CN1058626C - Application of sinomenine in the preparation of medicine for immunological eye diseases - Google Patents

Abstract

The present invention relates to a new application of sinomenine and pharmacological acceptable salts thereof, more specifically a new application of sinomenine and pharmacological acceptable salts thereof for preparing medicines for preventing and curing immunological eye diseases. The sinomenine and the pharmacological acceptable salt thereof can be used for preparing medicines which have the effects of suppressing the peroxidation of lipoid, decreasing inflammation infiltration and protecting the activity of antioxidant enzyme during treating autoimmune retinitis pigmentosa (EAU), and can be used for preparing medicines for treating endogenous uveitis.

Description

The application of sinomenine in treatment immunological eye diseases medicine

The present invention relates to the pharmaceutic usage of sinomenine, is the application of sinomenine in preparation treatment immunological eye diseases medicine specifically.The particularly application of sinomenine in the medicament of preparation control autoimmune retino-uveitis; sinomenine has the application in the middle of the medicament that suppresses lipid peroxidation, minimizing inflammatory infiltration, the effect of protection activities of antioxidant enzymes in preparation control autoimmune retino-uveitis (EAU) therapeutic process, and the application of sinomenine in preparation treatment endogenous uveitis medicament.

Sinomenine (sinomenine) is one of alkaloid that extracts from the stem of menispermaceae plant Sinomenium acutum Sinomenium acutum (Thunb.) R.et Wils and root, is a kind of known medical substance.Modern pharmacology studies show that sinomenine has multiple pharmacological effect.Put it briefly, the main pharmacological of the sinomenine of having known at present is as follows: effect such as analgesia, antiinflammatory, blood pressure lowering, inhibition maincenter, arrhythmia [Zhang Shishan etc., pharmacological action I. analgesia, antiinflammation and the acute toxicity testing of coculine, Acta Pharmaceutica Sinica 1960,8:177; Ling Xiuzhen, etc., Acta Pharmaceutica Sinica 1962,9 (7): 393; Zhao Dehua, etc., Acta Pharmaceutica Sinica, 1985,20 (11): 856; Zhang Mingfa, Shaanxi new medicine, 1981,10 (10): 57]; Function to body nonspecific immunity, cell has stronger inhibitory action, especially the lymphocytic function of T is had stronger inhibitory action [Peng Huimin, etc., the immunosuppressive action of sinomenine, Acta Pharmacologica Sinica 1988,9 (4) 377-380]; Sinomenine is again that (Feng Jingyi is etc., the pharmacological action VII of sinomenine in a kind of histamine liberators.To active influence of gastrointestinal and mechanism thereof, Acta Pharmaceutica Sinica 1965; 12:492); Be used for the treatment of rheumatoid arthritis (Zhang Xin etc., the Shaanxi traditional Chinese medical science, 1980,5:13 evident in efficacy clinically; Refined Pu, Hunan Province county institute of traditional Chinese medicine scientific research group, barefoot doctor's magazine, 1975,12:612); Someone also to the mechanism of action of sinomenine treatment rheumatoid arthritis carried out studying [Li Siying, etc., sinomenine to effect of immunologic function, Chinese herbal medicine, 1992,23 (2): 81-83].Up to the present, the report that does not also have relevant sinomenine treatment autoimmune retino-uveitis (EAU).

Uveitis is common multiple oculopathy, also is one of worldwide main diseases causing blindness.In China, uveitis accounts for the 2-6 position of diseases causing blindness.One of emphasis of the uveitis research of the medicine that therefore, seek effectively, toxic and side effects is little.In recent years, to the immune-mediated inflammation of eye, the attention of treatment concentrates on the immunomodulator, and its starting point is to find effective, specific inhibition to relate to the key link of immunopathogenesis, and the immunologic function of integral body is not had the medicine of very influence.For autoimmune retino-uveitis (EAU), ideal medicine preferably just works in the immunne response phase of autoimmune retino-uveitis (EAU), because exactly be parallel to the incubation period of clinical patient this moment.Because the Chinese medicine ingredients toxic and side effects is little, curative effect is lasting, is suitable for having the treatment of the uveitis that recurs feature.

In light of this situation, the inventor finds that sinomenine has better curative effect aspect the treatment autoimmune retino-uveitis (EAU), and is made into medicament through a large amount of experimentation and clinical observation.

The purpose of this invention is to provide the application of sinomenine in the medicament of preparation treatment immunological eye diseases; be that the application, particularly sinomenine of sinomenine in preparing the medicament of preventing and treating autoimmune retino-uveitis (EAU) has the central application of medicament that suppresses lipid peroxidation, minimizing inflammatory infiltration, protects the activities of antioxidant enzymes effect specifically in preparation autoimmune retino-uveitis (EAU) therapeutic process.

The object of the invention also is to provide the application of sinomenine in preparation treatment endogenous uveitis medicament.

Show that through our experimentation sinomenine, particularly sinomenine hydrochloride have the effect of good curing autoimmune retino-uveitis (EAU).Particularly sinomenine has the effect of aspects such as suppressing lipid peroxidation, minimizing inflammatory infiltration, protection activities of antioxidant enzymes in treatment autoimmune retino-uveitis (EAU) process.

Clinical research through us shows that sinomenine and pharmaceutically acceptable salt thereof have good medical effect aspect the treatment endogenous uveitis.

Sinomenine has had product to sell as a kind of medicine, for example, the sinomenine that area, Qianyang, Hunan pharmaceutical factory produces (sinomenine, SIN), the sinomenine hydrochloride powder that connection pharmaceutical factory produces in the Wuhan City, etc.

Therefore, sinomenine is as the medicine of treatment immunological eye diseases, according to the formulation method of routine, can be prepared into any pharmaceutical dosage form that is suitable for using clinically, for example, tablet, pill, capsule, electuary, oral liquid, Emulsion, suspensoid, injection, suppository, unguentum, etc.

Except the said sinomenine of the present invention; acceptable salt on the materia medica of sinomenine; for example; sinomenine hydrochloride; the effect that has the treatment immunological eye diseases equally; therefore, sinomenine and pharmaceutically acceptable salt thereof are used to prepare the medicine for the treatment of immunological eye diseases, all belong to protection scope of the present invention.

Acceptable salt is when being used to prepare the medicine for the treatment of immunological eye diseases on sinomenine and the materia medica thereof, can add excipient commonly used on the materia medica, as, binding agent, disintegrating agent, filler, lubricant, solubilizing agent, antiseptic, flavoring agent, solvent, emulsifying agent, etc.

Acceptable salt also can be made medicament by the medicine compatible with other together on sinomenine and the materia medica thereof when being used to prepare the medicine for the treatment of immunological eye diseases.

Embodiment 1

Sinomenine hydrochloride powder 25000mg, available from joining pharmaceutical factory in the Wuhan City, medical starch 250mg, an amount of pelletize of ethanol, drying is advanced the tablet machine tabletting, makes 1000, and every sheet heavily is 275mg, hydrochloric sinomenine 25mg.

Embodiment 2

Sinomenine hydrochloride powder 20000mg, starch 180mg, the two fully mixes, and is encapsulated, makes 2000 capsules, and every 0.1g is heavy, hydrochloric sinomenine 10mg.

