CN115645512B - Application of trichosanthin in preparing medicine for treating and/or preventing psoriasis - Google Patents
Application of trichosanthin in preparing medicine for treating and/or preventing psoriasis Download PDFInfo
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- CN115645512B CN115645512B CN202211095269.3A CN202211095269A CN115645512B CN 115645512 B CN115645512 B CN 115645512B CN 202211095269 A CN202211095269 A CN 202211095269A CN 115645512 B CN115645512 B CN 115645512B
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Abstract
The invention discloses application of trichosanthin in preparation of a medicament for treating and/or preventing psoriasis, and relates to the technical field of biological medicines. Through extensive and intensive studies, the inventor discovers that the trichosanthin for external use can effectively treat psoriasis for the first time. Compared with hormone medicines, the monomeric traditional Chinese medicine trichosanthin not only can inhibit local inflammation of psoriasis skin lesions, but also can inhibit hyperplasia of epidermal cells, has quicker effect, and has insignificant toxic and side effects such as drug dependence and anaphylactic reaction. Compared with compound traditional Chinese medicine decoction (radix trichosanthis decoction, etc.), the method of locally smearing and administering the skin surface of the lesion with the single traditional Chinese medicine trichosanthin as the carrier is more convenient to use, has quicker effect, prevents the medicine from entering the blood circulation by local administration, and has lower organ toxicity.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to application of trichosanthin in preparation of a medicament for treating psoriasis.
Background
Psoriasis is a chronic inflammatory papular squamous skin disease induced by various factors, and has the characteristics of high incidence, chronicity, intractable, easy recurrence after healing and the like. The pathological course involves complex interactions between the innate and adaptive immune systems, and common drugs for the treatment of psoriasis are steroid hormones, vitamin D3 analogues, retinoids, cyclosporin a and the like. These drugs mainly rely on broad spectrum inhibition of local immunity or inhibition of epidermal cell proliferation to relieve psoriasis symptoms, but have side effects such as drug dependence, organ toxicity or cancer.
The traditional Chinese medicine radix Trichosanthis is dried root of Trichosanthes kirilowii Maxim or Trichosanthes kirilowii Maxim of Cucurbitaceae, and has effects of clearing heat, purging pathogenic fire, expelling pus and detumescence. The traditional Chinese medicine formula radix trichosanthis Shang Fangji contains a plurality of traditional Chinese medicinal materials such as cortex phellodendri, cortex moutan, fringed pink, radix polygonati officinalis, endothelium corneum gigeriae galli, polygonum aviculare, radix linderae, bamboo leaves, fructus kochiae, cogongrass rhizome, rhizoma anemarrhenae, radix rehmanniae, radix trichosanthis, snakegourd fruit, gypsum, raw liquorice and the like, and is used for treating psoriasis vulgaris through compatibility of monarch, minister, assistant and guide. However, the traditional Chinese medicine compound decoction has complex processing, uncertain curative effect and undefined pharmacological action.
Trichosanthin (TCS) is a monomeric protein molecule extracted from the traditional Chinese medicine Trichosanthin. TCS belongs to II type ribosome inactivating protein, has various pharmacological activities of inducing labor, resisting virus, inhibiting tumor growth and the like, is a clinical labor inducing medicine, and is mainly used for terminating early and medium term pregnancy (national medicine standard H10870026). At present, no research on the application of monomeric trichosanthin in preparing medicaments for treating psoriasis exists. The novel drug effect and mechanism of traditional Chinese medicine are developed, and particularly the novel therapeutic effect of traditional Chinese medicine monomer molecules such as TCS is excavated, so that the novel use of old medicine is realized, and the novel drug effect has important significance for realizing 'accelerating the modernization and industrialization of traditional Chinese medicine'.
Disclosure of Invention
The invention aims to solve the technical problem of further researching the application of trichosanthin in preparing a medicament for treating psoriasis.
In order to solve the problems, the invention provides the following technical scheme:
in a first aspect, the invention provides an application of trichosanthin in preparing a medicament for treating and/or preventing psoriasis.
