CN115300574A - Application of eczema ointment containing seven kinds of ginseng and coptis in preparation of medicine for treating neurodermatitis - Google Patents
Application of eczema ointment containing seven kinds of ginseng and coptis in preparation of medicine for treating neurodermatitis Download PDFInfo
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- CN115300574A CN115300574A CN202210721568.7A CN202210721568A CN115300574A CN 115300574 A CN115300574 A CN 115300574A CN 202210721568 A CN202210721568 A CN 202210721568A CN 115300574 A CN115300574 A CN 115300574A
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Abstract
The invention relates to an application of a eczema ointment containing panax notoginseng and coptis chinensis in preparing a medicine for treating neurodermatitis, belonging to the technical field of biological medicines. The eczema ointment comprises the following raw materials in parts by weight: 45 parts of coptis root, 40 parts of sophora flavescens, 35 parts of rhizoma atractylodis, 40 parts of rhizoma paridis, 15 parts of pseudo-ginseng, 30 parts of cortex dictamni, 30 parts of calamine, 20 parts of pepper, 15 parts of borneol and 30 parts of liquorice. The research of the invention finds that the eczema ointment containing seven ginsengs and coptis chinensis can obviously improve the neurodermatitis, reduce the total length and the expression of skin nerve fibers, and reduce the expression of IL-6, TNF-alpha, NGF, PAR2 protein and mRNA thereof, which is one of the mechanisms of treating the neurodermatitis by the eczema ointment containing seven ginsengs and coptis chinensis.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an application of a eczema ointment containing panax notoginseng and coptis chinensis in preparation of a medicine for treating neurodermatitis.
Background
Neurodermatitis (neurodermatitis), also known as licheniplex chronicus, is a chronic inflammatory skin disease characterized by paroxysmal severe itching and lichenification of the skin, which is clinically mainly manifested by infiltration, hypertrophy, desquamation, severe itching, lichen of the skin and the like, and has the characteristics of severe itching, long course of disease and repeated attack. In recent years, the cross-sectional survey of multiple centers in China by experts shows that neurodermatitis is common in eczema and dermatitis and has complex etiology.
Topical glucocorticoids are the currently preferred treatment, and the mechanism of action may be to reduce capillary permeability and thus counteract the proliferation of epidermal cells, improving the clinical condition of the patient. However, because neurodermatitis needs to be treated for a long time, patients can generate a series of adverse reactions after long-term use of glucocorticoid, such as pigmentation, skin atrophy and the like, and even can generate inhibition effect on hypothalamus-pituitary-adrenal axis and the like. In the traditional Chinese medicine external treatment method, the effectiveness of the self-made traditional Chinese medicine preparation smearing, fumigating or acupuncture method is definite.
The ointment for treating eczema is prepared by Yunnan Yunan Yuankang pharmaceutical industry limited company, has the effects of clearing heat and drying dampness, activating blood and reducing swelling, and dispelling wind and arresting itching, is approved to be produced and marketed for many years, and has definite clinical curative effect on eczema caused by rheumatism, heat toxin and stasis.
Patent CN1125117A "eczema ointment" related to the present invention: the invention patent is reported by Wangdenke et al 1994.12.22 of the flare scientific and technological development industry company of Kunming city, the predecessor of the company. The eczema ointment with seven ginsengs produced by the company at present is a product which is preferably selected from the patents, the approval document number is the national drug standard B20020221, the execution standard is WS-5400 (B-0400) -2014Z, and the functions and the indications are as follows: clearing heat, eliminating dampness, promoting blood circulation, relieving swelling, dispelling pathogenic wind, and relieving itching. Can be used for treating eczema with excessive exudation due to stagnation of wind-damp-heat-toxin. Has been widely used in the market, and achieves better treatment effect. At present, no report related to the application of the eczema of seven ginsengs and coptis ointment in the preparation of the medicine for treating the neurodermatitis exists.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides application of a seven-ginseng eczema ointment in preparing a medicine for treating neurodermatitis.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
application of QISHENLIANeczema ointment in preparing medicine for treating neurodermatitis is provided.
Further, preferably, the eczema ointment containing panax notoginseng and coptis chinensis comprises the following raw materials in parts by weight:
45 parts of coptis chinensis, 40 parts of radix sophorae flavescentis, 35 parts of rhizoma atractylodis, 40 parts of rhizoma paridis, 15 parts of pseudo-ginseng, 30 parts of cortex dictamni, 30 parts of calamine, 20 parts of pepper, 15 parts of borneol and 30 parts of liquorice.
Further, preferably, the preparation method of the eczema ointment containing panax notoginseng and coptis chinensis comprises the following steps:
pulverizing Coptidis rhizoma, radix Sophorae Flavescentis, rhizoma Atractylodis, rhizoma paridis, cortex Dictamni Radicis, fructus Zanthoxyli and Glycyrrhrizae radix into coarse powder, adding 5 times (5 times of the total mass of Coptidis rhizoma, radix Sophorae Flavescentis, rhizoma Atractylodis, rhizoma paridis, cortex Dictamni Radicis, fructus Zanthoxyli and Glycyrrhrizae radix) of water, soaking for 1h, boiling for 1h, filtering, adding 3 times of water (3 times of the mass of the residue) into the residue, decocting for 2 times (1 h each time), filtering, mixing the three extractive solutions, and concentrating; the volume of the concentrated solution and the weight ratio of the cortex dictamni are 6mL:1g;
respectively pulverizing Notoginseng radix, galamina, and Borneolum Syntheticum into powder, and sieving with 100 mesh sieve;
heating the concentrated solution to 90 ℃ in a water bath kettle, adding 1 part of potassium hydroxide and 3.6 parts of triethanolamine by weight, and adding ethanol for dissolving to obtain a water phase; the volume ratio of the weight of the potassium hydroxide to the volume of the ethanol is 1g:15mL;
adding 45 parts of stearic acid, 24 parts of glycerol, 1.5 parts of stearyl alcohol and 21 parts of liquid paraffin into a water bath kettle in a container, and melting at 90 ℃ to obtain an oil phase; heating in water bath, introducing water phase, stirring, emulsifying, adding Calamina, notoginseng radix, and Borneolum Syntheticum powder, and mixing.
The invention also provides application of the eczema ointment containing panax notoginseng and coptis in preparing a medicine for treating neurodermatitis by reducing the expression of IL-6, TNF-alpha, NGF, PAR2 protein and mRNA thereof.
The invention also provides application of the eczema ointment containing panax notoginseng and coptis chinensis in preparing medicines for reducing expression of IL-6, TNF-alpha, NGF, PAR2 proteins and mRNA thereof.