The invention discloses the application of acceptable salt in the medicine of preparation treatment autoimmune retino-uveitis (EAU) on sinomenine and the materia medica thereof.According to the preparation technique of routine, sinomenine, sinomenine hydrochloride etc. can be made various medicaments, be used for the treatment of treatment autoimmune retino-uveitis (EAU).

Fig. 1 is used to illustrate that sinomenine is with EAU21 days curative effect comparision figure of 50mg/kg dosage treatment Wistar rat.Wherein Fig. 1 a is a matched group, and Fig. 1 b is the treatment group.

Fig. 2 is used to illustrate sinomenine with EAU21 days curative effect comparision figure of 100mg/kg dosage treatment Wistar rat, and wherein Fig. 2 a is a matched group, and Fig. 2 b is the treatment group.

Fig. 3 is used to illustrate sinomenine with EAU21 days curative effect comparision figure of 150mg/kg dosage treatment Wistar rat, and wherein Fig. 3 a is a matched group, and Fig. 3 b is the treatment group.

Experimental example 1 sinomenine is to the pharmacodynamic study of experimental autoimmune retinitis pigmentosa (EAU) preventive and therapeutic effect.

Materials and methods

(1) reagent and animal

Freund adjuvant (Complete Freund ' s Adjuvant) is purchased the company in GIBCO fully, 9,000,000,000/ml of bordetella pertussis vaccine (pertussis) is available from Beijing Biological Product Inst., hyclone (Fetus Bovin Serum) is purchased the company in GIBCO, the RPMI-1640 cell culture medium is available from Sigma company, and other reagent is homemade analytical reagent.Natural and bionical medicine National Key Laboratory provides sinomenine hydrochloride for the court.Laboratory animal is the Wistar rat that the court laboratory animal portion provides, and is male, the about 150g of body weight, healthy no oculopathy.

(2) experimental autoimmune retinitis pigmentosa rat model brings out.Rabbit retina S antigen 1 00 μ g (1mg/ml), with the complete freund adjuvant mixing and emulsifying of equal-volume, the two back of animal end subcutaneous injection, tail vein injection bordetella pertussis vaccine 0.2ml simultaneously.

(3) grouping of animal model and treatment

Animal model through the S antigen immune, be divided into three groups at random, use 50mg/kg respectively, (sinomenine hydrochloride solution is with the 0.9% fresh preparation of sodium chloride solution of sterilizing for the treatment of the sinomenine hydrochloride lumbar injection of 100mg/kg and 150mg/kg, Φ 0.25 μ micropore is filtered unconcerned degerming, uses at once).Matched group is with 0.9% sterilization sodium chloride lumbar injection.The normal control group is not done any treatment, and the raising condition is consistent with animal pattern.Treatment is beginning in second day behind antigen immune, and injected once every day, to the 21 day end.

The daily slit lamp observation of animal per behind the S antigen immune.Clinical grading is revised with reference to the standard of Wacker WB etc.

0 degree (-): normal.

I (+): the limbus of corneae blood vessel is significantly congested, and iris is obviously congested, contracted pupil, and photoreaction is slow, and there is tiny point-like exudate on lesser ring of Merkel anterior lens capsule film surface.

II (++): have obviously in anterior chamber or the vitreous body and ooze out.

III (+++): hypopyon or serous detachment of retina occur.

(4) enzyme linked immunological absorption method (ELISA) is measured the level of antibody

Fortnight and 21 days are respectively through tail vein blood behind the S antigen immune, and separation of serum is measured S antigen-antibody level.Method adopts enzyme linked immunological absorption method (ELISA), and simple is: the S antigen-antibody solution 300 μ l of 2 μ g/ml are added in the flat culture plate in 96 holes, and 4 ℃ are spent the night.After the antigen liquid sucking-off, use 0.02MoL/L, pH7.4 phosphate buffer (containing 0.05%TWeen-20) flushing three times.Every hole adds dilute serum sample 300 μ l (diluent is that above-mentioned buffer includes 0.5% bovine serum albumin).37 ℃ of insulations are after 1 hour, and the sucking-off sample is with phosphate buffer flushing three times.Add 300 μ l (HEP-LgG) horseradish peroxidase 1gG (200 times of dilutions), in 37 ℃ of insulations 1 hour.The sucking-off sample is with phosphate buffer flushing three times.Add 0.04% o-phenylenediamine (OPD) solution 0.3ml, room temperature 30 minutes, 8M sulphuric acid 0.05ml cessation reaction, 495nm measures the OD value.Normal Wistar rats serum is as normal control.

(5) the lymphocyte vitro conversion is measured

Behind the S antigen immune the 14th and 21 day, get rat eye socket blood, the isolated lymphocytes In vitro culture, with S antigen as stimulus object, cultivate and assay method the same.Observation index stimulation index (SI).Computational methods

The result

1, the preventive and therapeutic effect of sinomenine EAU that S antigen is brought out.

The sinomenine of three kinds of dosage has all reduced the occurrence rate of experimental autoimmune retinitis pigmentosa (EAU) to some extent.From showing 1-1 and Fig. 1 as can be seen, after the treatment of sinomenine 50mg/kg body weight, the occurrence rate of EAU drops to 80.56% by 90.91% of matched group, descended 10.35%, wherein three degree pathological changes persons about 4.3%, two degree pathological changes that descended has descended approximately 4.1%, and once pathological changes had descended about 1.9%.But on the statistics, matched group and treatment group compare, and difference is nonsignificance still.When the sinomenine therapeutic dose increased to the 100mg/kg body weight, the occurrence rate of EAU obviously descended, and the results are shown in Table 1-2 and Fig. 2.The occurrence rate of treatment back EAU drops to 68.18% by 93.48% of matched group.Wherein three degree pathological changes drop to 20.5%, two degree pathological changes by 47.8% of matched group and drop to 9.1% by 21.7%.Compare with matched group and treatment group, significant difference is arranged, P＜0.05 on the statistics.From table 1-3 and Fig. 3 as can be seen when the therapeutic dose of sinomenine increases to the 150mg/kg body weight, the same obviously decline of the occurrence rate of EAU.The occurrence rate of matched group is 90.48%, and the treatment group is 63.64%, has reduced 26.84%.Wherein three degree pathological changes drop to 4.5% of treatment group by 15.9%, the two degree pathological changes that 19.0% of matched group drops to the treatment group by 42.9% of matched group.Matched group and treatment group compare, and there were significant differences on the statistics, P＜0.05.Experimental result shows that the sinomenine treatment can obviously reduce the EAU occurrence rate that S antigen lures.

2. the sinomenine treatment is to the influence of the antibody horizontal of EAU rat anti S antigen.

Use enzyme linked immunosorbent assay, the results are shown in Table 1-4 what EAU rat anti S antigen levels was measured.Behind the S antigen immune, the antibody horizontal of anti-S antigen raises, and in the time of the 14th day, the OD value increases to 0.385 by 0.246 of normal control, raise about 56.5%, the OD value increases to 0.758 in the time of the 21st day to the immunity back, has raise about 3 times, compares with the normal control group, significant difference is all arranged on the statistics, P＜0.05 and P＜0.01. though the antibody horizontal of EAU rat antigen still is higher than the normal control group, have decline to some extent than not treatment group after the sinomenine treatment.Wherein the OD value is reduced to treatment group 0.291 by 0.385 of matched group the 14th day the time, both results relatively, there was no significant difference on the statistics, P＞0.05; In the time of the 21st day, reduce to treatment group 0.614 by 0.758 of matched group, both results relatively have significant difference, P＜0.05 on the statistics.Above result shows that the production of antibodies that sinomenine is treated the 3rd week back antagonism S antigen has the obvious suppression effect.

