Study On The Imitating Androgen Therapeutic Effects Of Buddleja Officinali On Induced Dry Eye In Castrated Rats

First part Study on the initial technology of extraction of buddleja officinali and preparation of the materiaObjective:Seek the best method to extract buddleja officinali, make the flavone higher than 80%.Method:Adopt the alcohol to propose water and sink the law, and then utilize the method that big hole resin gradient elutes, try to find out the alcohol and propose water in testing many times and sink the multiple with water, and the condition that big hole and resin elute, confirm that is best method to extract buddleja officinali.Result:The craft of extract have been confirmed through two times of test. 110g finished productwas gotten from 15kg native material. Take out two finished product parallel kind from it, measured with HPLC, the result show that the impurity of linarin have already been eluted basically. Controlled with the standard of linarin,calculate with external reference method, the content of linarin is 17.573% and 17.011%,average is 17.292%.The final total content of flavone is 81.488%.Conclusion:The method of adopt alcohol propose water sink law,then utilize big hole resin is the best,this law can make the content of material of extracts higher than 80%, accord with the fifth new medicine standard, can be used in this research.Second part The study on the role of buddleja officinali flavone on expression of Bax mRNA,bcl-2 mRNA and AR induced dry eye in castrated ratsObjective:Establishing the induced dry eye model in castrated rats; Observing the up-regulation of AR in lacrimal gland and preliminary appraisaling the effect of flavone on simulating androgen; Observing the basic tears secretory volume and tear film stability, and the ultra micro-structure change of lacrimal gland in the electric mirror of transmission; Observing therapeutic effects of extract of buddleja officinalis on apoptosis of lacrimal gland induced dry eye in castrated rats.Method:150 rats(male,healthy,0.2kg,1month) divided into A1, B1, Cl, D1, El, A3, B3, C3, D3, E3, A5, B5, C5, D5, E5 15 groups averagely and randomly. the normal group (A); the false operation group (B;) C the model group; therapy group with Testosterone (D); therapy group with Buddleja officinalis flavone (E); raise for one month (1); raise for 3 months (3); raise for 5 months (5). The dry eye model was established with ORX. Every group is checked by the same people, at the same time, place, lighting luminance, humidity and temperature. All rats were detected with SIT and BUT,before the operation and lavaging at the end time. It is abnormal that SIT and BUT were differential controlled with the normal group, cornea fluorescence staining for (+).Result:1.The cornea fluorescence staining:Group A, B had not obviously changed; Group C shew masculine after 4 months and aggravate as time; Group D, E the cornea fluorescence are usually dyed a bit and reduced after 4,6 month. 2.SIT:Group C (except group C1)the wet paper are shortenned, the difference is significant with before the castration(P<0.05), there is no significancewith the extension difference of time (P> 0.05). Group D (besides group D1) compare with C, the difference were significant (P＜0.05). Group E (besides group E1) compare with C, the difference were significant (P<0.05). There is significance in group D5 and group E5 (P＜0.05).3.BUT:Group C (besides group C1) breaks time is shortenned, the difference is significant with before the castration(P<0.05), and there is significant with the extension difference of time(P<0.05). Group D (besides group D1) compare with C, the difference is significant (P<0.05). Group E (besides group E1) compare with group C, the difference were significant (P<0.05). There is significance in group D5 and group E5 (P<0.05). The changes of the two above-mentioned indexes prove that model of dry eyes is successful; Buddleja officinalis flavone has effect on the induced dry eye in castrated rats, but the effect is reduced as the extension of the course of disease, and have differences with androgen.4.The electric mirror result shows:The texture of lacrimal gland arrange in regularity, the degenerate and necrosising part are deduced, the apoptosis of cells are deduced, and the came to disappeare. But the effect came to weak with the extension of the course of disease, and the difference is significant compared with the androgen.5.AR and bcl-2 mRNA expression:Group C (besides group C1) are expressed and risen, the difference is significant compared with before the castration (P<0.01), and the difference