The study of the invention finds that the eczema ointment with seven ginsengs and coptis has an improvement effect on neurodermatitis. The invention adopts the combination of dorsal skin friction stimulation and a chronic mild unpredictable stress method to establish a rat neurodermatitis model. The animal motor ability is evaluated by an open field method, the interest preference of the animal is detected by a sucrose water consumption test, the pathological change of a skin lesion tissue is observed by HE staining, the expression of skin nerve fibers is detected by an immunohistochemical method, the total length of the skin lesion nerve fibers is analyzed by adopting an image, and the expressions of interleukin 6 (IL-6), tumor necrosis factor a (TNF-a), nerve Growth Factor (NGF) and protease activated receptor 2 (PAR 2) protein and mRNA thereof of the skin lesion tissue are respectively detected by a Western Blot method and a Q-PCR method.
As a result: before modeling, rats in each group move freely, the times of leaving the ground with feet are frequent, the weight and the water consumption of cane sugar are normally increased, the horizontal displacement grid number, the standing times and the weight among the groups have no obvious difference, the rats after modeling do not move a little, the curiosity of the environment is weak, the times of leaving the ground with feet are reduced, and the weight and the water consumption of cane sugar are obviously reduced. After the model is made and the medicine is taken, compared with the normal control group, the horizontal displacement lattice number and the standing times of the model group are obviously reduced, and the weight and the water consumption of cane sugar are obviously reduced ( b P<0.01 Compared with the model group, the matrix and the 0.5 and 1g/kg eczema ointment of the root of Chinese ginseng and the rhizome of Chinese honeylocust abnormal fruit are used for the horizontal displacement lattice number, the standing times, the body weight and the water consumption of cane sugarWithout obvious influence, the 2g/kg group and the positive control group are both obviously increased ( c P<0.05， d P<0.01). Histopathology results show that horny layer keratosis of back skin of a model group is not clear in structure and is arranged closely, epidermis is thickened irregularly, local cells of a spinous layer are arranged disorderly and loosely, a dermis layer is infiltrated by a large number of inflammatory cells, a superficial layer fibrous structure is thin and light dyed, high-dose eczema heptadentate ointment and positive drug compound flumethasone ointment can improve pathological changes of skin lesions, and 0.5g/kg, 1g/kg of eczema heptadentate ointment and matrix thereof have no obvious influence. Immunohistochemistry shows that a great amount of dark brown positive expressions are generated on skin nerve fibers of a model group, the skin nerve fibers are mainly arranged on a dermis part and partially extend to an epidermis, the total length of the nerve fibers is obviously increased, most of the fibers extend to the epidermis part, the quantity of the nerve fibers of the dermis layer of the seven-ginseng eczema ointment and the positive control group is obviously less, the length of the nerve fibers is shorter, and the epidermis does not have the distribution of the nerve fibers. Western blot and qPCR detection results show that compared with a normal control group, the expression of IL-6, TNF-alpha, NGF, PAR2 proteins and mRNA thereof in a model group is obviously increased ( b P<0.01 2g/kg of eczema ointment, a positive control group and IL-6, TNF-alpha, NGF, PAR2 proteins and mRNA expression of the proteins are obviously reduced (compared with a model group) c P<0.05、 d P<0.01 The effect of the high dose group was weak compared with the positive control group ( e P<0.05)。
And (4) conclusion: the 2g/kg ointment has the treatment effect on the dorsal neurodermatitis and can improve the pathological changes of the skin. The reduction of the total length and expression of skin nerve fibers and the reduction of the expression of IL-6, TNF-alpha, NGF and PAR2 proteins and mRNA thereof are one of the mechanisms of the eczema ointment hepta-ginseng for treating neurodermatitis.
Drawings
FIG. 1 is a schematic view of an electrotransformation sandwich;
FIG. 2 is a diagram of the behavior of a rat in an open field before modeling;
FIG. 3 is an open field view of rat behavioristics after model creation;
FIG. 4 shows the behavioral changes of rats before and after the model preparation (open field method); wherein, (a) is the number of horizontally displaced lattices, and (b) is the number of upright lattices;
FIG. 5 shows the change in body weight (g) of rats before and after administration of the eczema ointment;
FIG. 6 is the effect of eczema of seven ginseng and coptis ointment on the pathology of the skin on the back of rats (x 200); wherein, a is a normal control group; B. neurodermatitis model group; C. model + vehicle control group; D. model + low dose group of eczema ointment of radix notoginseng and coptis chinensis; E. model + medium dosage group of eczema ointment of seven ginseng and coptis; F. model + high dose group of eczema ointment of seven ginseng; G. model + compound flumethasone ointment positive control group;
FIG. 7 shows the effect of eczema ointment on rat dermal nerve fiber expression (x 400); wherein, a is a normal control group; B. neurodermatitis model group; C. model + vehicle control group; D. model + low dose group of eczema ointment of seven ginseng; E. model + medium dosage group of eczema ointment of seven ginseng and coptis; F. model + high dose group of eczema ointment of seven ginseng; G. model + compound flumethasone ointment positive control group;
FIG. 8 shows the effect of the ointment of Qishen Lian eczema on the expression of nerve fibers in the skin of rats with neurodermatitis;
FIG. 9 shows the effect of the ointment of radix Ginseng and rhizoma Coptidis on the total length of nerve fibers in rat skin (x 400); wherein, a is a normal control group; B. neurodermatitis model group; C. model + vehicle control group; D. model + low dose group of eczema ointment of radix notoginseng and coptis chinensis; E. model + medium dosage group of eczema ointment of seven ginseng and coptis; F. model + high dose group of eczema ointment of radix notoginseng and coptis chinensis; G. model + compound flumethasone ointment positive control group;
FIG. 10 shows the effect of the ointment for treating eczema on the total length of nerve fibers on the skin of rats;
FIG. 11 shows a Western blot band; wherein, 1, normal control group; 2. neurodermatitis model group; 3. model + vehicle control group; 4. model + high dose group of eczema ointment of seven ginseng; 5. model + compound flumethasone ointment positive control group;
FIG. 12 is a graph of the effect of eczema ointment on IL-6, TNF- α, NGF and PAR2 protein expression (IOD) in skin lesion tissues; wherein (a) is IL-6, (b) is TNF-alpha, (c) is NGF, and (d) is PAR2;
FIG. 13 is total RNA agarose gel electrophoresis; wherein, lane 1: a normal control group; lane 2: neurodermatitis model group; lane 3: model + vehicle control group; lane 4: model + high dose group of eczema ointment of seven ginseng; lane 5: model + compound flumethasone ointment positive control group;
FIG. 14 is a melting curve for β -actin, IL-6, TNF- α, NGF and PAR2;
FIG. 15 is a graph of amplification curves for beta-actin, IL-6, TNF-alpha, NGF and PAR2;
FIG. 16 is a graph of the effect of eczema balm on skin IL-4, TNF- α, NGF and PAR2 mRNA expression; wherein (a) is IL-6, (b) is TNF-alpha, (c) is NGF, and (d) is PAR2.
Detailed Description
The present invention will be described in further detail with reference to examples.
It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials or equipment used are not indicated by manufacturers, and all are conventional products available by purchase.
The percentage numbers are volume percentages and the ratios are volume ratios unless otherwise specified.
1 Material
1.1 animals
The name of the product is: rats.
And (2) breeding: and (7) SD.