3. sinomenine sees Table 1-5 to the influence of EAU rat lymphocyte vitro conversion.Behind antigen immune the 14th day, the stimulation index of matched group was 3.94, and treatment group stimulation index is 2.29, descends about 41.9%.After the 21st day, the stimulation index of matched group is 4.69 and 3.21.Treatment group stimulation index descends about 31.6%.Both results relatively all have significant difference, P＜0.05 on the statistics.After above result showed that sinomenine treated for two weeks, the external breeder reaction that antigenic stimulus is taken place to S of EAU rat lymphocyte had obvious inhibitory action.

Table 1-1 treats the observation of curative effect of Wistar rat EAU with sinomenine (50mg/kg treated 21 days)

Example number occurrence rate

The group sum

p n p n

Matched group 30 3 33 90.91 9.09

Treatment organizes 29 7 36 80.56 19.44

Amount to 59 10 69 85.51 14.49

P: positive (+～+++)

N: negative (-)

The observation of curative effect of the table blue or green alkali of 1-2 (100mg/kg, 21 days courses of treatment) treatment Wistar rat EAU

Example number occurrence rate

The group sum

p n p n

Matched group 43 3 46 93.48 6.52

Treatment organizes 30 14 44 68.11 31.82

Amount to 73 17 90 81.11 18.88

P: positive (+～+++)

N: negative (-)

The observation of curative effect of table 1-3 sinomenine (150mg/kg, 21 days courses of treatment) treatment Wistar rat EAU

Example number occurrence rate

The group sum

p n p n

Matched group 38 4 42 90.48 9.52

Treatment organizes 28 16 44 63.64 36.36

Amount to 66 20 86 76.74 23.26

P: positive (+～+++)

N: negative (-)

The sinomenine that table 1-4 measures with ELISA is treated anti--antigenic antibody of S (100mg/kg) in the EAU Wistar rat blood serum

OD 492nm (X±SD)

Group

14 days (n) 21 days (n)

Matched group 0.385 ± 0.017 (4) 0.758 ± 0.013 (4)

Treatment group 0.291 ± 0.010 (4) 0.614 ± 0.056* (4)

Normally: 0.246 ± 0.027 (4)

*: matched group and treatment group p＜0.05

In the EAU Wistar rat of table 1-5 sinomenine treatment to the lymphproliferation response (100mg/kg) of S-antigenic stimulus

S-antigen (20 μ g/ml)

14 days 21 days

Group cpm (SI of X ± SD) (cpm of X ± SD) (SI of X ± SD) (X ± SD)

Matched group 17232 ± 3,310 3.94 ± 1.37 19331 ± 4,121 4.69 ± 1.93

Treatment group 11817 ± 3,767 2.29 ± 1.01* 11936 ± 3,673 3.21 ± 1.21*

SI: stimulation index

Sample number in every group is 4

*: matched group and treatment group P＜0.05

Discuss

The observed result of this experiment shows, sinomenine can suppress the generation of the rat EAU that S antigen brought out effectively.Behind the antigen immune, give animal lumbar injection sinomenine, the occurrence rate of its EAU and the order of severity of clinical pathological changes all obviously descend (100mg/kg and 150mg/kg dosage), and in therapeutic process, do not find that animal has untoward reaction such as weight loss thereupon.Report in the past, with Caulis Sinomenii dry extract treatment morphotropism degeneration bone disorder, each 300mg is oral, and every day three times, even SM hundred days does not see that the patient has side effect yet, urine, blood and blood biochemistry checking are all no abnormal.Medicine such as ciclosporin A and the FK506 of the treatment EAU that research at present is more, though can suppress the generation of animal EAU when heavy dose of fully, but its Toxicity of Kidney that causes, losing weight and toxic and side effects that the blood non-protein nitrogen raises, is to transit to the thorny problem that clinical practice institute must solution.By contrast, sinomenine is a safe and effective medicine, and the patient that its these characteristics may more suitablely have chronic recurrence process clinically uses.

Only can not determine still that with regard to the observation of this experiment sinomenine suppresses the rat EAU of S antigen induced, be to act on its immunoreactive afferent limb (afferent arm), (i.e. the induction and the stage of reaction) still acts on its immunoreactive efferent limb (afferent arm) (being the effective stage).Yet experimental result prompting, sinomenine has also suppressed the EAU rat to antigenic humoral immunization of S and cell immune response when suppressing EAU and taking place, and as if wherein the obvious inhibition of pair cell immunity is in time prior to the inhibition of humoral immunization.In the research of the immunomodulator of seeking treatment EAU, require ideal medicine except toxic and side effects is little at present, also wish and to suppress cellular immune function by specificity, whole immunity is not had very influence; And inhibitory action just produced in the immunne response phase.From this viewpoint, the specificity of the immunosuppressive action of sinomenine is still satisfied inadequately.From immunoregulatory network theory, each lymphocyte clone in the immune system is by oneself's identification, stimulate mutually or mutual restriction, constitute the network structure of a dynamic equilibrium, its material base that constitutes net connection is exactly idiotype and the antiidiotype structure of lymphocytic cell surface Ig.The effect of cellular immunization, humoral immunization and macrophage is to be mutually related in this theory explanation immune system.Studies show that in recent years, anti-idiotype antibody is relevant with the immune negative regulation of EAU.Because antibody can discern idiotypic determinant, therefore can with S antigenic competition binding site, to a certain extent the EAU animal is shielded.Idiotypic immunity can activate the adjusting path in addition, produces the suppressor T lymphocyte of antiidiotype, thereby suppresses the pathological process of EAU.We think how to regulate unbalance immune system thus, make it to recover dynamic equilibrium, are still the problem that needs are inquired into.

Discuss

We adopt the approach of intraperitoneal injection with retina S antigen induced rat experiment systemic autoimmune retinitis pigmentosa (EAU) model, have studied the preventive and therapeutic effect of Chinese medicine sinomenine to EAU.Experimental result shows that sinomenine can obviously suppress the generation of the rat EAU of S antigen induced.Growth of animal is had no adverse effects.When suppressing the EAU generation, sinomenine all has obvious inhibition to cellular immunization and the humoral immunization of rat.We think that sinomenine can effectively suppress the generation of EAU according to experimental result.

Experimental example 2 sinomenines are to the retina lipid peroxidation product of experimental autoimmune retinitis pigmentosa and the influence of antioxidant reductase.

Materials and methods

1. reagent and animal

Available from Fluka company, 3,3'-dimethoxy-4,4'-diaminobiphenyl. (O-dianisidine) is available from magnificent company for tetraethoxypropane (1,1,3,3-tetraethoxy propane TEP); Reduced glutathion (GSH glutathione), DPNH I (NADPH), glutathion reductase (glutathione reduetase) and sodium azide (NaN ₃) all available from Sigma company.Surplus reagent is homemade analytical pure.Sinomenine hydrochloride provides with natural drug National Key Laboratory purification for Beijing Medical University is bionical.

