The Study Of TCM Syndrome Of The Adenomyosis And The Influence Of HuaYuZhiTong Prescription On The Adenomyosis Cells

Objective:1. In this study, the clinical symptoms in patients with adenomyosis (AD) are collected and organized, then the characteristics of TCM symptoms are studied with a view to investigate the pathogenesis of AD, guide the clinical application, improve the curative effects, provide the clinical basis for the further research.2. The fingerprint of HuaYuZhiTong Prescription is established to provide the basis for the determination of the active components.3. By the primary culture and identification of AD cells, the study on mechanism of Chinese materia medica in treating for AD has an ideal experimental model.4. To explore the effect of HuaYuZhiTong Prescription on apoptosis, prostaglandin, subtype of estrogen receptor a, oxytocin receptor expression level of Adenomyosis Cells, observe the effective parts, and provide a more scientific basis for drug screening and streamlining the prescription.Methods:1.This study obtained the basic information and the four diagnostic data through the observation of the in-patients and out-patients from the First Affiliated Hospital of the Guangzhou University of Traditional Chinese Medicine from January of 2001 to February of 2011, then analyzed and summarized their age, calculated the distribution of each syndrome and the distribution of each common symptoms:dysmenorrhea, menstruation, menorrhagia, and the incidence of infertility.2. HPLC was used to establish a finger print of HuaYuZhiTong Prescription, which was applied with SB-C18 as the chromatic column, methanol-H3PO4 solution (gradient elution) as the mobile phase, detection was conducted at a wavelength of 230nm, flow rate being 1.0 mL/min, and column temperature at 35℃.GC-MS was used to establish a finger print analysis method of HuaYuZhiTong Prescription.GC was applied with DB-1 capillary as the chromatic column, Helium as the carrier gas, flow rate being 1.0 mL/min; MS was applied with El source (70ev), dual filament; mass range from 40 to 400 m/z with full scan per 0.5 seconds.3. Adenomyosis cells were digested with collagenase in primary culture and identified by immunocytochemistry.4. MTT was performed to observe the inhibitory rate of adenomyosis cells, which were interved by HuaYuZhiTong Prescription and its decomposed formulas, the main chemical composition. The concentration of drug intervention was selected through the IC50.5. Flow cytometry, real-time fluorescent RT-PCR and ELISA were used to observe the expression and changes on apoptosis, PGF2α, PGE2, ER a mRNA, OTRmRNA of AD cells, which were treated with HuaYuZhiTong Prescription, the decomposed formulas, and the main chemical composition, respectively, and RU486 was used as reference drug.Results:1.294 cases with final diagnosis of AD have been collected. The high-incidence age of the investigated AM patients is 31-50, accounting for 86.06% of total patients. the first syndrome is Qi stagnation and blood stasis type (44.56%), the second syndrome is Kidney deficiency and blood stasis type(20.75%), the third is Qi deficiency and blood stasis type (17.01%); the incidence of dysmenorrhea was 73.5%, prolonged menstruation was 11.9%, menorrhagia was 55.8%, and infertility cases was 10.5%. The AM patients who were 31-year-old were prone to be the qi stagnation and blood stasis type, hot burning blood stasis. Then the AM patients who were 41-year-old were prone to be the kidney deficiency and blood stasis type, phlegm accumulating with blood stasis type, blood stasis due to cold, qi deficiency and blood stasis type. Dysmenorrhea and menorrhea be easy appear in qi stagnation and blood stasis type, and kidney deficiency and blood stasis type was the second; And prolonged menstruation was more seen in kidney deficiency and blood stasis type, was followed by qi stagnation and blood stasis type. 2. The finger print of HPLC:According to the results of 10 batches samples, 10 common Peaks were marked. Root of herbaceous peony and HuaYuZhiTong Prescription have two overlapping peaks:3th Peak and 5th Peak, while licorice and HuaYuZhiTong Prescription also have two overlapping Peaks:7th Peak and 10th Peak. In