Sex: and (4) male.
The age of the month: 2 months old.
Weight: about 200-240 g.
Certificate number: SCXK (Xiang) 2019-0004.
The source is as follows: lake south slyke Jingda laboratory animals Co Ltd
1.2 test drugs and Positive controls
Test drug
The name is as follows: qishen Lian eczema ointment.
The formula is as follows: coptis root, lightyellow sophora root, rhizoma atractylodis, rhizoma paridis, pseudo-ginseng, cortex dictamni, calamine, pepper, borneol and liquorice.
Approval document No.: the national drug standard B20020421.
Production batch number: 20200202.
matrix: potassium hydroxide, triethanolamine, ethanol, stearic acid, glycerol, stearyl alcohol and liquid paraffin. Produced in 2020, 6 months and 1 day.
The clinical indications are: is used for clearing heat and drying dampness, promoting blood circulation and detumescence, dispelling wind and relieving itching. Can be used for treating eczema exudation caused by rheumatism and toxic heat stagnation.
Source and manufacturer: the eczema ointment and the base material are provided by Yunnan blue-green health pharmaceutical limited company.
Storage conditions are as follows: sealing, and storing in a cool and dry place.
2 method
2.1 Effect of Qishen Lian eczema ointment on local skin neurodermatitis of rats
2.1.1 preparation of dorsal cutaneous neurodermatitis model: fixing rat, selecting diameter of 4cm on its back, depilating with depilatory until skin is exposed, and rubbing skin with electric friction instrument until congestion and hemorrhage point appear locally, 5min each time, 1 time per day; chronic mild unpredictable stress: the stimulation mode adopted comprises: the method comprises the following steps of 24h fasting, water prohibition, ultrasonic stimulation (110dB, 1h), overnight illumination, thermal stimulation (5 min in an electric thermostat with the temperature of 45 ℃), and in order to prevent the rats from generating adaptation, more than 1 stimulation mode is randomly given every day, so that the rats cannot expect the stimulation. The molding method of local skin rubbing stimulation combined with chronic mild unpredictable stress lasted 8 weeks.
The molding method lasts for 8 weeks, and the behavioral indexes of the rats are detected by an open field method before and after molding. The successful molding standard is as follows: the body mass is reduced, the activity is reduced, the interest is reduced, and the pathological detection of local skin damage of rats shows cutin thickening, desquamation and the like.
2.1.2 the setting basis of the external medicine dosage is as follows: the prescription of the eczema ointment containing seven kinds of ginseng and coptis root indicates that the clinical application dosage is 'external application, and the eczema ointment is applied to the affected part in a proper amount for 3 to 4 times a day'. The existing skin irritation and anaphylaxis tests suggest that the 4g/kg eczema ointment containing radix scrophulariae and coptis chinensis has a skin irritation effect, and 1g/kg and 2g/kg eczema ointment containing radix scrophulariae and coptis chinensis have no obvious irritation and anaphylaxis, so that the dosage adopted in the pharmacodynamics experiment is set to be the safe dosage of skin medication, namely 0.5g/kg, 1g/kg and 2g/kg of eczema ointment containing radix scrophulariae and coptis chinensis are respectively coated. In the experiment, 0.5, 1, 2g/kg of eczema-treating ointment containing seven ginseng and coptis chinensis is smeared in a skin lesion range with the diameter of 4cm (for example, rats with the weight of 200g, and 100mg, 200mg and 400mg of ointment are respectively smeared on each rat at low, medium and high doses), and the skin lesion area can be uniformly smeared. The usage amount of the instruction book of the positive control drug compound flumethasone ointment is that the ointment is applied to an affected part in a thin layer and is 1-2 times per day according to symptoms, the dose of the compound flumethasone ointment in the research is 1g/kg (namely the dose of the flumethasone is 200mg/kg, and the dose of salicylic acid is 3000 mg/kg), and the ointment can be uniformly applied to the skin damage area.
2.1.3 animal grouping and administration: rats were divided into a normal control group (diameter 4cm selected from the back, depilatory to expose skin, but without electric friction instrument friction skin, chronic mild unpredictable stress stimulation), a neurodermatitis model group (short for model group), a model + matrix group (1 g/kg) (short for matrix group), a model + eczema patch low dose group (0.5 g/kg) (short for low dose group), a model + eczema patch medium dose group (1 g/kg) (short for medium dose group), a model + eczema patch high dose group (2 g/kg) (short for high dose group), a model + compound flumethasone ointment positive control group (1 g/kg) (short for positive control group), and 10 rats/group.
After the model is successfully copied, normal control groups and neurodermatitis model groups are coated with physiological saline, a matrix control group (a model and a matrix group) is coated with a matrix of the seven-ginseng and eczema ointment, a test sample group (the model and the seven-ginseng and eczema ointment low-dose group (0.5 g/kg), the model and the seven-ginseng and eczema ointment medium-dose group (1 g/kg), the model and the seven-ginseng and eczema ointment high-dose group) and a positive control group (the model and the compound flumethasone ointment positive control group) are respectively coated with the seven-ginseng and eczema ointment and the compound flumethasone ointment with different doses, affected parts are fixedly covered by gauze, the test sample is taken 2 times per day for 14 days continuously, and the animals are sacrificed 24 hours after the last use of the test sample and are prepared for inspection.
2.1.4 detection indexes:
open field experiments were conducted to evaluate the behavioral changes induced by neurodermatitis and the changes in animal body weight.
And (3) pathological detection: anesthetized rats were examined histologically at the corresponding time points by taking a dorsal skin biopsy with a skin loop.
Immunohistochemical technique detects the expression of nerve fiber and total length of nerve fiber in epidermis.
IL-6 and TNF- α expression: the expression of IL-4 and TNF-alpha protein and mRNA thereof at the skin lesion is respectively detected by adopting an RT-qPCR and western blot method.
2.2 histopathological examination of the skin: fixing the back skin in 10% neutral buffered formalin; 4 mu m paraffin sections were stained with HE and examined under 200-fold microscope for pathological changes in skin tissue and inflammatory cell infiltration. The existing method is adopted for detection, and for example, the method specifically comprises the following steps:
1. paraffin section
The following procedure was followed:
(1) The fixed skin tissue was placed in an embedding cassette and rinsed with tap water for 24h (to remove excess paraformaldehyde).
(2) And (3) dehydrating: the skin tissue after fixation is still full of moisture. Since paraffin is a water-insoluble substance and does not fuse with water, it is necessary to remove water from the tissue with a dehydrating agent before waxing, a process called dehydration. The method comprises the following steps: 80% ethanol 40min → 90% ethanol 400min → 95% ethanol 50min → absolute ethanol 45min.
(3) And (3) transparency: xylene 20min → xylene 30min.
(4) Wax dipping: and (3) placing the transparent tissue block into the paraffin wax which is melted in the incubator at the temperature of 58-60 ℃, and gradually discharging the transparent agent xylene in the tissue for the paraffin wax to be easy to permeate, wherein the paraffin wax is three cups of wax, namely 1h of first wax, 1h of second wax for 20min and 1h of third wax for 30min.