Laboratory animal is the Wistar pure lines rat of the court animal portion, and is male, the about 150g of body weight, healthy no oculopathy.Animal EAU model bring out and route of administration the same.Rat after disconnected neck is put to death, is taken out eyeball at different observing time of point immediately, strips off its whole retinal tissue under the ice bath, and it is stand-by that preparation tissue homogenate or put is preserved in-80 ℃ of refrigerators.

(1) mensuration of retinal tissue conjugated diene

Improve a little with reference to methods such as Buege.Carefully separate retinal tissue at microscopically,, be prepared into the homogenate of 10% (W/V) with glass homogenizer with ice-cold 0.05mol/L phosphate buffer pH7.2 (EDTA that contains 0.5mmol/L), standby.With 3ml chloroform methanol (1: 2V/V) add the 0.2ml homogenate in the mixed liquor, vibrated 2 minutes, add 1 milliliter of chloroform methanol mixed liquor again, vibrated 1 minute.Add 1ml acidifying water (distilled water transfers to pH2.5 with 0.1N hydrochloric acid), vibrated again 1 minute, centrifugal 2000 rev/mins, 10 minutes; Subnatant 1ml is taken out, feed nitrogen and dry up, add 1ml ethanol, after shaking up, survey the OD value at the 233nm place, phosphate buffer is done blank.

(2) mda content is measured

Get above-mentioned homogenate 0.2ml, add 8.1%SDS solution 0.2ml, 20% acetum 1.5ml and 0.8% thiobarbituricacid solution 1.5ml after vibration shakes up 5 minutes, place 95 ℃ of water-baths, take out after 1 hour, put into the ice bath cessation reaction; Centrifugal 2000 rev/mins of cooling back 5 minutes, adds n-butyl alcohol pyridine 5ml, vibration extracting 2 minutes, centrifugal 2000 rev/mins, layering in 15 minutes; Draw n-butyl alcohol pyridine layer, measure fluorescence intensity on fluorophotometer, excitation wavelength (Ex) is 515nm, and wavelength of transmitted light (Em) is 553nm.Simultaneously with tetraethoxypropane 1nmoL/L solution as standard.Calculate mda content.The Bradford rule is decided protein content (down together).

(3) fluorescent color lipid (flurescent chromolipid) Determination on content

Get the 0.2ml homogenate, and adding methanol chloroform mixed liquor (1: 2V/V) 3.5ml, vibrated 5 minutes, add 2ml acidifying water (ditto) again.Vibrated 5 minutes; Centrifugal 2000 rev/mins, 5 minutes.After the layering methanol aqueous layer is taken out.Measure fluorescence intensity, excitation wavelength (Ex) is 360nm, and wavelength of transmitted light (Em) is 430nm.(1 μ g/ml is dissolved in 0.1NH to use quinine sulfate solution simultaneously ₂SO ₄In) do criterion calculation color lipid content.

(4) mensuration of lipid hydroperoxides (lipid hydroperoxides) is with reference to methods such as Peter A.ward.

Get the 0.5ml homogenate, and adding 3.5ml methanol chloroform mixed liquor (1: 2V/V), vibrated 2 minutes, centrifugal 2000 rev/mins, 5 minutes; Draw subnatant 2ml after the layering, logical nitrogen dry up add then acetic acid chloroform mixed liquor (3: 2/V/V) 1.0ml, under the lucifuge condition, add 12.8% liquor kalii iodide 0.05ml, vibrate after 5 minutes, add 0.5% cadmium acetate 3.0ml, vibrated 1 minute; Centrifugal 1000 rev/mins, 5 minutes, get upper strata liquid, measure light absorption value at 353nm.

(5) mensuration of activity of myeloperoxidase

Use 50mmol/L, the kaliumphosphate buffer of pH6.0 (containing 0.5% cetab), in glass homogenate device, the homogenate that retinal tissue is made 10% (W/V), 10 seconds of ultrasonication under the ice bath.After ice melted 3 times repeatedly, ultrasonication was 10 seconds once more.Centrifugal 4000g, 4 ℃, 15 minutes.Get the 0.2ml supernatant, add the 2.8ml phosphate buffer (contain 0.167mg/ml dianisidine dihydrochloride and 0.005% hydrogen peroxide, after shaking up promptly in the continuous monitoring of 460 places.Activity of myeloperoxidase: under 25 ℃ of conditions, it is 1 active unit that per minute consumes 1 μ moL hydrogen peroxide).

(6) mensuration of superoxide dismutase activity

Method with reference to Deng Biyu etc.Get above-mentioned homogenate, centrifugal 4000 rev/mins, 20 minutes, get supernatant and do extracting solution, get an amount of extracting solution, add 4.5ml 50mmol/L, pH8.30K ₂HPO ₄Buffer, 10 μ 150mmol/L 1,2,3,-thrihydroxy-benzene solution (with the fresh preparation of 10mmol/L HCl) are mixing rapidly, the change of continuous measurement 325nm place absorbance.Reaction picks up counting to add 1,2,3,-thrihydroxy-benzene solution, 25 ℃ of reaction temperatures, and total reaction volume 4.6ml.1 unit enzymatic activity is defined as.Under 25 ℃ of conditions, every milliliter of reactant liquor per minute suppresses 1,2,3,-thrihydroxy-benzene autoxidation rate and reaches 50% enzyme amount.

(7) hydrogen peroxide decomposes, catalase can catalyzing hydrogen peroxide be decomposed, and make that content of hydrogen peroxide constantly reduces in the system, and hydrogen peroxide utilizes this principle can measure catalatic activity in the decline gradually thereupon of 240nm place light absorption value.Contain 0.05mol/L Tris-HCl buffer in the sample analysis liquid, pH7.4 adds a certain amount of hydrogen peroxide and an amount of zyme extract, and reaction volume is 1ml, with add extracting solution for instead with regard to the time, observe the variation of 240nm place absorbance continuously.1 unit enzymatic activity is defined as under 37 ℃ of conditions, the required enzyme amount of per minute catalysis 1 μ mol actor.

(8) mensuration of activity of glutathione peroxidase

Method with reference to the coupling glutathion reductase of Paglia etc.Contain 0.05mol/L phosphate buffer (containing 3mmol/L EDTA) in the sample analysis liquid, pH7.2,10mmol/L reduced glutathion, 0.3mmol/L DPNH I, 1mmol/L sodium azide, the glutathion reductase of 1 unit and an amount of zyme extract.30 ℃ of insulations behind the mixing, adding special butane to the final concentration of peroxidating is 1mol/L, reaction cumulative volume 1ml.Reaction picks up counting to add the special butane of peroxidating, measures the variation of 340nm place absorbance.1 unit enzymatic activity is defined as under 30 ℃ of conditions, the required enzyme amount of DPNH I of per minute oxidation 1 μ mol.