The further technical proposal is that the medicine for treating and/or preventing psoriasis comprises trichosanthin and a pharmaceutically acceptable carrier, excipient or diluent.
The further technical scheme is that the trichosanthin is applied by adopting an in vitro local administration mode.
The further technical proposal is that the content of trichosanthin in the medicine for treating and/or preventing psoriasis is 0.001-1wt%.
In a second aspect, the present invention provides a pharmaceutical composition for the treatment and/or prophylaxis of psoriasis, comprising trichosanthin, and a pharmaceutically acceptable carrier, excipient or diluent.
The pharmaceutical composition provided by the invention can be prepared into a pharmaceutical preparation according to a conventional method. In the preparation process, the antibody is preferably mixed with or diluted with a carrier, or is packed into a carrier in the form of a container.
In view of the fact that trichosanthin injection is clinically used as a labor inducing agent, in order to reduce the administration risk, the pharmaceutical composition of the present invention is preferably in a topical pharmaceutical dosage form, and the form of the pharmaceutical composition may be liquid, solid or semisolid. The preparation can be emulsion, gel preparation, suspension, etc. Examples of suitable carriers, excipients, or diluents include petrolatum, glycerin (propylene glycol), polyethylene glycol, and the like. The formulation may also include fillers, anticoagulants, lubricants, wetting agents, emulsifiers, preservatives, novel nanomaterials for aiding the specific entry of TCS into cells, and the like.
In some embodiments, the pharmaceutical compositions or formulations may be used alone or in combination.
In some embodiments, the pharmaceutical composition is a trichosanthin cream made from trichosanthin and petrolatum.
In some embodiments, the pharmaceutical composition is a trichosanthin ointment made from trichosanthin, propylene glycol, polyethylene glycol.
Experiments prove that under the condition of safe use, the effective dose of the medicine composition is 0.5ug/g to 50ug/g (trichosanthin amount/weight of a drug administration person) and 5ug/cm 2 To 500ug/cm 2 (trichosanthin amount/area of skin application).
In a third aspect, the present invention also provides a preparation method of the trichosanthin, which comprises: the nucleotide sequence containing the encoded trichosanthin is used for constructing a recombinant expression vector for expressing the trichosanthin; introducing into a host cell; culturing host cells under the expression condition, expressing, separating and purifying to obtain the trichosanthin.
In the embodiment of the invention, the trichosanthin comprises the whole trichosanthin length or fragments thereof, as shown in GenBank: M34858.1. The trichosanthin usable in the present invention is not particularly limited and may include wild-type and recombinant trichosanthin and active fragments thereof.
In the examples of the present invention, the method for preparing trichosanthin is not particularly limited, and those skilled in the art can prepare trichosanthin by biological process techniques using well known methods, and the steps of constructing vector plasmid and purifying protein expression are described in reference (PMID: 1999291).
Alternatively, the preparation method of the trichosanthin comprises the following steps: is prepared from Chinese-medicinal materials including trichosanthes root through extracting and purifying.
In the examples of the present invention, the trichosanthin is derived from dried root of trichosanthes kirilowii Trichosanthes kirilowii maxim or trichosanthes kirilowii Trichoman-thes rosthornii Herms, a plant of the family trichosanthis.
Compared with the prior art, the invention has the following technical effects:
through extensive and intensive studies, the inventor discovers that the local external trichosanthin can effectively treat psoriasis for the first time. Compared with hormone medicines, the monomeric traditional Chinese medicine trichosanthin not only can inhibit local inflammation of psoriasis skin lesions, but also can inhibit hyperplasia of epidermal cells, has quicker effect, and has insignificant toxic and side effects such as drug dependence and anaphylactic reaction. Compared with compound traditional Chinese medicine decoction (radix trichosanthis decoction, etc.), the method of locally smearing and administering the skin surface of the lesion with the single traditional Chinese medicine trichosanthin as the carrier is more convenient to use, has quicker effect, prevents the medicine from entering the blood circulation by local administration, and has lower organ toxicity.
The invention discovers that the trichosanthin protein has the function of treating psoriasis, provides a new application of the trichosanthin in treating psoriasis, and further expands the application range of trichosanthin monomers.