(5) Embedding: the filtered fresh wax is poured into the embedding device and the wax-impregnated tissue mass is placed inside as soon as possible. The section of the tissue block is downward, no gap can be left between the tissue and the wax, the tissue tries to be placed in a straight and flat way, the tissue is contacted with the bottom plate of the embedding device as much as possible, and finally the marking paper is put on.
(6) And (3) continuous slicing: the paraffin blocks were cut into sections of 5 μm thickness with a paraffin slicer.
(7) Patching: the cut paraffin tissue slices are firstly put into 40 percent alcohol and then are transferred into warm water with the temperature of 40 ℃. (this is a two-exhibition method, it means in the section in the process of exhibition, float the tissue slice in 30-40% ethanol solution first, carry on the first exhibition slice then take the section out, put into 45-50 duC warm water again and carry on the second exhibition slice, because there is a force difference between water and the alcoholic solution, so process and can get the tissue slice of the level and wrinkle-free, the alcohol concentration and water temperature can be adjusted from the line according to the different levels of the melting point of paraffin of the tissue, reuse the glass slide processed by polylysine wrap up tissue slice take out and affix to glass slide finally.
(8) Baking slices: the paraffin-sliced glass slide was placed on a glass slide rack and baked in an oven at 60 ℃ overnight.
2. HE staining
(1) Paraffin section is subjected to conventional dewaxing and hydration: xylene 10min → absolute ethanol 10min → 95% ethanol 5min → 90% ethanol 5min → 80% ethanol 5min → 70% ethanol 5min → distilled water 5min.
(2) Washing with distilled water for 5min.
(3) Hematoxylin staining for 16min.
(4) Washing with tap water to remove loose color, and differentiating with 1% ethanol (concentrated hydrochloric acid 1ml added into 75% anhydrous ethanol 99 ml) for 2 s.
(5) The tap water turns blue.
(6) Eosin was stained for 15s at room temperature.
(7) The tap water stopped developing color.
(8) Gradient alcohol dehydration, 75% → 80% → 85% → 90% → 100% → 100%. Each for 10min.
(9) Xylene transparent, 30min.
(10) The neutral gum was mounted and the developed color was observed under a normal optical microscope and photographed.
2.3 immunohistochemical technique to detect the expression of Nerve Fibers (NF): the back skin is continuously sliced by a constant-temperature slicer, and the expression of nerve fibers is detected by a streptavidin-biotin technology (LAB-SA method) combined with horseradish peroxidase. The primary antibody is a mouse anti-human Neurofilament (NF) monoclonal antibody (abcam, ab 207176), and the secondary antibody is biotin-labeled goat anti-mouse IgG (CST 7074). The display system adopts a Histostatin-SP kit and DAB color development. The existing method is adopted for detection, and for example, the method specifically comprises the following steps:
1. paraffin section
The following procedure was followed:
(1) The fixed skin tissue was placed in an embedding cassette and rinsed with tap water for 24h (to remove excess paraformaldehyde).
(2) And (3) dehydrating: the skin tissue after fixation is still full of moisture. Since paraffin is a water-insoluble substance and does not fuse with water, it is necessary to remove water from the tissue with a dehydrating agent before waxing, a process called dehydration. The method comprises the following steps: 80% ethanol 40min → 90% ethanol 400min → 95% ethanol 50min → absolute ethanol 45min.
(3) And (3) transparency: xylene 20min → xylene 30min.
(4) Wax dipping: and (3) placing the transparent tissue block into the melted 58-60 ℃ paraffin in the incubator for three cups of paraffin, namely 1h first cup of paraffin, 20 h second cup of paraffin and 10 h30min, so that the transparent xylene in the tissue is gradually discharged, and the paraffin is easy to permeate.
(5) Embedding: the filtered fresh wax is poured into the embedding device and the wax-impregnated tissue mass is placed inside as soon as possible. The section of the tissue block is downward, no gap can be left between the tissue and the wax, the tissue tries to be placed in a straight and flat way, the tissue is contacted with the bottom plate of the embedding device as much as possible, and finally the marking paper is put on.
(6) And (3) continuous slicing: the paraffin blocks were cut into sections of 5 μm thickness with a paraffin slicer.
(7) Surface mounting: the cut paraffin tissue slices are firstly put into 40 percent alcohol and then are transferred into warm water with the temperature of about 40 ℃. (this is a two-exhibition method, it means in the section in the process of exhibition, float the tissue slice in 30-40% ethanol solution first, carry on the first exhibition slice then take the section out, put into 45-50 duC warm water again and carry on the second exhibition slice, because there is a force difference between water and the alcoholic solution, so process and can get the tissue slice of the level and wrinkle-free, the alcohol concentration and water temperature can be adjusted from the line according to the different levels of the melting point of paraffin of the tissue, reuse the glass slide processed by polylysine wrap up tissue slice take out and affix to glass slide finally.
(8) Baking slices: the paraffin-sliced glass slide was placed on a glass slide rack and baked in an oven at 60 ℃ overnight.
2. IHC staining
(1) The slices were placed in a constant temperature oven at a temperature of 65 ℃ for at least 30min.
(2) The slices were dewaxed conventionally to water: the process comprises the steps of washing with xylene (10 min), absolute ethyl alcohol (5 min), 95% alcohol (5 min), 90% alcohol (5 min), 85% alcohol (5 min), 80% alcohol (5 min), 70% alcohol (5 min) and tap water (3 min).
(3) Rinsing with 0.01mol/L PBS solution for 5min multiplied by 3 times.
(4) And (3) antigen restoration, namely immersing the slices into citrate buffer solution, heating the slices to boiling by microwave, treating the slices for 10min, and naturally cooling the slices.
(5) Rinsing with 0.01mol/L PBS solution for 5min multiplied by 3 times.
(6) Blocking with 3% hydrogen peroxide, protecting from light, and incubating at room temperature for 30min.
(7) 0.01mol/L PBS solution rinsing, 5min 3 times.
(8) After the water around the section is sucked dry by the filter paper, a circle is drawn around the section by a grouping pen to prevent the liquid from leaking in the subsequent dyeing process.
(9) Blocking with 5% sheep serum, 25 deg.C, 1h.
(10) The excess blocking solution was spun off and primary antibody was added and the freezer was left overnight at 4 ℃.
(11) 0.01mol/L PBST solution rinse, 5min x 4 times.
(12) PV9000 reagent (polymer adjuvant, proteitech) was added dropwise and incubated at 37 ℃ for 30min.
(13) 0.01mol/L PBST solution rinsing, 5min x 3 times.
(14) PV9000 reagent (Universal enzyme-labeled goat anti-mouse/rabbit, proteitech) was added dropwise and incubated at 25 ℃ for 1h.
(15) 0.01mol/L PBST solution rinse, 5min x 4 times.
(16) DAB color development, 5-10min at room temperature, and stopping color development with distilled water.
