Experimental Study Of Rukanghe Decoction In Treating Mammary Gland Hyperplasia In Rats

Objective: Rukanghe decoction which consists of Rhizoma Curculigins, Radix Rehmanniae Preparata, Radix Bupleuri, Rhizoma Cyperi, Pericarpium Citri Reticulatae, Sargassum, etc has therapeutical effect of dispersing stagnated liver qi and invigorating kidney qi, promoting blood circulation for removing blood stasis and softening and resolving hard mass. We established mammary gland hyperplasia (MGH) rat models by injecting estradiol benzoate and progestin to observe the effect of Rukanghe decoction for morphological, level of serum estradiol(E₂), progestogen(P), testosterone(T), prolactin(PRL) and activity of SOD, level of MDA, the expression of ER and AT₁ on MGH in rat models and discuss the pathogenesis of MGH and the therapeutic effect mechanism of the Rukanghe decoction, for providing the reliable basis data for the wider clinical use.Method: 50 depuratory and healthy Wistar female rats, 170±10g in bodyweight, were randomly divided into 5 groups: Blank control group (Blank group), Model control group (Model group), Rupixiao group (RPX group), high dose of Rukanghe decoction group (high dose group) and low dose of Rukanghe decoction group (low dose group). MGH rat models were established by injecting estradiol benzoate and progestin. Except the rats of blank group, all the other rats were injected estradiol benzoate, in dose of 0.5 mg/kg, once a day for 25 days, then injected progestin, in dose of 4 mg/kg, once a day for 5 days. The rats in blank group were injected normal saline with the same volume, once a day for 30 days. After modeling successfully, each group adopted group therapy. High dose group: The rats were administrated rukanghe decoction, in dose of 28.94g/kg·d, once a day for 30 days. Low dose group: The rats were administrated rukanghe decoction, in dose of 14.47g/kg, once a day for 30 days. RPX group: The rats were administrated rupixiao, in dose of 0.67g/kg, once a day for 30 days. The rats in blank group and model group were given the same volume distilled water for 30 days. Each group was administrated in the dosage of 10ml/kg. Fasting 12 hours after last administration, intraperitoneal anesthesia 1% sodium pentobarbital, measured the second mammary height and diameter with sliding caliper, collected blood, separated serum, detected the serum levels of E₂, P, T and PRL by radioimmunology. Detected the serum SOD activity by chemiluminescent and the serum MDA content by TBA. Cut the second mammary of rats quickly, fixed mammary in 4% polyoxymethylene, paraffin embedded, slice in 3um. HE dyeing, observed the mammary histomorphology. Cut the third mammary of rats quickly, fixed mammary in 4% polyoxymethylene, paraffin embedded, slice in 3um, immunohistochemistry dyeing, detect the expression of ER and AT₁ in mammary glandular tissue. Observe the expression of positive cells: the positive cells of ER expression were found yellow and light brown grain in nuclear; the positive cells of AT₁ expression were found yellow and light brown grain in cytolymph and use the Motic 6.0 digital image analysis system to detect the optical density of positive cells for analyzing the expression of ER and AT₁. All date are statistical analysis by SPSS11.5 and expressed with X±S.Result:1 Changes of rats after MGH models were established.The rats mammary were protruding, hyperemia, dark red and hard. Compared with blank group(height: 1.02±0.04, diameter: 1.05±0.05), the height(1.92±0.06) and diameter(1.42±0.06) of mammary in model group were increased significantly (P<0.01). Observe the mammary histomorphology by optical microscope: Hyperplasia was diffusivity. The number of mammary lobule and acinus were increased. Lumen of catheter and alveolar cavity were enlarged. Epitheliums of catheter were increased and secretion and exfoliated cells were found in acinus. The fibrillar connective tissue was hyperplasia too. The level of serum E₂ and MDA increased higher (P<0.01). P , T and SOD activity were lower (P<0.01). PRL has no obviously changed(P>0.05). The positive cells of ER expression were found yellow and light brown grain in nuclear; the positive cells of AT₁ expression were found yellow and light brown grain in cytolymph. The expression of ER and AT₁ were increased. And their optical density