Drawings
FIG. 1 is a photograph and staining of skin tissue of mice from different treatment groups;
FIG. 2 shows the statistical results of the cell thickness of the epidermal layer of mice in different treatment groups, the statistical significant reduction of the cell thickness of the epidermal layer of mice after treatment in experimental groups 1, 2 and 3, with P < 0.05;
fig. 3 is a photograph of spleens of mice from different treatment groups.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments. It will be apparent that the embodiments described below are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Trichosanthin TCS
The trichosanthin TCS is a plant protein extracted from traditional Chinese medicine trichosanthin, is clinically used as a midterm labor induction medicine, and has very pure extraction and preparation methods. The common preparation method comprises extracting and purifying radix Trichosanthis; or the nucleotide sequence of trichosanthin expressed by host cells is obtained by adopting a biological engineering technology and separation and purification.
In the embodiment of the invention, the trichosanthin comprises the whole trichosanthin length or fragments thereof, as shown in GenBank: M34858.1. The trichosanthin usable in the present invention is not particularly limited and may include wild-type and recombinant trichosanthin and active fragments thereof.
In the examples of the present invention, the method for preparing trichosanthin is not particularly limited, and the skilled person can prepare trichosanthin by a biological process technique by using a well-known method, and generally comprises the following steps: the nucleotide sequence containing the encoded trichosanthin is used for constructing a recombinant expression vector for expressing the trichosanthin; introducing into a host cell; culturing host cells under the expression condition, expressing, separating and purifying to obtain the trichosanthin.
In the embodiment of the invention, the gene sequence of the trichosanthin is shown in GenBank:M34858.1 in GenBank database. The construction of vector plasmids and the purification of protein expression are described in the reference (PMID: 1999291).
The trichosanthin used in the invention is obtained by purification after expression of bioengineered escherichia coli.
The trichosanthin used in the embodiment of the invention is obtained by extracting and purifying bioengineering escherichia coli, and the extraction method comprises the following steps:
(1) construction of bioengineered E.coli expressing trichosanthin: plasmid carrying trichosanthin expression sequence is constructed according to the information listed in the reference document (the gene sequence of trichosanthin is shown as GenBank: M34858.1 in GenBank database); the plasmid is introduced into engineering colibacillus, and colibacillus with stable expression vector target gene is selected and amplified in great amount.
(2) Amplification and lysis of E.coli: amplifying a large amount of escherichia coli stably expressing trichosanthin, collecting bacterial liquid, centrifuging to obtain bacterial precipitate, re-suspending the bacteria in a lysis buffer solution, primarily lysing the bacteria by repeatedly freezing and thawing bacterial suspension, then placing the bacterial liquid under an ultrasonic cell lysing instrument to fully lyse the escherichia coli, and centrifuging to obtain escherichia coli lysate supernatant. (3) Collecting and purifying trichosanthin: e.coli lysate supernatant was transferred to a dialysis bag and dialyzed at 4℃for more than 48 hours, and the E.coli lysate solvent was replaced with protein purification buffer. And (3) using a protein purifier to replace escherichia coli lysate suspension, taking a target liquid flow section to obtain purified recombinant expression trichosanthin solution by utilizing a His peptide chain tag carried by recombination, and freeze-drying the trichosanthin solution by adopting a low-temperature freeze quick-drying instrument to obtain trichosanthin powder.
Because the process for extracting and purifying trichosanthin by bioengineering is very mature, the preparation method is only briefly summarized, and the related reagents and process parameters are conventional technical means in the field.
In order to verify that the trichosanthin monomer has the effect of treating psoriasis, the trichosanthin and a medicinal carrier are prepared into an external medicament for verification, and the following trichosanthin medicinal composition examples for treating psoriasis are as follows:
EXAMPLE 1TCS cream
Preparation of 0.05% trichosanthin cream: weighing 10g of Vaseline, heating to 50deg.C for dissolving completely, adding 5mg of trichosanthin powder, stirring thoroughly, mixing well to obtain emulsion, and cooling at 4deg.C to obtain 0.05% trichosanthin vaseline cream.