line with the reference substance, single herb and literature, four Peaks were identified:5th Peak has been paeoniflorin,6th Peak has been ferulic acid,7th Peak was mixing Peak:Liquiritin and Tetrahydropalmatine. The largest Peak was paeoniflorin, and the scond Peak was glycyrrhizin.3. The characteristic finger print of volatile oil which was extracted from HuaYuZhiTong Prescription was established by GC-MS, and a total of 107 Peaks were marked.. According to the information which was obtained by mass spectrometry, the relative retention time, the computer with the standard MS libraries (NIST and WILEY spectrum library) and standard search patterns, the 26 large Peaks were identified:53th Peak was ketone Curcuma and the highest, the 12th Peak was theδ-elemene and the scond highest, the 61th Peak was Spartak alcohol and the third, the 36th Peak was root geranium ene and the fourth big, and the 76th Peaks was ligustilide and the fifth.4. Six cases of adenomyosis cells were successfully cultured and were identified by immunocytochemistry. The multiplication time of AD cells is 39.3h, and the growth curve of 1st generation and 8th cells was the almost same.5. Apoptosis:Compared with the blank group, the AD cells'rate of apoptosis in RU486 groups was significantly higher (P<0.05); Compared with the blank group, the AD cells'rate of apoptosis in all Experimental groups was also significantly higher (P<0.05), and the SGD group, Remaining medicine group, Volatile oil group's difference which was a dose-dependent was statistically significant (P<0.05). There was significant difference between the HuaYuZhiTong Prescription group and the Volatile oil groups in the rate of apoptosis (P<0.05). However, compared with the RU486 group, the rate of apoptosis in the HuaYuZhiTong Prescription group was significantly higher (P <0.05).Compared with the blank group, the rate of apoptosis in the HuaYuZhiTong Prescription group, SGD group, Root of herbaceous peony group, Licorice group, Paeoniflorin group, Glycyrrhizin group, Paeoniflorin and Glycyrrhizin group was significantly higher (P<0.05). Compared with the HuaYuZhiTong Prescription group, the rate of apoptosis in the low concentrations of SGD group, Root of herbaceous peony group, Licorice group, the high concentrations of Paeoniflorin group, Glycyrrhizin group was significantly higher (P<0.05). Compared with the low concentrations of SGD group, the rate of apoptosis in the Root of herbaceous peony group, Paeoniflorin group, Glycyrrhizin group, Paeoniflorin and Glycyrrhizin group was significantly higher (P<0.05). However, Compared with the high concentrations of SGD group, the rate of apoptosis in the Root of herbaceous peony group, the low concentrations of Licorice group, the high concentrations of Paeoniflorin group, Glycyrrhizin group was significantly higher (P<0.05).6. PGE2:Compared with the blank group, the expression of PGE2 in the RU486 group was no statistical significance in other Experimental group (P>0.05). Compared with the blank group, the expression of PGE2 in the HuaYuZhiTong Prescription group, the SGD group, the Volatile oil group was statistically significant (P<0.05), and there was no statistical significance in other Experimental group(P>0.05). There was significant difference between the SGD group, the Volatile oil group and the Remaining medicine group in the expression of PGE, (P<0.05).Compared with the blank group, the expression of PGE, in the HuaYuZhiTong Prescription group, SGD group, Root of herbaceous peony group, the high concentrations of Paeoniflorin and Glycyrrhizin group was significantly higher (P<0.05), and there was no statistical significance in other Experimental group (P>0.05). Compared with the HuaYuZhiTong Prescription group, the expression of PGE2 in the Glycyrrhizin group and RU486 group was significantly higher (P<0.05), and there was no statistical significance in other Experimental group (P>0.05). Compared with the SGD group, the expression of PGE2 in the Glycyrrhizin group was significantly higher (P<0.05), and there was no statistical significance in other Experimental group (P>0.05).7. PGF2α:Compared with the blank group, the expression of PGF2αin the HuaYuZhiTong Prescription