation⁴Da。

2.1 rat radioactivity model establishment

192 SPF-grade SD rats purchased from Experimental animals center of Guangxi medical university are adaptively raised in an SPF-grade environment for one week, and then are divided into 8 groups by a complete random numerical method, wherein the 8 groups comprise a 0Gy blank control group, a 0Gy administration control group, an 18Gy irradiation experiment group and an 18Gy administration experiment group (a high dose group is 400mg/kg, a medium dose group is 200mg/kg, and a low dose group is 100mg/kg), and each group comprises 24 rats (divided into 4 time points and 6 rats at each time point). The rats in each group are injected with 2% sodium pentobarbital (50mg/kg) in an abdominal cavity, after complete anesthesia, 60Co gamma rays are used as a radiation source, the head and neck are used as a radiation field, the rats are in a supine position, the radiation area is 2cm multiplied by 5cm, other parts are protected by lead blocks, the skin source distance is 50cm, the radiation center is 1cm under the skin, the radiation speed is 81.4cGy/min for split radiation, the method for simulating human body radiation is improved, 18Gy radiation amount is given at one time [39], after radiation, the rats are raised in an SPF level environment of the experimental animal center of Guangxi medical university for further experiment, the test is carried out in the morning after 1 week before radiation and 4 weeks after radiation, 9 am each day: 00-11: during the period of 00, rats of the administration group are given corresponding dose DCP1A by a gastric lavage method.

2.2 radiation rat Whole body situation Observation

2.2.1 body weight

Rat body weights before and after irradiation were measured and values were recorded, measured every 7 days for 12 weeks.

Measuring the weight of the rats before and after irradiation, recording numerical values, measuring once every 7 days for 12 weeks, and calculating the weight increase average value drawing result as shown in figure 2-1, wherein in the adaptive breeding stage before ① irradiation, the increase value of each group of 0Gy administration control is slightly higher than that of the other groups, in one week after ② irradiation, the increase average value of each group is reduced, which is probably related to the reduction of the diet and water intake of the rats caused by the change of the living environment of the rats in the process of manufacturing the rat radiation injury model, but the 18Gy irradiation experimental group is in a negative increase state, the other groups are in a positive increase state, and the difference of the increase level is small, and in the 18Gy irradiation experimental group after ③ irradiation for 2 weeks, the positive increase state is recovered, but the increase value is obviously lower than that of the other groups, and the difference of the 18Gy administration experimental group and the other control groups is small, which indirectly indicates that the weight increase value of the rats with the radioactivity injury can be influenced by a certain.

2.4 Radioactive morphological Observation of submaxillary gland in rat

2.4.1 specimen processing method

Rapidly dividing the submaxillary gland with the measured submaxillary gland index into three parts at 4 time points of 1, 4, 8 and 12 weeks after irradiation, respectively, putting one part into paraformaldehyde for fixation, and then preparing into wax block for HE staining method and immunohistochemistry experiment; weighing one part, putting the weighed part into a freezing tube, and preserving the part in liquid nitrogen for an immunoblotting experiment; and the other part is weighed and put into the retention solution containing RNA degradation, and put into a refrigerator at the temperature of 80 ℃ below zero for freezing storage to be used for RT-qPCR experiments.

2.4.2HE staining

And conventionally slicing the wax blocks to be detected after 1, 4, 8 and 12 weeks of irradiation, wherein the thickness of the wax blocks is 4 mu m, baking the wax blocks, dewaxing, rehydrating, conventionally HE dyeing and sealing the wax blocks.

As shown in FIG. 5, it can be seen from the light microscope that the submandibular gland of the 0Gy control group had a clear structure, had full acini, had round or oval acinar nuclei, and were arranged relatively neatly (FIGS. A-D, I-L), and the 18Gy administration groups had smaller acinar cells, increased nuclear deformation, shrinkage, and vacuole-like changes, but had a lower degree of damage than the 18Gy irradiation group (FIGS. E-H, M-P), compared to the 0Gy control group.

2.4.3PAS dyeing

Respectively slicing the wax blocks to be tested after irradiation for 1, 4, 8 and 12 weeks, conventionally slicing to a thickness of 4 mu m, baking, dewaxing, washing with distilled water, soaking in clean periodic acid liquor, washing with tap water, soaking in Schiff's solution for 10min, conventionally dehydrating, transparentizing and sealing after hematoxylin staining nuclei.