(17) And (4) performing hematoxylin counterstaining (the stained nuclei are blue) (soaking in a hematoxylin solution for 2min, removing the floating color by using tap water, decoloring by using 1% hydrochloric acid ethanol for 1-2s, and stopping washing by using the tap water).
(18) Drying, and drying in a constant temperature oven at 60 ℃ for 1h.
(19) Transparent and sealing sheet. Xylene is transparent for 10min, and the gel is sealed with neutral gum.
(20) And (6) photo collection.
2.4 Total Length of nerve fibers in epidermis: and (3) carrying out data acquisition and analysis on the total length of the epidermal nerve fibers by using an Olympus image analysis system. Each slice was randomly selected for 5 fields. The length of the intraepidermal nerve fibers was measured under a 40-fold objective, all punctate cross sections were not counted.
And opening a picture to be analyzed by using Olympus image analysis software, drawing a ruler as a standard for calculating the length, drawing a corresponding curve along the nerve fiber to be measured, and obtaining the length of the curve corresponding to the standard length. And selecting 5 visual fields from each section to measure the fiber length of each section, and superposing the measured fiber lengths to obtain the total length of the nerve fibers. The total length of each slice was used to calculate the mean and standard deviation.
2.5qPCR and Western blot to detect the expression of IL-4, TNF-alpha, nerve Growth Factor (NGF) and protease activated receptor 2 (PAR 2) proteins and their mRNAs in skin.
2.5.1qPCR assay
1. Primer design and Synthesis
The target gene mRNA sequence of the corresponding species is queried on pubmed, and a primer is designed according to the CDS sequence.
Q-PCR primers were designed using beacon designer 7.90 and the primer sequences are shown in Table 1.
TABLE 1
2. Extraction of Total RNA
(1) 20mg of skin tissue is taken, ground by liquid nitrogen, then added with 500 mul of TRIZOL lysate, continuously lysed for 20min in ice bath, centrifuged at 12000r/min at 4 ℃ for 10min, and the supernatant is sucked into a new EP tube.
(2) Adding 200 μ l chloroform, shaking vigorously for 10-15s, standing at room temperature for 15min, centrifuging at 12000r/min at 4 deg.C for 15min in a refrigerated centrifuge, collecting the supernatant in the top layer of tube.
(3) Collecting supernatant to a new EP tube, adding 500 μ l isopropanol, shaking for 10-15s, standing at-20 deg.C for 30min, centrifuging at 12000r/min at 4 deg.C for 8min (to see transparent colloidal precipitate at the bottom of the tube), and discarding the supernatant.
(4) 0.5ml of 75% ethanol (prepared with DEPC-treated water) at 4 ℃ was added to wash the RNA, and after shaking gently several times, the RNA was centrifuged at 12000r/min at 4 ℃ for 5min, and the supernatant was discarded.
(5) After discarding the supernatant, the EP tube was held down and the ethanol was aspirated as dry as possible on filter paper.
(6) RNA was dissolved by adding 20. Mu.l of DEPC-treated water in a reverse transcription kit (GenCopoeia, 20 RT). After repeated pipetting with a gun, 2. Mu.l of the pipette was used to measure the RNA concentration.
3. Determination of RNA concentration
Mu.l of total RNA samples were taken and their concentration was measured in an ND-1000 spectrophotometer.
4. Calculating the volume of total RNA required in RT
Calculated from the volume of total RNA required for the synthesis of 20. Mu.l of cDNA in a 2. Mu.g sample, the formula is as follows:
volume amount of total RNA required in reverse transcription =2 μ g/measured RNA concentration
Synthesis of first Strand cDNA by reverse transcription of RNA
First strand cDNA synthesis was performed according to the FastKing RT Kit (With gDNase) FastKing cDNA first strand synthesis Kit (GenCopoeia, 20 RT).
(1) The following reactions were added to the PCR tube in order (operating on an ice bath):
total RNA volume amount μ l calculated for RNA sample
5×gDNA Buffer 2μl
RNase-Free ddH 2 O xμl
Until the total volume of the three is 10 μ l, gently shaking, and slightly centrifuging for 3-5s to mix the reactants.
(2) The following reactions were added in an ice bath:
the final reaction volume was 20. Mu.l, incubated at 42 ℃ for 15min, heated at 95 ℃ for 3min, and then the reaction was terminated, and cooled in an ice bath to obtain the reverse transcribed cDNA first strand.
6.Q-PCR amplification of the target Gene
(1) To a 96-well PCR plate, the following reagents were added in order per well:
to a final reaction volume of 10. Mu.l.
(2) Placed in a LightCycle 96 instrument.
(3) Setting PCR cycling reaction conditions:
denaturation at 95 deg.C for 10min;
95℃ 15s；
annealing & extension at 60 ℃ for 30s;
and (3) melting curve analysis: 95 ℃:15s;60 ℃ below zero: 30s;95 ℃:15s.
(4) After the reaction, ct value was recorded and calculated by the following equation 2 -△△Ct 。
Δ Ct = Ct value of target gene-Ct value of reference gene;
Δ Ct = experimental-control Δ Ct.
2.5.2Western blot detection
Protein sample preparation: adding 500 μ l RIPA lysate (containing 50 μ l protease inhibitor) (Shanghai alatin) into skin tissue 100mg, shearing tissue with sterile ophthalmic scissors, homogenizing with ultrasonic cell disruptor for 20s in ice bath, and standing on ice bath for disruption for 20min. The mixture was centrifuged at 12000rpm for 10min at 4 ℃ to collect the supernatant.
And then carrying out protein concentration determination, SDS-PAGE electrophoresis, membrane transfer, immunoreaction, chemiluminescence, development, fixation and the like according to a conventional method so as to finish Western blot detection.
The detection is carried out by adopting the existing method, for example:
protein concentration determination (operating according to BCA kit instructions)
a. Preparing an AB mixed solution, multiplying the sum of the number of samples and the gradient number of the standard protein concentration by 200 mu l, wherein A: B = 50;
gradient protein standards were formulated in 96-well plates, as in table 2.
TABLE 2
| Number of |
0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| Protein Standard solution (μ L) | 0 | 1 | 2 | 4 | 8 | 12 | 16 | 20 |
| Ripa lysate (. Mu.L) | 20 | 19 | 18 | 16 | 12 | 8 | 4 | 0 |
| Corresponding protein concentration (μ g/. Mu.L) | 0 | 0.025 | 0.05 | 0.1 | 0.2 | 0.3 | 0.4 | 0.5 |
Note: the concentration of the protein standard solution added into the wells with the numbers of 0-7 is 0.5 mu g/mu l
c. In another row, 19. Mu.l of RIPA lysate and 1. Mu.l of protein sample (20-fold dilution) were added to each well
d. Adding 200 μ l BCA working solution (Biyuntian biotechnology, inc.) into each well, incubating at 37 deg.C for 30min, and measuring OD value in spectrophotometer 562 nm;
e. the sample loading volume was adjusted for each sample protein concentration, as shown in table 3.