were increased significantly(P<0.01).Result display: MGH rats endocrine were disturbance, the ER and AT₁ were over expression and occurred peroxidative damage at the same time.2 Effect of rukanghe decoction on mammary morphologic.The rats'mammary of high dose group and low dose group were decrease and were covered by fur. Compared with model group (height: 1.92±0.06, diameter: 1.42±0.06) , the height and diameter of mammary of high dose group (1.11±0.05, 1.08±0.04) and low dose group (1.23±0.07, 1.10±0.05) were decrease(P<0.01). There were no statistical significance compared between high dose group and RPX group(height: 1.09±0.06, diameter: 1.08±0.06) (P>0.05). Observe the mammary histomorphology by optical microscope, every treatment group were improvements. The number of mammary acinus and ductal were reduced. The dilatation of them got improved. The hyperplasia of catheter epitheliums were alleviates and the secretions were decreased too. The high dose group was the best in the treatment group.Result display: Rukanghe decoction has the effect to improve the rats MGH.3 Effect of rukanghe decoction on sex hormone in serum.Compared with model group(E₂ 18.48±2.66,P 34.71±2.36,T 0.07±0.02,PRL 2.57±0.89), E₂: Its level in high dose group(13.17±2.87) and low dose group(14.61±1.88) was markedly decreased(P<0.01). P: Its level in high dose group(44.39±3.50) and low dose group(43.34±3.61) was markedly increased(P<0.01). T:Its level in high dose group(0.10±0.02)was markedly increased (P < 0.01) and in low dose group it was of nostatistical significance(P>0.05). PRL: Its level in high dose group(3.37±0.69)was markedly increased (P<0.05) and in low dose group it was of nostatistical significance(P > 0.05). Compared with Rupixiao group(E₂ 12.10±1.59,P 40.31±4.66; T 0.11±0.03,PRL 2.82±0.71), E₂: Its level in high dose group was of nostatistical significance(P>0.05) in low dose group was markedly decreased(P<0.05). P: Its level in high dose group was markedly increased(P<0.05) and in low dose group it was of nostatistical significance(P>0.05). T:Its level in high dose group was of nostatistical significance(P>0.05). PRL: Its level in high dose group and low dose group was of no markedly changed. Compared with low dose group, E₂, P, PRL in high dose group were of nostatistical significance(P>0.05), and T was markedly increased(P<0.01).Result display: Rukanghe decoction can adjust endocrine disturbance.4 Effect of rukanghe decoction on SOD activity and MDA content in serum.Compared with model control group(SOD 217.43±5.44,MDA 16.20±1.65): the activity of SOD(223.67±2.97) in serum of high dose group was markedly increased (P<0.05), the contents of MDA(13.87±0.89) in serum of high dose group was markedly decreased (P<0.01); the activity of SOD(220.97±4.31) and contents of MDA(15.27±1.23) in serum of low dose group were of no statistical significance(P>0.05). Compared with RPX group (SOD 223.76±5.36,MDA 13.99±1.62): there were of no statistical significance between the high dose group and RPX group(P>0.05).Result display: Rukanghe decoction has effect of inhibiting lipid peroxidation and scavenging oxygen free radical. Its treatment effect was equivalent to RPX group.5 Effect of rukanghe decoction on the expression of ER and AT₁ in mammary glandular tissue.Compared with model control group(ER 0.1608±0.0161, AT₁ 0.1706±0.0267), ER: Its expression in high dose group(0.1232±0.0126) and low dose group(0.1331±0.0208) was markedly lower(P<0.01); AT₁: Its expression in high dose group(0.1279±0.0302) and low dose group(0.1302±0.0227) was markedly lower(P<0.01). Compared with RPX group (ER 0.1340±0.0152,AT₁ 0.1211±0.0191): there were of no statistical significance between the high dose group, low dose group and RPX group(P>0.05).Result display: Rukanghe decoction can down regulation the expression of ER and AT₁.Conclusion:1 The occurrence of MGH probably include over expression of AT₁ and peroxidative damage in addition to endocrine disturbance and over expression of ER.2 Rukanghe decoction on MGH, can adjust some genital hormones, down regulation expression of ER to diminishing sensitivity of E to mammary glandular tissue, down regulation expression of AT₁ to inhibit cell proliferation and improve blood circulation of mammary glandular tissue, relieve the peroxidative damage to achieve curing MGH.