As shown in FIG. 6, it can be seen from the optical microscope that the 0Gy blank submaxillary gland has a clear structure, plump acini, round or oval acinar cell nucleus and is arranged in order, while the 18Gy administration groups have smaller acinar cells than the 0Gy control group, cell nucleus deformation, shrinkage and vacuole change increase, but the damage degree is lighter than that of the 18Gy irradiation group, PAS staining result shows that the number of acinar cells containing glycogen in the cytoplasm of the 0Gy blank group is more, and the 18Gy administration groups show that the number of acinar cells containing glycogen is less than that of the 0Gy control group but more than that of the 18Gy irradiation group, which indicates that the Canitis liver vegetable sperm polysaccharide (DCP1A) has protection effect on the radioactively injured rat, the damage degree of the acinar cells of the submaxillaryx gland can be partially restored, and the morphological injury of the radioactively injured rat can be partially recovered, and, in conclusion, the Canitis liver vegetable sperm polysaccharide has protection effect on the radioactively injured rat and the, can lay a foundation for the treatment of radioactive salivary gland injury.

2.1 immunohistochemistry method for measuring AQP5 expression change in submaxillary gland after rat radiation

Continuously slicing a sample wax block 1, 4, 8 and 12 weeks after irradiation, baking the slice in an oven with the thickness of 4 mu m and the temperature of 60 ℃ for 3 hours, soaking the slice in dimethylbenzene I, II, III and IV solutions for 5min respectively, dewaxing by gradient alcohol (100%, 95%, 85% and 75%) for about 10 seconds each time, washing the slice with clear water for 3 times, placing the slice in a diluted EDTA (1:50) antigen repairing solution, repairing the antigen for 4 minutes at high temperature and high pressure, naturally cooling, washing the slice with distilled water for 3 times, 2 minutes each time, incubating the slice in 3% hydrogen peroxide solution for 10 minutes at 37 ℃ to block the activity of endogenous peroxidase, washing the slice with PBS for 3 times, 2 minutes each time, dripping diluted AQP5 polyclonal antibody (1:125) for incubation, standing overnight at 4 ℃, washing the PBS for 3 times for 2 minutes each time for the next day, dripping polymer auxiliary agent (reagent A) in the incubation kit, 20 minutes at 37 ℃, washing the slice with PBS for 3 times, 2 minutes each, adding horseradish enzyme labeled anti-rabbit IgG polymer (reagent B) in the kit dropwise for incubation, incubating at 37 ℃ for 20 minutes, washing with PBS for 3 times, washing for 2 minutes each time, developing with DAB color development solution for 2 minutes, washing with flowing clear water for 10 minutes, counterstaining with hematoxylin, dehydrating, transparentizing, and sealing. After the sheet was dried, it was analyzed by Image-pro plus 6.0 software, and the average optical density value of each specimen was measured at 6 different sites at random under a 200-fold microscope, and the average optical density was used to represent the expression amount of AQP 5.

TABLE 2DCP1A Effect on AQP5 mean optical density values (x + -s, n-6) in the submandibular glands of radiation-injured rats

Note that ① 0Gy was administered to each group vs0Gy as a blank group, p > 0.05: ② 18Gy4 weeks, a high dose group vs18Gy8 weeks, a high dose group vs18Gy12 weeks, a high dose group, p > 0.05: ③, and p < 0.05

As shown in figure 7, AQP5 in 0Gy blank group and 0Gy administration group has strong expression in apical membrane, lateral membrane and duct of acinus (figures A-D, I-L), single-factor variance analysis and LSD test are carried out between 0Gy blank group and 0Gy administration group, P is more than 0.05, statistical difference between control groups can not be considered, therefore, we can see that the AQP5 expression quantity in submaxillary gland tissue can not be increased by simply administering rat DCP1A, therefore we can exclude DCP1APositive incidence; as shown in table 2: after irradiation, the expression quantity of AQP5 in the submaxillary gland tissue of a rat in an 18Gy irradiation group is in a negative correlation relationship with time (figure E, M), the expression quantity of AQP5 is gradually reduced (P is less than 0.05), the expression quantity of each 18Gy administration group is higher than that of the 18Gy irradiation group (P is less than 0.05) at the same time point, the difference has statistical significance, and the fact that the DCP1A acts on a radioactive injury rat to increase the expression quantity of AQP5 in the submaxillary gland tissue of the rat is prompted; the dose group in 18Gy4 week is compared with the dose group in 18Gy1 week, and the high dose group in 18Gy4 week is compared with the high dose group in 18Gy1 week, it can be seen that the expression level of AQP5 in rat submandibular gland tissue is reduced (P < 0.05), but single-factor anova and LSD test is carried out in 18Gy4 week, high dose group and 18Gy8 week, high dose group and 18Gy12 week, and high dose group,