is significant compared with the extension of time(P<0.01). Group D (besides group D1) relatively increases, obviously, the difference is significant compared with group C(P<0.01). Group E (besides group C1), the difference has significance compared with group C (P<0.05).There is significance in group D5 and group E5 (P<0.01).6.Bax mRNA:Group C (besides group C1) is expressed and risen, the difference is significant compared with before the castration(P<0.01), and there is significance with the extension difference of time(P<0.01). Group D (besides group D1),r the difference is significant compared (P＜0.01). Group E (besides group C1) relatively increases with group C obviously, the difference has significance (P<0.05). There is significance in group D5 and group E5 (P<0.01). The above-mentioned result indicate Buddleja officinalis flavone can increase the quantity of AR, and increase the expression of bcl-2 mRNA,and reduce the expression of Bax mRNA. But the difference is significant with the extension of the course of disease,and compared with androgen.Conclusion:1.With the method of castration,the animal's model was established successfully. This method is simple and make the damage to rats slightly, and establish the foundation for studying further.2.There are androgen receptors in the lacrimal gland of rats.3. Buddleja officinalis flavone can raise androgen level,and create the similar effect compared with androgen. But the effect is relative to the course of disease.4. The therapeutic mechanism of buddleja officinalis flavone on the induced dry eye in castrated rats, may be relative to up-regulation of bcl-2 mRNA and down-regulation of Bax mRNA.The third part The study on the role of blood plasma including buddleja officinali flavone on expression of AR mRNA and the phosphorylation of STAT1 in lacrimal gland epithelial cellObjective:Establish the cell model that androgen level induce the dry eye; Try to find out the best addition of the blood plasma interfere in the cells; Observing the influence of the blood plasma on the expression of AR in the lacrimal gland epithelial cell;Approaching the influence of activating the STAT1 signal of cells conducting route; With the androgen blocker Fluorine, confirming buddleja officinali flavone create the effect by cohering with AR,withouting other receptor.Method:Separating and training the the lacrimal gland epithelial cell from the gland of Wistar rats(150-180g,1 month). Inducting apoptosis of the lacrimal gland epithelial cells with H202. Preparing the plasma include the medicine, and trying to find out the best addition of the blood plasma interfere in the cells with the MTT; the group were interfering with blank plasma, medicine plasma including buddleja officinali flavone and Testosterone, the expression of AR and phosphorylation of STAT1 should be observed. The effect of simulating androgen buddleja officinali flavone should be investigated with Fluorine.Result:1. After 10d,the lacrimal gland epithelial cells are round or oval in shape, cell bodys are loose and bright, the nucleus lie in the central, the cell membranes are clear, it is active to grow.2.MTT:the best addition of the blood plasma interfere in the cells is 8.95x 10"2.3. Western-blot:The group interfere with buddleja officinali flavone, the expression of AR increase, difference is significant compared with the blank group (P<0.01); The group interfere with Testosterone, the expression of AR increase, difference is significant compared with the blank group (P＜0.01),difference is significant compared with the group interfere with buddleja officinali flavone (P<0.01); The group interfere with buddleja officinali flavone, the expression of STAT1 increase, difference is significant compared with the blank group (P<0.01); The group interfere with Testosterone, the expression of STAT1 increase, difference is significant compared with the blank group (P＜0.01),difference is significant compared with the group interfere with buddleja officinali flavone (P<0.01); AR and STAT1 among every group have no difference to express with Fluorine.Conclusion:1. Separated and cultivated the lacrimal gland epithelial cell is successful. This practical method has improved the purity of the cell on the, it establishes the foundation for studying further.2.The androgen receptor exists in lacrimal gland epithelial cell. Plasma including buddleja officinali flavone cohere with AR, without other receptor. It can up-regulate AR.3.It can promote the phosphorylation of STAT1 to express with the combination of AR, and activate the STAT1 signal of cells conducting route.