TABLE 3
| Sample numbering | Total protein concentration (mg/ml) |
| 1-1 | 29.835 |
| 1-2 | 32.94 |
| 1-3 | 30.105 |
| 1-4 | 33.135 |
| 1-5 | 25.2 |
| 2-1 | 27.975 |
| 2-2 | 27.3 |
| 2-3 | 29.535 |
| 2-4 | 30.27 |
| 2-5 | 25.95 |
| 3-1 | 29.145 |
| 3-2 | 22.425 |
| 3-3 | 27.615 |
| 3-4 | 27.675 |
| 3-5 | 28.95 |
| 4-1 | 28.545 |
| 4-2 | 23.085 |
| 4-3 | 28.12 |
| 4-4 | 37.74 |
| 4-5 | 21.15 |
| 5-1 | 28.83 |
| 5-2 | 28.71 |
| 5-3 | 25.965 |
| 5-4 | 28.815 |
| 5-5 | 26.745 |
SDS-PAGE electrophoresis
(1) The amount of protein loaded in each sample was adjusted to 80. Mu.g (normalized total protein loaded),
the loading volume was filled to 15. Mu.l with RIPA lysis buffer to ensure that the protein loading volume and the loading volume were consistent. Add 3. Mu.l 6 × loading buffer to each tube, blow and mix well, and denature in boiling water for 10min. Instantaneous centrifugation for 5 seconds, short storage at 4 deg.C, and long-term storage at-20 deg.C.
(2) Cleaning the glass plate: the glass plate was fastened with one hand and gently scrubbed with washing powder with one hand. Fully washing with tap water, washing with double distilled water, and drying in a baking oven.
(3) Pouring glue and loading sample:
a) The glass plate is vertically clamped on the frame after being aligned to prepare glue pouring.
b) After TEMED was added, the mixture was immediately shaken up and filled with 4ml of glue by a 1ml loading gun. Slowly add 0.5ml isopropanol to seal.
c) After 20min, when clear refraction lines appear between the isopropanol and the glue, pouring off the isopropanol on the upper layer of the glue and sucking the isopropanol with absorbent paper.
d) The TEMED was added and the gel was poured immediately, 2ml for each gel. After the space of the glass plate is filled, the comb is inserted into the concentrated glue to slowly avoid generating bubbles.
e) After the concentrated gel is fully solidified, pouring electrophoresis buffer solution into the gel, and slightly pulling out the gel by respectively pinching two sides of the comb vertically upwards with two hands. The wells were blown 3-4 times with a 200. Mu.l gun to remove a small amount of messy concentrated gel.
f) The processed sample is accurately sucked by a 10 mu l micro-sample-adding gun, and the gun head is vertically downwards and slowly injected into the glue hole.
g) Switching on a power supply, carrying out electrophoresis at a constant voltage of 60V until the lower edge of the sample contacts the separation gel (25 min), continuing electrophoresis for nearly 3h by using a constant voltage of 90V until bromophenol blue just overflows the lower edge of the separation gel, and stopping electrophoresis;
h) The plate was removed, the concentrated gel carefully sheared off, and the gel was placed in wet-transfer buffer.
And (5) transferring the film (transferring the film by a wet transfer method).
(1) And (3) measuring the length and width of the next glue, firstly cutting the PVDF film, cutting the glue into a large film and a small film, and then cutting the filter paper according to the film. Soaking the PVDF membrane in methanol for several seconds, and then soaking the PVDF membrane, the filter paper and the sponge pad in a wet-conversion buffer solution;
(2) The discharge to sandwich conversion was stacked in the following order: negative pole (black surface) → sponge mat → 3-layer filter paper
→ glue → film → 3-layer filter paper → sponge pad → positive electrode (white) as in fig. 1.
(3) The sandwich clip was placed in the transfer tank with the black side facing the black side of the tank and the white side facing the red side of the tank. The transfer tank is placed in a basin, water is poured in, and ice blocks are added around the transfer tank. Turning off the power supply after the film is rotated for 1h at 200mA, and taking out
A PVDF membrane.
Note: transferring the membrane for 1h by using a PVDF membrane with the molecular weight of more than 12kD and the thickness of 0.45 mu m; the molecular weight <12kD was transferred for 50min using 0.22 μm PVDF membrane.
(4) Placing in ponceau staining solution for 3-5min, observing protein band condition after membrane transfer, and rinsing with ultrapure water to remove the staining solution. The subsequent experiments were continued.
Immune response
(1) Placing the PVDF membrane in 5% Bovine Serum Albumin (BSA), and sealing for 1h at room temperature (20-30 ℃);
(2) Rinsing with TBST for 5min 3 times on a decolorizing shaker;
(3) BSA solution was blotted as dry as possible, 5ml of primary antibody (IL-6, abcam, abc62351, TNF-. Alpha., abcam, ab205587, NGF, abcam, ab52918; PAR2, abcam, ab 180953.) was added, respectively, and the mixture was packed in a hybridization bag, and the cut PVDF membrane was placed therein, bound to a silent mixer, and left in a refrigerator at 4 ℃ overnight.
(4) Taking out the hybridization bag, re-warming at room temperature for 10min, removing primary antibody liquid, and washing the membrane with TBST buffer for 3 times, each time for 10min;
(5) Adding enzyme-labeled secondary antibody, and incubating for 2h at room temperature. Washing the membrane for 3 times by TBST, 10min each time;
chemiluminescence, development, and fixation
ECL color development: after water on the PVDF film is absorbed to the greatest extent by water absorption paper, the PVDF film is flatly placed in a gel imager for collecting chemiluminescence signals, and air bubbles do not exist; sucking 50 mul of solution A and solution B in the ECL kit, respectively, adding into 900 mul of double distilled water, mixing uniformly, and dripping onto the membrane uniformly; click on live aquire and start to acquire images.
Determining the relative expression quantity of the target protein (gray value of the target protein) by taking the same tube GAPDH as an internal reference
GAPDH grey values).
3 results
3.1 behavioral indexes of rats detected by open field method
Before modeling, rats in each group move freely, the times of feet leaving the ground are frequent, the horizontal displacement grid number and the standing times among the groups have no obvious difference (figures 2 and 4 and table 4), the rats after modeling move less, the curiosity of the environment is weaker, and the times of feet leaving the ground are reduced. After the model is made and used, compared with the normal control group, the horizontal displacement lattice number and the standing times of the model group are obviously reduced ( b P<0.01 Compared with the model group, the matrix group, the low and medium dosage groups have no obvious influence on the horizontal displacement grid number and the standing times, and the high dosage group and the positive control group are both obviously increased ( c P<0.05， d P<0.01 Weaker than that of the positive control group (c) e P<0.05 (FIGS. 3, 4, table 4).
TABLE 4 behavioral changes in rats before and after model building (open field method)
Note: n =10, and n is a linear variable of,
b P<0.01, comparing the model group with a normal control group; c P<0.05， d P<0.01, comparing the eczema ointment group with the model group; e P<0.05, the eczema ointment with seven ginsengs and coptis root group is compared with a positive control group.