it can not be considered that there is a statistical difference between the control groups, so we can see that, although the expression level of AQP5 in the submaxillary gland tissue of the rat is reduced in 4 weeks compared with 1 week after radiation, the reduction of the expression level of AQP5 in the submaxillary gland tissue of the rat can tend to a stable state with the continuous administration, and no drug rebound phenomenon appears after observation, so we can find that: the DCP1A can increase the expression of AQP5 in submandibular gland tissue of a rat after radiation, and the medium and high dose DCP1A is more suitable for acting on a radiation-damaged rat.

2.3RT-qPCR assay of AQP5 expression changes in the submaxillary gland after rat irradiation

Collecting irradiated submaxillary gland tissue 1, 4, 8, 12 weeks, placing into precooled mortar, pouring liquid nitrogen, grinding tissue into fine powder, adding 1ml RNAiso Plus solution, standing at room temperature for 5min, 12.000g, centrifuging at 4 deg.C for 5min, collecting supernatant, transferring into DEPC-treated 1.5ml centrifuge tube, adding 200ul chloroform, shaking, standing at room temperature for 5min, 12.000g, centrifuging at 4 deg.C for 15min, collecting supernatant, transferring into new 1.5ml centrifuge tube, adding 500ul isopropanol, standing at room temperature for 10min, 2.000g, centrifuging at 4 deg.C for 10min, collecting supernatant, standing precipitate, rinsing with 1ml 75% ethanol (prepared from DEPC-treated water), precipitating at 12.000g, centrifuging at 4 deg.C for 5min, discarding supernatantRemoving precipitate, naturally drying, dissolving in appropriate amount of non-enzyme water, performing purity and concentration analysis with ultraviolet visible light photometer, reverse transcribing the sample with OD260/OD280 value of 1.8-2.0 into cDNA according to the method of PrimeScript RT reagent Kit, specifically comprising ① removing genome DNA reaction, preparing mixed solution of 5 XgDNA Eraser Buffer2 μ l and gDNA Eraser 1 μ l on ice, calculating the total RNA volume of 1 μ g of each sample according to the concentration, adding into the mixed solution, adding RNase FreedH₂O to make the reaction system reach 10 mul, 42 deg.C, making reaction for 2 min on PCR instrument, ② preparing reverse transcription reaction mixed liquor, adding 4 mul RNase freedH in turn₂O, 4 mul 5 XPrimeScript Buffer2(for Real Time), 1 mul PrimeScript RT Enzyme MixI and 1 mul RT Primer Mix reverse transcription reagent are put in a 0.2ml PCR tube, ③ reverse transcription is carried out to cDNA, 10 mul of the reverse transcription reaction mixed solution prepared in the step ② is taken to be added into a reaction tube in the step ① to be reacted in a PCR instrument, the reaction conditions are 15 minutes at 37 ℃ and 5 seconds at 85 ℃, then the reaction tube is placed under the condition of-20 ℃ to be stored, the expression condition of AQP5mRNA is detected by using a RT-qPCR method in the later period, a Primer is designed according to the whole gene sequence of rat AQP5 in GenBank, the Primer is synthesized by TaKaRa biotechnology limited company (Table 1), the delta Ct method is adopted after amplification, and the formula is 2^{- [ Ct (radiation group-internal reference) -Ct (control group-internal reference)}]And (3) calculating the expression quantity of the AQP5 of the submandibular gland of the relative control group of 0Gy blank control group, and drawing a histogram to know the expression change condition of the AQP5 in the submandibular gland of the radioactive injury rat after the DCP1A acts on the radioactive injury rat. Step by step

Premix Ex TaqTMII kit instruction is carried out, specifically as follows, ① is 10.0 mu