The result

One, sinomenine treatment EAU rat is to the influence of its retina lipid peroxidation product level

1. conjugated diene: the result is shown in table 2-1, and behind the S antigen immune the 9th day, the conjugated diene level obviously increased in the retina, be about 4.2 times of normal rat, be about 4.8 times of normal rat during fortnight, by 21 o'clock, the conjugated diene level increased to 6.8 times of normal rat.Find out that thus its level is in the observation period, and is in rising trend, with normal group utmost point significant difference is arranged more all, P＜0.01; After sinomenine treatment, the level of retina conjugated diene descends to some extent than matched group, and it is about 8.9% to have descended in the time of the 9th day, significant difference is arranged, P＜0.05 on the statistics.Above result shows that in rat EAU pathological changes, retina conjugated diene level raises, and the sinomenine treatment can suppress the rising of EAU rat retina conjugated diene level.

2. malonaldehyde: from table 2-2 as can be seen, EAU rat view MDA level changes and the conjugated diene synchronised, all is rising trend.In the time of the 9th day, be 2.27 times of normal group, in the time of the 14th day, for normal group 3.28 times during by the 21st day, are increased to 3.39 times of normal group.Compare with normal group, utmost point significant difference is all arranged, P＜0.01 on the statistics.After the sinomenine treatment, retina MDA level descends in various degree than matched group.In the time of the 9th day, descended about 8.4%, the 14 day the time, descended about 41.2%, the 21 day the time, descended about 45.8%.Treatment group and matched group result relatively, except that the 9th day organized, other two groups all had utmost point significant difference statistically, and P is all＜0.01.Above result shows that in rat EAU pathological changes, retina malonaldehyde level raises, and the sinomenine treatment can suppress the degree that EAU rat retina malonaldehyde level raises.

3. fluorescent color lipid: fluorescent color lipid content changes shown in table 2-3, the therefrom rising of EAU rat retina fluorescent color lipid content as can be seen, lag behind the variation of conjugated diene and MDA from the time, when the 9th day and the 14th day, though matched group raises to some extent than normal group, but the statistics there was no significant difference, P＜0.05.When the 21st day of EAU, its content obviously raises, and is about 7.3 times of normal group, and both results relatively have utmost point significant difference, P＜0.01 on the statistics.After the sinomenine treatment, EAU rat retina fluorescent color lipid descends to some extent than matched group, is respectively 12% (the 9th day), 6.3% (the 14th day) and 5.10% (the 21st day).Both results relatively, the 9th day and the 14th day group, there was no significant difference on the statistics, p＞0.05, the 21 a day group has utmost point significant difference, p＞0.01 statistically.Above result shows that in rat EAU pathological changes, beginning 2 all retina fluorescent color lipids does not have significant change, just obviously raises afterwards, and the sinomenine treatment can suppress the rising of EAU rat retina fluorescent color lipid content.

4, lipid hydroperoxides: the change of lipid hydroperoxides level is different from first three and plants product.As can be seen, in the time of the 9th day, its level obviously raises, and is about normal 2.49 times from table 2-4, when the 14th day and the 21st day, is respectively 2.03 and 1.84 times of normal group, and both results relatively all have significant difference, p＜0.05 on the statistics.After sinomenine treatment, the lipid hydroperoxides level of each time group has to a certain degree and descends, and is respectively 19.7% (the 9th day), 25.2% (the 14th day) and 5.3% (the 21st day), but there are no significant on statistics difference, p＞0.05.Above result shows that retina lipid hydroperoxides level raises in big EAU pathological changes, and EAU rat retina lipid hydroperoxides level rising degree in sinomenine treatment back lowers, but there was no significant difference is arranged on the statistics.

Two, sinomenine treatment EAU rat is to the influence of its retina relevant enzyme level

1, myeloperoxidase (MPer): as can be seen, in back 14 days of immunity, EAU rat retina activity of myeloperoxidase constantly increases from table 2-5, wherein increases the most significantly in second week, descends gradually again afterwards.After the sinomenine treatment, activity of myeloperoxidase descends in various degree than matched group, and wherein descending in the 9th day descended in 9.7%, the 14 day descended 17.1% in 36.5%, the 21 day.Both results relatively, the 14th day group has utmost point significant difference on the statistics, p＜0.01, all the other two groups all do not reach the degree that there were significant differences on the statistics, p＞0.05.Above result shows that in rat EAU pathological process, the retina activity of myeloperoxidase raises, and is the peak in the time of the 14th day, and the sinomenine treatment can reduce the degree that increases of its enzymatic activity.

2, superoxide dismutase: the variation of EAU rat retina superoxide dismutase activity sees Table 2-6.After immunity the 9th day, the activity of superoxide dismutase increases, and is about normal 1.29 times.Descend afterwards, reduced the most significantly in the time of the 14th day, activity has only 46.2% of normal value.More preceding slightly rising in the time of the 21st day, activity has only 69.5% of normal value.After the sinomenine treatment, in the time of the 9th day, relatively there is not significant change with matched group.Apparently higher than matched group, both results relatively have significant difference in statistics on this when the 14th day and the 21st day, and p all＜0.05.The result shows that in rat EAU pathological process, the of short duration rising earlier of retina superoxide dismutase activity descends afterwards, and in sinomenine treatment 2 thoughtful 3 weeks of back, the decline degree of its enzymatic activity is inhibited.

3, catalase: the results are shown in Table 2-7.Therefrom as can be seen, EAU rat retina catalase activity is in time gradually falls trend, compares 22.4% (the 9th day) that descended respectively, 45.6% (the 14th day) and 52.0% (the 21st day) with normal group.Matched group and treatment group result relatively in each time point, the latter's catalase activity all is higher than the former, and wherein when the 14th day and the 21st day, both results relatively all have significant difference, p＜0.01 and p＜0.05 on the statistics.The above results shows that in rat EAU pathological process, the retina catalase activity descends.The sinomenine treatment can reduce the degree that its enzymatic activity descends.

4, glutathion peroxidase

The variation of EAU rat retina activity of glutathione peroxidase sees Table 2-8.Different with the catalase activity variation is, (the 9th day and the 14th day) its activity relatively changes not quite with the normal control group observing in early days, its variation side is more obvious during to the 21st day, descended than normal control group and to be about 20.9%, but both results relatively, significant difference does not reach significant degree as yet.p＞0.05。The variation tendency of the activity of glutathione peroxidase of sinomenine treatment group is similar in appearance to not treatment group.Though a little higher than not treatment group of the enzymatic activity of its each time point does not have significant difference on the statistics, p＞0.05.Experimental result shows, the activity of EAU rat retina glutathion peroxidase is being observed early stage no significant change, and the 3rd week descended to some extent.There is not significant difference between sinomenine treatment group and the not treatment group.

