Modulatory Effect Of Ruxianning Plaster (乳腺宁贴片) On Mammary Gland Hyperplasia Induced By Estrogen And Progestin In Female Rats

Objective:Hyperplasia of mammary glands is the most common breast disease in women of childbearing age,accounting for more than 75%of all breast diseases.At present,there is no research on the treatment of hyperplasia of mammary glands by external application of mineral drugs at home and abroad,and there is still a lack of effective treatment methods in clinic.Although the traditional endocrine therapy（such as tamoxifen,toremifene,etc.）or oral medication（such as Chaixuan Xiaopi decoction,Danggui Shaoyao Powder,etc.）is short-term,it is difficult to treat hyperplasia of mammary glands It can relieve breast pain and other symptoms,but the curative effect is not exact,and long-term use is easy to produce endocrine disorders or hepatorenal toxicity and other serious side effects.In our clinical application,the mineral drug Ruxianning patch has obvious effect in relieving pain in the treatment of hyperplasia of mammary glands,which is still lack of objective experimental basis.At the same time,the specific molecular mechanism of hyperplasia of mammary glands and the mechanism of action of Ruxianning patch on hyperplasia of mammary glands are still unclear.The purpose of this project is to prove the effect of external application of Ruxianning patch on the treatment of hyperplasia of mammary glands,and to test the efficacy of Ruxianning patch And then to explore its mechanism of action,to provide experimental basis for further clinical application.Methods:1.30 SD rats were randomly divided into control group,mammary gland hyperplasia group and Ruxianning patch group,with 10 rats in each group.The mammary gland hyperplasia model of SD rats in mammary gland hyperplasia group and Ruxianning patch group was established by intramuscular injection of estradiol benzoate followed by progesterone.From the beginning of intramuscular injection,the second and third nipples of rats in Ruxianning patch group were externally applied with 3 g Ruxianning patch plaster,while the control group and mammary gland hyperplasia group were given the same amount of styling agent,changing dressing every 24 hours.After each external application,the trunk of rats was wrapped with a strap,and the rats were fed in a single cage.The diameter and height of the second and third nipples of the three groups were measured by electronic vernier caliper.The pathological manifestations of mammary gland were observed.The expressions of estrogen receptorα（ERα）,progesterone receptor（PR）and Ki-67were detected by immunohistochemistry.2.24 SD rats were randomly divided into control group,low dose group,medium dose group and high dose group with 6 rats in each group.The high,medium and low-dose groups were treated by intragastric administration of 10 ml/kg Ruxianning patch（the dosage was calculated by body surface area method according to 2,1 and 0.5 times of the clinical equivalent dose）.The control group was treated by intragastric administration of equal volume distilled water once a day for 2 weeks.One hour after the last administration,the blood was taken from the submandibular region of rats,and the counts of white blood cell（WBC）,red blood cell（RBC）,hemoglobin（HGB）and platelet（PLT）in plasma were detected by automatic hematology analyzer.Blood samples were centrifuged at 3000r/min for10min.Plasma and serum were separated.Liver function indexes including aspartate aminotransferase（AST）,alanine aminotransferase（ALT）,total protein（TP）,triglyceride（TG）,albumin（ALB）,total bilirubin（TBIL）,alkaline phosphatase（ALP）,total cholesterol（Chol）and renal function indexes were determined by automatic biochemical analyzer Including urea nitrogen（BUN）,serum creatinine（crea）and urea（UA）,to analyze the toxic and side effects of Ruxianning patch.In addition,Ruxianning patch was sent to Zhejiang University for X-ray diffraction analysis.Result:1.The diameter and height of the nipple in the control group were 1.40±0.07mm and 1.24±0.09,1.64±0.05mm and 2.24±0.12mm in the hyperplasia of mammary glands group,and 1.51±0.03mm and 1.87±0.10mm in the Ruxianning patch group.Compared with the control group,the diameter and height of the nipple in the hyperplasia of mammary glands group increased significantly（P<0.05 and P<0.01）The diameter and height of nipple in Ruxianning patch group were decreased（P<0.05）,which indicated that the model was successfully established and Ruxianning patch was effective in the treatment of hyperplasia of mammary glands.Under the light microscope,compared with the control group,the number of acini in the mammary gland lobule of the Ruxianning patch group increased significantly,the mammary gland cavity expanded and a large number of secretions appeared at the same time,the ductal epithelial cells proliferated significantly and arranged disorderly;compared with the mammary gland hyperplasia group,the number of acini in the mammary gland lobule of the Ruxianning patch group decreased significantly,and the secretion in the official cavity weakened,indicating that Ruxianning patch can improve the mammary gland hyperplasia Pathology.Compared with the control group,the expressions of ERα,PR and Ki-67 were significantly increased in the mammary gland hyperplasia group（P<0.01）;compared with the mammary gland hyperplasia group,the expressions of ERrα,PR and Ki-67 were decreased in the Ruxianning patch group（P<0.05）,indicating that the activity of ERα,PR and Ki-67 could be inhibited after administration.2.From the blood routine index,WBC,RBC,HGB and PLT of rats in the large,medium and small dose groups had no statistical significance（P>0.05）.From the liver function index,the differences were not statistically significant between the two groups（P>0.05）;from the liver function index,the differences were not statistically significant（P>0.05）;from the view of renal function index,there was no significant difference between the two groups（P>0.05）;from the perspective of renal function index,there was no significant difference between the two groups（P>0.05）The difference between the two groups was statistically significant（P>0.05）,indicating that there was no toxic and side effects on rats.The results showed that the contents of oxygen ion,sulfur ion,sodium ion,chloride ion,silicon ion and aluminum ion were 49.94%,17.88%,31.10%,00.51%,00.27%and 00.31%respectively.Conclusion:1.From the physical level,the Ruxianning patch can improve the changes of the diameter and height of the nipple in the rats with breast hyperplasia;From the cell level,the Ruxianning patch can improve the number of lobular acini,the secretion of mammary duct and lumen in the mammary gland of the rats with breast hyperplasia;At molecular level,the Ruxianning patch can reduce anti-estrogen receptor alpha antibody,progesterone receptor and Ki-67 in rats with mammary gland hyperplasia receptor expression.2.From the side effects,there was no damage of liver and kidney function and Hemogram function in rats.It is a kind of Polysulphate,which is composed of sulfate ion and many metal element cations.The main components include Na₂SO₄、Si O₂、Al₂（SO4）₃.