Premix Ex Taq II (Tli RNaseH Plus), 0.8. mu.l PCRForward Primer (10. mu.M), 0.8. mu.l PCR Reverse Primer (10. mu.M), 0.4. mu.l ROX Reference Dye, 2.0. mu.l DNA template, 6.0. mu. ldH₂Preparing PCR reaction mixture on O (sterilized distilled water) ice in a dark place, repeating 3 tubes for each specimen, ② performing qPCR reaction in StepOnePlusTM Real-TimeThe PCR instrument is loaded and amplified by adopting a two-step method, the specific reaction conditions are ① 95 ℃ for 30s and 1 cycle, ② 95 ℃ for 5s and 60 ℃ for 30s and 40 cycles, ③ 95 ℃ for 15s and 60 ℃ for 1h and 95 ℃ for 15s and 1 cycle, the amplification curve and the melting curve of RT-qPCR are analyzed and confirmed after the reaction is finished, and the Ct value of each sample is recorded.

TABLE 1AQP5 and GAPDH primer Steps

After the set program in a StepOnePlus Real-Time PCR System instrument is finished, a target gene AQP5 and an internal reference gene GAPDH (figure 9) which can form a good amplification curve and have a unimodal dissolution curve and good specificity are selected, Ct values of the target gene AQP5 and the internal reference gene GAPDH are recorded, expression amounts of samples in the experimental group relative to a 0Gy blank group (figure 8) are calculated according to a formula, as can be seen from table 3, the results show that the expression amounts of AQP5mRNA in rat submaxillary gland tissues of ① Gy administration groups are about 1.0000 relative to the 0Gy blank group, whether normal rat DCP1A is administered and does not affect the expression of AQP5mRNA in rat submaxil gland tissues, the observation Time after radiation is increased for ① Gy administration groups, the AQP5mRNA expression amounts of 18 irradiation groups relative to the 0Gy blank group are about 1.0000, whether the normal rat DCP1A is administered and the mRNA expression amounts of the rat submaxillaryx 5mRNA in rat submaxillary gland tissues are about 42 + -0000.0010.0010.8, the 18Gy irradiation groups are increased along with the observation Time after radiation, the 18Gy irradiation Time, the 18 irradiation groups, the 18 mRNA dosage amounts of the rat submaxillary irradiation groups are about 0.0010.8, the AQ irradiation groups, the AQP5mRNA dosage values of the rat submaxil irradiation groups are gradually reduced from the rat submaxil groups, the mRNA dosage values of the rat submaxillary groups of the rat submaxil groups are about 0000.0010.8, the rat submaxil groups, the rat submaxillary groups, the rat submaxil groups, the mRNA dosage values of the rat submaxillary groups are about 0000.0010.0010.8, the mRNA dosage of the rat mice are gradually reduced from the rat mice 7 + -0.8, the mRNA dosage of the rat mice 7 + -0.The AQP5mRNA expression value in the tissue is reduced, but the numerical value is reduced little after 4 weeks of irradiation, ③ at the same observation time point, the AQP5mRNA expression relative value of the 18Gy administration group is higher than that of the 18Gy irradiation group, and the AQP5mRNA expression relative value of the 18Gy administration group is from high to low as that of the high dose group>Middle dose group>The low dose group, but the difference from the high dose group to the medium dose group after 4 weeks was not statistically significant

The results show that the effect of the medium and high treatment doses on the AQP5mRNA expression in the submaxillary gland tissue of the radioactive injury rat is the same after 4 weeks of radiation, and the phenomenon of drug rebound is not found.

TABLE 3DCP1A action on AQP5mRNA relative expression level (x + -s, n ═ 6) in the submaxillary gland of radiation injured rats

Note that ① represents the 0Gy dosing of each group vs0Gy blank groups,

②

represents 18Gy4 week middle, high dose group vs18Gy8 week middle, low dose group vs18Gy12 week middle, high dose group vs18Gy12 week middle, high dose group,

the above examples are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but not to be construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims (3)

1. An application of herba Sedi Aizoon polysaccharide in preparing radioactive xerostomia medicine is characterized by that its molecular weight is 2.40-2.48X 10⁴Da's rhizoma et radix Bergeniae polysaccharide is used for preparing medicine for preventing and treating radioactive xerostomia.

2. The application of the kohlrabi polysaccharide in preparing the medicine for treating the radioactive xerostomia according to claim 1, wherein the medicine is prepared into a clinical common preparation according to a conventional method.

3. The application of the rabdosia koenigii polysaccharide in the preparation of the medicine for treating the radioactive xerostomia according to claim 1, wherein the preparation comprises tablets, capsules, pills, granules, powder and injection.

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