Yoke diene (D) in the EAU rat retina in table 2-1 sinomenine treatment group and the not treatment group

CD X ± SD (nmol/mg albumen)

Not treatment group of group n treatment group

9 days 8 6.19 ± 0.52 5.63 ± 0.95

14 days 8 7.10 ± 0.40 6.19 ± 1.25

21 days 8 10.04 ± 3.10 8.06 ± 1.15* normal group (4): 1.47 ± 0.36*:p＜0.05

The change of malonaldehyde (MDA) in the EAUwistar rat retina in table 2-2 sinomenine treatment group and the not treatment group

MDA X ± SD (nmol/mg albumen)

Not treatment group of group n treatment group

9 days 8 0.427 ± 0.035 0.391 ± 0.042

14 days 8 0.617 ± 0.102 0.361 ± 0.033*

21 days 8 0.738 ± 0.105 0.400 ± 0.065* normal group (4): 1.88 ± 0.013*:p＜0.01

The variation of iridescent lipid (FL) in the EAUwistar rat retina in table 2-3 sinomenine treatment group and the not treatment group

FL X ± SD (μ g/mg albumen)

Not treatment group of group n treatment group

9 days 8 0.025 ± 0.007 0.022 ± 0.006

14 days 8 0.016 ± 0.005 0.015 ± 0.005

21 days 8 0.102 ± 0.035 0.050 ± 0.013** normal group (4): 0.014 ± 0.004*:p＜0.01

(LH) of lipid hydroperoxides variation in the EAUwistar rat retina in table 2-4 sinomenine treatment group and the not treatment group

LH X ± SD (nmol/mg albumen)

Not treatment group of group n treatment group

9 days 4 0.386 ± 0.100 0.310 ± 0.120

14 days 4 0.314 ± 0.140 0.235 ± 0.085

21 days 4 0.285 ± 0.084 0.270 ± 0.064 normal group (4): 0.155 ± 0.046

The activity of myeloperoxidase (MPO) (MPO) in the EAUwistar rat retina in table 2-5 sinomenine treatment group and the not treatment group

Activity of myeloperoxidase (u/mg albumen)

(X±SD)

Not treatment group of group n treatment group

9 days 6 0.031 ± 0.006 0.028 ± 0.005

14 days 6 0.115 ± 0.015 0.073 ± 0.007**

21 days 6 0.041 ± 0.004 0.034 ± 0.004*:p＜0.01

SOD activity in the EAUwistar rat retina in table 2-6 sinomenine treatment group and the not treatment group

SOD activity (u/mg albumen)

(X±SD)

Not treatment group of group n treatment group

9 days 7 9.92 ± 0.82 9.8 ± 0.74

14 days 7 3.56 ± 0.93 4.83 ± 0.77*

21 days 7 5.36 ± 1.69 6.27 ± 1.54* normal group (4): 7.71 ± 0.25*:p＜0.05

Catalase activity in the EAUwistar rat retina in table 2-7 sinomenine treatment group and the not treatment group

Catalase activity (u/mg albumen)

(X±SD)

Not treatment group of group n treatment group

9 days 8 0.097 ± 0.027 0.109 ± 0.014

14 days 8 0.068 ± 0.006 0.099 ± 0.016*

21 days 8 0.060 ± 0.006 0.076 ± 0.006* normal group (4): 0.125 ± 0.016*:p＜0.05**p＜0.01

GSH Peroxidase activity in the EAUwistar rat retina in table 2-8 sinomenine treatment group and the not treatment group

GSH peroxidase activity (u/mg albumen)

(X±SD)

Not treatment group of group n treatment group

9 days 4 0.315 ± 0.031 0.324 ± 0.030

14 days 4 0.307 ± 0.014 0.314 ± 0.050

21 days 4 0.261 ± 0.041 0.274 ± 0.039 normal group (4): 0.330 ± 0.041

Discuss

In this experiment, our observed conjugated diene, malonaldehyde, lipid hydroperoxides and fluorescent color lipid are considered to the product of different phase in the lipid peroxidation process.

In the starting stage of lipid peroxidation, free radical seizes hydrogen from unsaturated fatty acid, make it to form conjugated diene (it has characteristic absorption peak at the 233mm place), and the latter and oxygen react thereupon, form lipid peroxide, further change class matter endoperoxide into.The lipid endoperoxide can form alkyl diradical and lipid hydroperoxides with multiple chemical compound (comprising the hydroxyl class) reaction, or formation propionic aldehyde and malonaldehyde, the amino of the latter and phospholipid and fragrant aniline, and it is proteinic amino in conjunction with forming the conjugation Schiff's base, this material has special fluorescent in addition, and hydroperoxides can react the generation malonaldehyde.

Our experimental result shows, in the EAU of S antigen induced rat retina, multiple lipid peroxidation product is increasing in various degree all, reflects in the course of disease of EAU, the process aggravation of the lipid peroxidation of free radical mediated, the damaging action of its generation also increases the weight of thereupon.

To discovering of the pathology damage mechanism of EAU, although the T cell is the mediation factor of pathological changes, amphiblestroid oxidative damage then is due to the polymorphonuclear leukocyte (PMNs).The polyunsaturated fatty acid (PUFAs) all higher than other any organ-tissue content arranged in the retina photoreceptor cell, thereby be vulnerable to the damage of the lipid peroxidation of free radical mediated most.In the acute stage of EAU, PMNs builds up to inflammation part, is activated simultaneously, and respiratory burst (Respiratory burst) process of feature takes place to be released to oxygen consumption increase, pentose shunt activation and active oxygen product thereupon.Wherein the active oxygen product mainly is a ultra-oxygen anion free radical, and ultra-oxygen anion free radical can produce H through dismutation ₂O ₂, react by Haber-Weiss thereupon: And Featon reaction: , the hydroxy radical that toxigenicity is stronger (.OH).These active oxygens increase in tissue, can cause the damage of lipid peroxidation, protein and DNA etc.

In recent years when the EAU animal model brings out, widespread usage bacillus calmette-guerin vaccine and bordetella pertussis or bordetella pertussis toxin are as two adjuvants and the antigen immune animal of coming together.The animal model behind two adjuvant immunities is found in experiment, and its PMNs infiltration degree increases, retina lipid peroxidation damage aggravation [159].Utilize the reflective method of chemistry to find that peak period ultra-oxygen anion free radical and the hydroxy radical of EAU obviously increase.No matter think initial period at EAU thus, or the duration, oxygen-derived free radicals plays a major role in the retina oxidative damage, and finally causes retinal degeneration.

This result of experiment shows, the increasing of EAU rat retina conjugated diene and malonaldehyde level, and basic synchronization in time, hundred increasing of fluorescent color lipid levels lag behind the above two, just significantly increase after second week.Increasing at first weekend of lipid hydroperoxides level just reaches the peak, though afterwards apparently higher than normal control, be continuous downward trend.

Infer from the process of lipid peroxidation, because fluorescent color lipid is the product that is generated by malonaldehyde and phospholipid, fragrant aniline and protein amino reaction back, the speed that may react is slow relatively, to the active metabolism of aldehydes, all may become fluorescent color lipid lagging reasons in organizing in addition.

Initiating stage at lipid peroxidation produces lipid hydroperoxides (LHOOH), and cracking (fragmentation) can take place, and produces alkoxy radical (.LHO) and hydroxy radical (.OH): 。In the extension phase of lipid peroxidation, ferrous ion (Fe ⁺⁺) but catalysis LHOOH is reacted into alkoxy radical: 。We infer thus, greater than generation, may cause its content to descend gradually after first week in the decomposition of a certain stage LHOOH of EAU pathological changes.