3.2 sucrose Water consumption
Before molding, the sugarcane water consumption of each group of rats has no obvious difference; after the model is made and the medicine is taken, compared with a normal control group, the cane sugar water consumption of rats in the model group is obviously reduced ( b P<0.01 Compared with the model group, the substrate group, the low and medium dosage groups have no obvious influence on the sucrose water consumption, and the sucrose water consumption of the high dosage group and the sucrose water consumption of the positive control group are both obviously increased ( c P<0.05， d P<0.01 And the water consumption of sucrose was less increased in the high dose group compared with the positive control group ( e P<0.05 (Table 5).
TABLE 5 comparison of sucrose water consumption in rats before and after administration of the ointment
| Group of | Dosage (g/kg) | Before modeling (mL) | After making model and using medicine (mL) |
| Normal control group | - | 50.35±3.48 | 54.61±4.23 |
| Model set | - | 51.24±4.16 | 24.28±4.33 b |
| |
1 | 49.62±3.14 | 25.27±3.18 |
| Low dose group | 0.5 | 49.58±5.26 | 23.62±4.47 e |
| |
1 | 51.22±4.67 | 26.26±4.53 e |
| |
2 | 52.28±5.24 | 31.57±5.41 c e |
| |
1 | 51.82±3.61 | 41.52±6.43 d |
Note: n =10, and n is a linear variable of,
b P<0.01, comparing the model group with a normal control group; c P<0.05， d P<0.01, comparing the eczema ointment group with the model group; e P<0.05, seven ginseng and coptis eczema ointment groupCompared with a positive control group.
3.3 weight changes
Before modeling, the body weight of each group of rats has no obvious difference; after model making, the weight of the rats in the model group is obviously reduced compared with that in the normal control group ( b P<0.01 Compared with the model group, the matrix, the low and medium dosage groups have no obvious influence on the body weight, and the weight average of the high dosage group and the positive control group is obviously increased ( c P<0.05， d P<0.01 And the weight gain of the high dose group is less compared with that of the positive control group (b) ((b)) e P<0.05 Table 6, fig. 5.
TABLE 6 weight change of rats before and after administration of the ointment
| Group of | Dosage (g/kg) | Before modeling (g) | After the model is made and the medicine is used (g) |
| Normal control group | - | 225.4±12.28 | 247.0±14.63 |
| Model set | - | 223.8±4.15 | 190.0±4.06 b |
| |
1 | 219.5±8.26 | 195.2±18.74 |
| Low dose group | 0.5 | 222.2±10.96 | 190.6±4.31 e |
| |
1 | 226.8±9.01 | 193.0±4.24 e |
| |
2 | 224.8±11.97 | 205.0±7.91 c e |
| |
1 | 225.3±20.23 | 219.2±7.01 c |
Note: n =10, and n is a linear variable of,
b P<0.01, comparing the model group with a normal control group; c P<0.05, comparing the eczema ointment group with the model group; e P<0.05, the group of eczema ointment of seven ginsengs and coptis was compared with the positive control group.
3.4 skin histopathological examination
The results of the detection are shown in FIG. 6.
Normal control group: the horny layer of the back skin is thin and is in a parallel net structure, the cells of the epidermal layer are regularly arranged, the collagen fiber structure of the dermal layer is clear, and inflammatory cell infiltration is not seen.
Neurodermatitis model group: the cuticle is not keratinized completely, the structures among the cuticles are unclear and are arranged compactly, the epidermis is thickened irregularly, local cells of a spinous layer are arranged disorderly and are loose, the dermis layer is infiltrated by a large number of inflammatory cells, and the superficial fibrous structure is delicate and lightly dyed.
Model + vehicle control group: the stratum corneum is locally mild parakeratosis, the epidermis is thickened, part of cells are solidified and shrunk to be transparent, the superficial dermis has obvious inflammatory cell infiltration, and simultaneously, the fiber structure near the basal layer is relatively disorderly arranged and is lightly colored.
Model + low dose group of eczema ointment with seven ginseng and coptis: the stratum corneum is in horizontal direction mild parakeratosis, the epidermis is thickened, part of cells are in a solid state and are transparent, the dermis superficial layer is accompanied by inflammatory cell infiltration, and the fibrous structure level is unclear.
Model + medium dose group of eczema ointment containing seven ginsengs and coptis: the cuticle is mild parakeratosis, the epidermis is irregularly thickened locally, cells are regularly arranged, and inflammatory cells infiltrate into the superficial dermis.
Model + high dose group of eczema ointment with seven ginsengs and coptis: the epidermal layer cells are regularly arranged and slightly thickened, and the dermal layer fiber structure is clear with a small amount of inflammatory cells.
Model + positive control group of compound flumethasone ointment: the cuticle is mild parakeratosis, the epidermal layer cells are regularly arranged, the local part has slight thickening phenomenon, the dermal layer fiber structure is clear, and a small amount of inflammatory cells are seen.
3.5 expression of cutaneous Nerve Fibers (NF)
The immunohistochemical results of fig. 8 show:
normal control group: a small positive expression in the dermis was seen.
Neurodermatitis model group: there was a large amount of dark brown positive expression, mainly in the dermis, extending partially into the epidermis.
Model + vehicle control group: the positive expression is strong, and the positive expression is mainly distributed in dermis and partially extends to epidermis.
Model + low dose group of eczema ointment with seven ginseng and coptis: the positive expression is strong, and the positive expression is mainly distributed in dermis and slightly extends to epidermis.
Model + medium dosage group of eczema ointment containing seven ginseng and coptis: the positive expression is slightly weaker, and is mainly distributed in the dermis and a little in the epidermis.
Model + high dose group of eczema ointment with seven ginsengs and coptis: the dermis is seen to have a small amount of positive expression, and the epidermis is not seen to have positive expression.
Model + positive control group of compound flumethasone ointment: the dermis layer had a small amount of positive expression in brown color, and the epidermis showed positive expression.
As can be seen from Table 7 and FIG. 8, the nerve fibers in the skin lesion sites in the model group were highly expressed as compared with those in the normal control group: ( b P<0.01 And compared with the model group, the low and medium dose groups have no obvious influence, and the expression quantity of the high dose group and the positive control group is lower (1) d P<0.01 The effect of the high dose group was weak compared with the positive control group ( e P<0.05)。
TABLE 7 Effect of Qishen Lian eczema ointment on the expression of dermal nerve fibers in rats with neurodermatitis
Note: n =5, and n is a linear chain,
b P<0.01, comparing the model group with a normal control group; d P<0.01, drug group versus model group; e P<0.05, the test drug was compared with the positive control group.
3.6 Total Length of cutaneous nerve fiber
Immunohistochemical staining and Olympus image analysis showed (figure 9):
normal control group: the fibers are small and short, mainly in the dermis.
Neurodermatitis model group: there are a large number of nerve fibers and most of the fibers extend toward the epidermis.