EXAMPLE 2TCS ointment
Preparation of 0.05% trichosanthin ointment: weighing propylene glycol 0.5g, adding distilled water 0.5g, mixing well, adding trichosanthin 5mg, mixing well to paste for use; mixing polyethylene glycol 80002.495g and polyethylene glycol 4006.5g, heating to 85deg.C in water bath, melting, mixing, cooling to below 40deg.C, adding the above paste, and stirring.
The using mode is as follows: the trichosanthin pharmaceutical composition of example 1 or example 2 is applied to the psoriasis patient. Specifically, 0.05% of trichosanthin cream/ointment is added according to 0.5ug/g to 50ug/g (trichosanthin amount/weight of drug administration person) or 5ug/cm 2 To 500ug/cm 2 (trichosanthin quality/area of skin application) is applied to psoriasis patient uniformly, once every two days, and is used continuously until symptoms subside.
In the embodiment of the invention, common medicinal carriers of Vaseline, glycerol and polyethylene glycol are taken as examples to prepare the trichosanthin milk/ointment, so that the therapeutic effect of trichosanthin on psoriasis is verified, and the medicinal activity is trichosanthin. The use of trichosanthin as an active agent for the treatment of psoriasis by those skilled in the art is within the scope of the present invention.
Verification of therapeutic effect of trichosanthin on skin psoriasis
1. Construction of mice psoriasis model
The method comprises the following steps: male Balb/c mice with body weight of 18-21g and 5-6 weeks were selected and divided into 4 groups, namely a control group, an experimental group 1, an experimental group 2 and an experimental group 3.
The back hair of the mice is removed, and the mice with psoriasis are obtained by applying a 62.5mg dose of a commercial 5% IMQ cream (imiquimod cream) to induce skin to show psoriasis-like skin loss for 7 consecutive days in the experimental group 1, the experimental group 2 and the experimental group 3, and successfully establishing an IMQ-induced psoriasis-like mouse model. The skin status of the mice was recorded daily by photography. And the severity of back skin inflammation was measured by PASI scoring criteria. PASI scores include erythema, flaking and infiltration and total score. The range of erythema, flaking and infiltration scores was 0-4, representing none, mild, obvious, very obvious, respectively.
The control group was shaved off the back hair of the mice and only applied with petrolatum.
2. Trichosanthin for treating psoriasis in mice
The psoriasis-like lesions of mice of experimental group 1 were treated at a rate of 12.5mg/cm 2 Dose spread example 1, 0.5ug/g TCS cream;
the psoriasis-like skin lesions of mice of experimental group 2 were treated at 50mg/cm 2 Dose spread example 1, amount of 2ug/g TCS cream;
the psoriasis-like lesions of mice of experimental group 3 were treated at 50mg/cm 2 Dose spread example 2, dose 2ug/g TCS ointment;
the exposed skin of the back of the mice in the control group was treated at a concentration of 50mg/cm 2 Dose spread example 1, amount 2ug/g TCS cream.
The frequency of administration was applied once every two days for 6 days (3 times total administration). The mice were scored for psoriasis severity index (PASI), and the skin was removed from the back, fixed with 4% paraformaldehyde solution, and embedded in paraffin. Paraffin-embedded (5-10 μm) sections were prepared and stained with HE and examined with an optical microscope.
Meanwhile, since psoriasis affects spleen organs, the spleen is enlarged, and the experiment also comprises the steps of obtaining spleen forms of normal mice, psoriasis mice and mice treated by different experiment groups, weighing spleen quality and recording.
The experimental results are shown in tables 1-2 and figures 1-3.
TABLE 1 trichosanthin treatment of psoriasis model psoriasis skin area and severity index (PASI score) and body weight in mice
Table 2 spleen quality (mg) of mice treated in different experimental groups
| Normal mice | Psoriasis mice | Experiment group 1 | Experiment group 2 | Experiment group 3 | Control group |
| 70.57±6.11 | 100.42±10.45 | 102.57±9.55 | 78.57±13.43 | 87.33±10.07 | 70±4.24 |
As shown in fig. 1, mice treated with trichosanthin had reduced epidermal layer thickness, reduced inflammatory cell infiltration, reduced hyperemia, and reduced skin scaling symptoms (experimental groups 1, 2, 3). Normal mouse skin showed no apparent skin damage after application of trichosanthin (control).