After the sinomenine treatment, the generation of EAU rat retina lipid peroxidation product is subjected to inhibition in various degree, and pathologic finding confirms that the infiltration degree of polymorphonuclear leukocyte also alleviates simultaneously.Find that from our front experiment in vitro sinomenine itself just can be removed ultra-oxygen anion free radical and hydroxy radical, and the snperoxiaized effect of lipotropism class is arranged.Former experiments proved already, the oxidative damage of cell membrane, may cause flow of calcium ions, activate phospholipase, cause that free arachidonic acid generates increase, the latter can further change into prostaglandin, the lipid hydroperoxides also can cause arachidonic oxidation in addition, in these two kinds of courses of reaction, all can facilitate the inflammatory cell chemotactic factor to produce, increase the weight of the local inflammation infiltration degree.Therefore, we think sinomenine alleviate the EAU inflammatory reaction effect may with its removing free radical lipotropism class peroxidating, produce relevant thereby suppress inflammatory chemokine.

Myeloperoxidase (MPO) (MPO) neutrophilic leukocyte to have a liking in the aniline granule concentration very high, be used as the marker enzyme of neutrophilic leukocyte always.In the observation of this experiment, the retina MPO activity of EAU rat just raise after immunity on the 9th day, reached the peak in the time of the 14th day, descended afterwards, still was higher than the 9th day in the time of the 21st day.This result is similar in appearance to the laboratory observation of Goto.H etc.

As everyone knows, in normal ocular tissue, exist the protection mechanism of three kinds of anti-oxidative damages: the one, the antioxidase system can prevent that active oxygen from producing; The 2nd, free radical scavenger; The 3rd, the repair mechanism of oxidative damage.In three kinds of mechanism, what stand in the breach is the antioxidase system, and it mainly comprises: superoxide dismutase (SOD), catalase and peroxidase system.

SOD is considered to the principal component of antioxygen enzyme system, and it can remove ultra-oxygen anion free radical (.O in specificity ground ₂ ^-).The F catalase not only can be removed hydrogen peroxide (H ₂O ₂), and can reduce free its generation of hydroxyl, glutathion peroxidase is a selenoenzyme, but catalysis glutathion and hydrogen peroxide and peroxide react, with the toxic action of elimination oxide.

In this experiment, we have observed the variation of SOD, catalase and activity of glutathione peroxidase.Experimental result shows that the activity of SOD is at first rising in the 9th day behind animal immune, descends rapidly with the course of disease afterwards, and activity reduces as far as possible in the peak period (the 14th day) of inflammation, gos up to some extent later on.Have and experiment showed, that retina SOD mainly is positioned the activity of the inner segment of photoreceptor cell and retina SOD much larger than crystalline lens.Therefore we infer, at the EAU pathological changes initial stage, retina SOD active reaction raises, the .O that is on the increase with elimination ₂ ^-, work as O ₂Generation surpass that SOD is active obviously to descend.Discover that in addition too much hydrogen peroxide generation can cause the important prothetic group Cu of Cu-Zn SOD in the tissue ⁺⁺Reduction, thereby suppress the activity of SOD.

We show that to the result of EAU rat retina catalase and activity of glutathione peroxidase mensuration the two activity change is different from SOD, and on a declining curve always in the observation period, it is more obvious that its catalase activity descends.The two active decline, too much H in certainly will causing organizing ₂O ₂And peroxide can not effectively be removed.This not only can directly bring the damage of tissue, and the more important thing is too much H ₂O ₂Can react the stronger hydroxy radical of generation toxicity, thereby live the lipid peroxidation process, increase the weight of tissue injury.

Utilize after the sinomenine treatment, the rising of the activity of myeloperoxidase of neutrophilic infiltration degree has obtained inhibition in the reflection tissue, and SOD and catalase activity decline degree obviously alleviate.As everyone knows, oxidation and antioxygen mechanism are being kept dynamic equilibrium in the normal body.In the antioxidant system, the antioxidase cording responds rapidly, active high characteristics, therefore becomes composition main in the antioxidation mechanism.From our front as can be seen, retina lipid peroxidation process aggravation in the EAU process to the observed result of EAU rat retina lipid peroxidation product.The lipid peroxidation of this free radical mediated is a chain reaction, and continuous enhanced trend is arranged, and its damaging action can constantly increase the weight of, and has only by antioxidation mechanism and removes to interrupt reaction chain, could end or alleviate its damage.Therefore we think in EAU, and the activity of antioxidase reaches from external antioxidant or the antioxidase of giving in the protective, are the effective ways that alleviate the damage of EAU lipid peroxidation.Some authors also utilize has iron ion chelating agent and antioxidase (as SOD) the treatment EAU that removes free radical and lipotropism class peroxidation, obtains obvious curative effects.

Generally speaking, retina lipid peroxidation increased response in the course of disease of EAU, the activity of antioxidase system descends simultaneously.The sinomenine treatment is effective to suppressing lipid peroxidation, reducing inflammatory infiltration, protect antioxidant enzyme activities.

Experimental example 3 sinomenine hydrochlorides are to endogenous uveitis patient's therapeutical effect

One, material and method

(1) general clinical data

1, the object of observation; The ophthalmology outpatient service uveitis patient of Beijing Tongren Hospital, just send out and do not limit with the recidivist, the patient is divided into three groups at random: first group is simple sinomenine treatment, second group is simple 17-hydroxy-11-dehydrocorticosterone treatment group, the 3rd group is that sinomenine adds the cortex steroid group, the patient mainly selects the anterior uveitis patient of endogenous, sets up case history archive.

2, therapeutic scheme:

1) sinomenine group: three months courses of treatment, acute stage, is (in morbidity two weeks of back, as follows) give sinomenine hydrochloride injection (trade name: ZHENGQINGFENGTONGNING injection, the accurate word (1985) the 023025th of medicine is defended in Hunan, lot number 960404C) 1ml (25mg) subconjunctival injection, at 1 continuous 3-7 days every day, drip simultaneously with 1% sinomenine hydrochloride eye drop (eye grinds inner preparation), per hour 1 time, oral hydrochloride sinomenine sheet (trade name: ZHENGQINGFENGTONGNING PIAN, the accurate word of medicine (92) 23-092, lot number 960404C are defended in Hunan); First three days, each 40mg, every day three times, 80mg/ is each afterwards, every day three times, after the symptom control, stop subconjunctival injection, eye dripping changes every 2-4 hour into once, and oral medicine is constant, continues for two weeks, eye dripping changes every day 3-4 time into, and it is each that oral medicine changes 40mg/ into, every day three times, finishes to the course of treatment.

2) 17-hydroxy-11-dehydrocorticosterone group: three months courses of treatment, oral prednisone 20-30mg of acute stage, once a day, FD eye liquid, meticortelone+0.1% dexamethasone (eye grinds inner preparation), or 1%QDSL (acetic acid meticorten suspension), per hour eye dripping is 1 time, dexamethasone 2-3mg subconjunctival injection, once a day, 3-7 days continuously, symptom control back meticorten changes 10mg every day 1 time into, eye dripping changes every 2-3 hour into once, continues for two weeks, afterwards, meticorten changes 5mg into, every day 1 time, eye dripping every day 2-3 time, finish to the course of treatment.

3) therapeutic alliance group: treated three months, in acute stage oral meticorten 20-30mg once a day, sinomenine hydrochloride sheet 80mg/ time, every day three times, local dripping with FD and 1% sinomenine hydrochloride eye drop, per hour 1 time, indivedual severe patient give dexamethasone 2-3mg subconjunctival injection, or sinomenine hydrochloride subconjunctival injection 1ml (25mg), once a day, for three days on end, after the symptom control, meticorten decrement 10mg, once a day, sinomenine sheet oral dose, eye drip are kept to 2-3 hour 1 time, continued for two weeks, afterwards, meticorten is kept to 5mg, once a day, sinomenine sheet oral dose is constant, eye drip changes 3-4 time/day into, consolidate one month after, it is oral to stop meticorten, eye drip 2-3 time/day continues to finish oral sinomenine to the course of treatment.