Model + vehicle control group: there are a large number of fibers, and most of the nerve fibers extend toward the epidermis.
Type + qishen lian eczema ointment low dose group: the dermis is seen to have a greater number of fibers and extends toward the epidermis.
Model + medium dose group of eczema ointment containing seven ginsengs and coptis: the dermis layer has a high number of fibers, and a few fibers extend toward the basal layer.
Model + high dose group of eczema ointment with seven ginsengs and coptis: the number of nerve fibers in the dermis is obviously less, and the distribution of fibers in the epidermis is not seen.
Model + compound flumethasone ointment positive control group: the dermis has a small number of nerve fibers and the epidermis has no fiber distribution.
As can be seen from Table 8 and FIG. 10, the length of the skin nerve fibers in the model group was significantly increased as compared with the normal control group ( b P<0.01 And the low and medium dose groups had no significant effect, and the high dose group and the positive control group had shorter nerve fiber lengths, as compared with the model group (1) ((ii)) d P<0.01 And the effect of the high dose group is weaker than that of the positive control group (C) ((C)) e P<0.05)。
TABLE 8 Effect of QISHENLIANeczema ointment on Total Length of epidermal nerve fibers in rats
| Group of | Dosage (g/kg) | Total length of epidermal nerve fiber (μm) |
| Normal control group | - | 92.79±7.22 |
| Model set | - | 184.40±36.03 b |
| |
1 | 167.22±26.94 |
| Low dose group | 0.5 | 166.30±10.33 e |
| |
1 | 165.86±13.51 e |
| |
2 | 129.41±2.17 de |
| |
1 | 96.50±2.71 d |
Note: n =5, and n is a linear variable number,
b P<0.01, comparing the model group with a normal control group; d P<0.01, drug group and model group comparison; e P<0.05, the test drug was compared with the positive control group.
3.7 expression of IL-6, TNF- α, NGF and PAR2 proteins in dermal lesions
As can be seen from FIG. 11, table 9, and FIG. 12, the expression of IL-6, TNF-. Alpha.NGF and PAR2 proteins was significantly increased in the model group as compared with the normal control group ( b P<0.01 In comparison with the model group, the expression of IL-6, TNF-alpha, NGF and PAR2 proteins was significantly reduced in the high dose group and the positive control group: ( c P<0.05、 d P<0.01 And) withThe effect of the high dose group is weaker than that of the positive control group: (A) e P<0.05)。
TABLE 9 Effect of eczema ointment on IL-6, TNF-alpha, NGF and PAR2 protein expression in skin lesions (IOD)
Note: n =5, and n is a linear chain,
b P<0.01, comparing the model group with a normal control group; c P<0.05， d P<0.01, drug group versus model group; e P<0.05, the test drug was compared with the positive control group.
3.8 expression of IL-6, TNF- α, NGF and PAR2 mRNA in skin tissue
3.8.1RNA integrity
The detection result shows that the OD260 reading is between 0.1 and 1.0, and the OD260/OD280 reading is between 1.8 and 2.0, which indicates that no obvious protein pollution exists. The results of 1% agarose gel electrophoresis show that 18S and 28S bands in the total RNA sample are clear, and have a brightness of about 1 (18s.
3.8.2 melting and amplification curves
Melting and amplification curves are shown in FIGS. 14 and 15.
As can be seen from Table 10 and FIG. 16, mRNA expression was significantly increased in the model group for IL-6, TNF-. Alpha.NGF and PAR2, as compared with the normal control group ( b P<0.01 And the expression of IL-6, TNF-alpha, NGF and PAR2 mRNA was significantly reduced in the high dose group and the positive control group, compared with the model group (see d P<0.01 The effect of the high dose group was weak compared with the positive control group ( e P<0.05)。
TABLE 10 Effect of eczema cream with rhizoma Coptidis on skin IL-6, TNF- α, NGF and PAR2 mRNA expression (2) -ΔΔCt )
Note: n =5, and n is a linear variable number,
b P<0.01, comparing the model group with a normal control group; d P<0.01, drug group and model group comparison; e P<0.05, test drug compared to positive control group.
The foregoing shows and describes the general principles, principal features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (5)
1. Application of QISHENLIANeczema ointment in preparing medicine for treating neurodermatitis is provided.
2. The application of the eczema ointment containing panax notoginseng and coptis chinensis as claimed in claim 1 in preparing a medicament for treating neurodermatitis is characterized in that: the eczema ointment containing the radix codonopsitis, the coptis chinensis and the coptis chinensis comprises the following raw materials in parts by weight:
45 parts of coptis chinensis, 40 parts of radix sophorae flavescentis, 35 parts of rhizoma atractylodis, 40 parts of rhizoma paridis, 15 parts of pseudo-ginseng, 30 parts of cortex dictamni, 30 parts of calamine, 20 parts of pepper, 15 parts of borneol and 30 parts of liquorice.
3. The application of the eczema ointment containing radix notoginseng and coptis chinensis as claimed in claim 1 in preparing a medicine for treating neurodermatitis is characterized in that: the preparation method of the eczema ointment containing seven ginsengs and coptis comprises the following steps:
pulverizing Coptidis rhizoma, radix Sophorae Flavescentis, rhizoma Atractylodis, rhizoma paridis, cortex Dictamni Radicis, fructus Zanthoxyli and Glycyrrhrizae radix into coarse powder, adding 5 times of water, soaking for 1 hr, boiling for 1 hr, filtering, adding 3 times of water into residue, decocting for 2 times, each for 1 hr, filtering, mixing the three extractive solutions, and concentrating; the volume of the concentrated solution and the weight ratio of the cortex dictamni are 6mL:1g of a compound;
respectively pulverizing Notoginseng radix, galamina, and Borneolum Syntheticum into powder, and sieving with 100 mesh sieve;
heating the concentrated solution to 90 ℃ in a water bath kettle, adding 1 part of potassium hydroxide and 3.6 parts of triethanolamine by weight, and adding ethanol for dissolving to obtain a water phase; the weight of potassium hydroxide to volume ratio of ethanol was 1g:15mL;
adding 45 parts of stearic acid, 24 parts of glycerol, 1.5 parts of stearyl alcohol and 21 parts of liquid paraffin into a water bath kettle in a container, and melting at 90 ℃ to obtain an oil phase; heating in water bath, introducing water phase, stirring, emulsifying, adding Calamina, notoginseng radix, and Borneolum Syntheticum powder, and mixing.
4. Application of eczema ointment containing rhizoma coptidis and rhizoma coptidis in preparing medicines for treating neurodermatitis by reducing expression of IL-6, TNF-alpha, NGF, PAR2 protein and mRNA thereof.
5. Application of the eczema ointment containing rhizoma coptidis and rhizoma coptidis in preparing medicines for reducing expression of IL-6, TNF-alpha, NGF, PAR2 protein and mRNA thereof.
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| CN116159104A (en) * | 2023-03-20 | 2023-05-26 | 朱明浩 | Application of Qishenlian eczema ointment in preparation of medicine for treating diabetic skin itch |
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