As shown in fig. 2, the thickness of the epidermis of the mice treated by trichosanthin is remarkably reduced, which is statistically significant.
As shown in fig. 3, psoriatic mice showed enlargement of spleen, and after trichosanthin treatment, the spleen color of the mice changed to bright red, and the volume was restored to normal state (the volume change effect of experimental groups 2, 3 was remarkable). Normal mice were smeared with trichosanthin and no significant changes in spleen quality and morphology were seen (control).
The results show that the psoriasis skin PASI scores of the mice in the experimental group 1, the experimental group 2 and the experimental group 3 after three times of trichosanthin cream/ointment treatment are obviously reduced (table 1), the weight is increased, the psoriasis skin damage and the scale symptoms are obviously reduced (figure 1), the thickness of the epidermis layer is reduced by more than 20% compared with the thickness of the psoriasis skin epidermis layer (figure 2), and the splenomegaly symptoms of the psoriasis mice are obviously reduced (figure 3). Meanwhile, the control group showed no significant damaging change in the skin of the mice (fig. 1). By comparing spleen quality of psoriatic mice with normal mice and mice treated by different experimental groups (table 2), spleen quality of mice treated by the drug is obviously reduced, and the treatment effect is obvious.
The results prove that the trichosanthin can effectively treat the psoriasis skin, and the psoriasis skin hyperplasia, scale, itching and other psoriasis symptoms are obviously reduced after the trichosanthin is applied and administrated by local external application, and the trichosanthin has the characteristics of safety and effectiveness without obvious weight reduction, spleen abnormality and other organ toxicity.
In the foregoing embodiments, the descriptions of the embodiments are focused on, and for those portions of one embodiment that are not described in detail, reference may be made to the related descriptions of other embodiments.
While the invention has been described with reference to certain preferred embodiments, it will be understood by those skilled in the art that various changes and substitutions of equivalents may be made and equivalents will be apparent to those skilled in the art without departing from the scope of the invention. Therefore, the protection scope of the invention is subject to the protection scope of the claims.
Claims (5)
1. The application of trichosanthin in preparing medicine for treating psoriasis includes that trichosanthin is full length, and the gene sequence encoding trichosanthin is shown in GenBank: M34858.1.
2. The use according to claim 1, wherein the medicament for treating psoriasis comprises trichosanthin, and a pharmaceutically acceptable carrier, excipient or diluent.
3. The use according to claim 2, wherein the content of trichosanthin in the medicament for treating psoriasis is 0.001-1wt%.
4. The use according to claim 1, wherein the method for preparing trichosanthin comprises: preparing a nucleotide sequence for encoding trichosanthin, and constructing a recombinant expression vector for expressing the trichosanthin; introducing into a host cell; culturing host cells under the expression condition, expressing, separating and purifying to obtain the trichosanthin.
5. The use according to claim 1, wherein the method for preparing trichosanthin comprises: is prepared from Chinese-medicinal materials including trichosanthes root through extracting and purifying.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5376546A (en) * | 1991-11-04 | 1994-12-27 | Xoma Corporation | Analogs of ribosome-inactivating proteins |
| CN103724409A (en) * | 2012-10-15 | 2014-04-16 | 上海交通大学医学院 | Trichosanthin effective epitope peptide fragment with immunosuppression effect and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US8926990B2 (en) * | 2009-10-13 | 2015-01-06 | Rutgers, The State University Of New Jersey | Treatment and diagnosis of inflammatory disorders and HIV |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5376546A (en) * | 1991-11-04 | 1994-12-27 | Xoma Corporation | Analogs of ribosome-inactivating proteins |
| CN103724409A (en) * | 2012-10-15 | 2014-04-16 | 上海交通大学医学院 | Trichosanthin effective epitope peptide fragment with immunosuppression effect and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 夏丽英.《中药毒性手册》.赤峰：内蒙古科学技术出版社,2006,(第1版),第69-71页. * |
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