3, observation index:

1) congested degree (ciliary congestion and mixed congestion).

2) the muddy degree of corneal edema.

3) K _PWhat.

4) room dodges and oozes out (comprising empyema) degree.

5) iris tuberosity quantity.

6) caligo pupillae degree.

7) strong vitreous opacity degree.

8) vision part patient detects HLA-B27, eye ultrasound diagnosis and the contrast examination of fluorescent optical fundus.

4, criterion of therapeutical effect:

1) ciliary congestion or mixed congestion disappearance person are produce effects, reduce ++ the person is effectively, and minimizing+person is for taking a turn for the better, and no alleviator is a no change.

2) Kp disappears or all transfers the brown produce effects that is to, reduces ++ and the person is effectively, and minimizing+person is for taking a turn for the better, and no minimizing person is a no change.

3) room dodges or anterior chamber's fibroid is oozed out or empyema complete obiteration or be absorbed as produce effects, reduces ++ and the person be effectively, and minimizings+person is improvement, and no minimizing person is a no change.

5, respectively organize patient:

1) sinomenine group:

The patient is totally 30 examples, 39 eyes.Women's 14 examples, 20 eyes.Male's 16 examples, 19 eyes.Age 9-51 year, average 37 years old.Female age 9-51 wherein, average 34.0 years old.Male 20-57 year, average 40.0 years old.Eyes patient 9 examples, simple eye patient's 21 examples.25 examples of anterior uveitis among the 30 routine patients (wherein Reitr ' s syndrome 1 example, Fuch ' s syndrome 1 example), panuveitis 5 examples, the time of following up a case by regular visits to is 2 thoughtful 1 year three months, average 4 months.

2) 17-hydroxy-11-dehydrocorticosterone group:

The patient is totally 15 examples, 21 eyes.Women's 9 examples, 14 eyes.Male's 6 examples, 7 eyes.Age 14-70 year, average 37.7 years old.Women 14-70 year wherein, average 34.8 years old.Male 20-70 year, average 42.2 years old.Eyes patient 6 examples, simple eye patient's 9 examples.13 examples of anterior uveitis among the 15 routine patients, panuveitis 2 examples.Follow up a case by regular visits to 1 thoughtful 8 weeks of time, average 4 weeks.

3) therapeutic alliance group:

The patient is totally 34 examples, 46 eyes.Women's 20 examples, 28 eyes.Women 19-66 year wherein, average 42.1 years old, male 11-69 year, average 34.1 years old, eyes were suffered from patient's 12 examples, simple eye patient's 22 examples.Anterior uveitis 27 examples among the 34 routine patients, panuveitis 7 examples are followed up a case by regular visits to 2 to 28 weeks of time, average 6.7 weeks.

Two, result

(1) total effective rate statistics

The results are shown in Table 3

Table 3 Caulis Sinomenii treatment endogenous uveitis patient effective percentage is observed the routine digital display of grouping and is imitated the effective routine number of the routine number total effectively sinomenine group 30 12 of routine number no change example number that takes a turn for the better, (40%) 5, (16.6%) 3, (10%) 10, (33.6%) 66.6% 17-hydroxy-11-dehydrocorticosterone group 15 8, (53.3%) 03, (20%) 4, (26.6%) 73.3% therapeutic alliance group 34 28, (82.4%) 4, (11.8%) 02, (5.9%) 94.2%

Add up to 79

Annotate total effective rate: the total routine number of the produce effects example number+effective routine number+routine number that takes a turn for the better/respectively organize

Three, discuss

From our Clinical results, sinomenine is for endogenic uveitis, and especially anterior uveitis has therapeutical effect really.Utilize this medicine total effective rate 66.6% separately.And the topical application safety of this medicine is big.In our observation, drip with 1% patient of sinomenine eye liquid for 1 year its corneal transparency, the no abnormal change of the crystal transparency.When having 4 routine patients to have different eyes not fall ill, give the result that sinomenine and hormone therapy all reach produce effects respectively in the case that we observe, the eyes morbidity simultaneously of a female patient is wherein arranged, right eye gives meticorten in subconjunctival injection, FD eye drop eye; Left eye gives the sinomenine subconjunctival injection, and the sinomenine eye drip all reaches the result of produce effects.

The sinomenine topical therapeutic is without any the effect of rising intraocular pressure, to sinomenine group patient intraocular pressure to chase after survey all no abnormal, even if individual patient is because seclusion of pupil generation secondary glaucoma, the application of sinomenine its intraocular pressure that also do not raise, this point may be bigger to uveitis patient's meaning of secondary glaucoma, the most course of disease of these patients is longer, belong to chronic process, need the longer-term medication, should avoid simultaneously exciting eye voltage rise height again, so sometimes just limited the application of hormone, and utilized the sinomenine treatment, or drug combination (minimizing hormone dosage) just shows good indication.In the drug combination group, a patient is arranged, eyes panuveitis more than two decades, hormone has been taken more than two decades, the hormone of at every turn attempting to stop using all causes the recurrence of eyes inflammation, after taking the sinomenine sheet, hormone has been removed, so far several months, the eyes inflammation is recurrence not, the patient feels skin pruritus and finds the little pimple of back extremity behind clothes sinomenine sheet, but fails to stop using, and hormone still adheres to taking.

In the sinomenine group, there is 40% patient that rheumatic or rheumatoid arthritis history (comprising tetanic property post inflammation) are previously arranged, after using the sinomenine treatment, remove eye inflammation control extrinsic articulation inflammation shape and also alleviate.Therefore, we think the uveitis patient of clearer and more definite rheumatism and rheumatoid history is arranged, and use sinomenine treatment (using separately or the application of associating other drug), aspect control whole body and the local symptom very big benefit are being arranged.

Unconverted in the sinomenine treatment group have 10 examples, accounts for 33.3%.Wherein chronic granuloma type uveitis accounts for 7 examples, the hormone therapy history is all arranged, so we thinks: should adopt sinomenine associating other medicines to use for chronic granuloma type uveitis.

Sinomenine or hormone person are used in sinomenine associating hormone therapy more merely, for the uveitic inflammation of control, better effect are arranged.Total effective rate reaches 94.2%.Wherein definite mechanism it be unclear that, but its synergism displays in preliminary observation.And in the patient of 34 routine therapeutic alliances, have 25 examples only on the sinomenine therapeutic scheme, to add FD eye liquid, just can reach good effect and need not intralesional injection of steroid, because endogenous uveitis cause of disease complexity.And along with its pathological changes mechanism of the course of disease also is not quite similar, so drug combination is the uveitic direction of treatment endogenous place.

Claims (2)

1, the application of acceptable salt in the uveitic medicament of preparation treatment endogenous on sinomenine and the materia medica thereof.

2, according to the purposes of claim 1, the acceptable salt of the materia medica of wherein said sinomenine is sinomenine hydrochloride.

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