group, the SGD group, the Volatile oil group and the Remaining medicine group was statistically significant (P<0.05), and their dose-dependent were statistically significant (P<0.05), but there was no statistical significance between the RU486 group and the blank group(P >0.05). Compared with the HuaYuZhiTong Prescription group, the expression of PGF2αin the SGD group, the Volatile oil group and the Remaining medicine group was statistically significant(P<0.05). The difference in the SGD group, the Volatile oil group and the Remaining medicine group was statistically significant (P<0.05), the SGD group was the greatest impact group, the Volatile oil group was the scond, the Remaining medicine group was the third.Compared with the blank group, the expression of PGF2αin the HuaYuZhiTong Prescription group, the SGD group, the Root of herbaceous peony group, the Licorice group, the Paeoniflorin group, the Glycyrrhizin group, the Paeoniflorin and Glycyrrhizin group was significantly higher (P<0.05), and their dose-dependent were statistically significant(P<0.05), but there was no statistical significance between the RU486 group and the blank group(P >0.05). Compared with the HuaYuZhiTong Prescription group, the expression of PGF2αin the SGD group, the Root of herbaceous peony group, the Licorice group, the Paeoniflorin group, the Glycyrrhizin group, the Paeoniflorin and Glycyrrhizin group was statistically significant (P<0.05). The difference in the SGD group, the Root of herbaceous peony group and the Paeoniflorin group was statistically significant (P<0.05), the SGD group was the greatest impact group, the Root of herbaceous peony group was the scond.8.OTR mRNA:Compared with the blank group, the expression of OTR mRNA in the RU486 group was no statistical significance in other Experimental group(P >0.05). But compared with the blank group, the expression of OTR mRNA in the HuaYuZhiTong Prescription group, the high concentrations of Volatile oil group and the high concentrations of Remaining medicine group was statistically significant(P<0.05), and there was no statistical significance in other Experimental group(P>0.05). There was significant difference between the HuaYuZhiTong Prescription group and the RU486 group in the expression of OTR mRNA(P<0.05). There was significant difference between the HuaYuZhiTong Prescription group and the low concentrations of the SGD group in the expression of OTR mRNA(P<0.05).Compared with the blank group, the expression of OTR mRNA in the HuaYuZhiTong Prescription group, the high concentrations of the Paeoniflorin and Glycyrrhizin group was statistically significant (P<0.05), and there was no statistical significance in other Experimental group (P>0.05). There was significant difference in the HuaYuZhiTong Prescription group and the low concentrations of the SGD group, the low concentrations of the Root of herbaceous peony group in the expression of OTR mRNA(P<0.05), and there was no statistical significance in other Experimental group(P>0.05). there was no statistical significance between the Paeoniflorin and Glycyrrhizin group and other Experimental group(P>0.05).9. ERαmRNA:Compared with the blank group, the expression of OTR mRNA in the RU486 group was no statistical significance in other Experimental group(P >0.05). But Compared with the blank group, the expression of ER mRNA in the HuaYuZhiTong Prescription group and the SGD group was statistically significant(P<0.05), and there was no statistical significance in other Experimental group (P>0.05). Compared with the RU486 group, the expression of ER mRNA in the HuaYuZhiTong Prescription group and the high concentrations of the SGD group was statistically significant (P<0.05), and there was no statistical significance in other Experimental group (P>0.05). Compared with the high concentrations of the HuaYuZhiTong Prescription group, the expression of ER mRNA in the high concentrations of Remaining medicine group and volatile oil group was statistically significant (P<0.05), and there was no statistical significance in other Experimental group(P>0.05).Compared with the blank group, the expression of ER mRNA in the HuaYuZhiTong Prescription group, the SGD group and the Root of herbaceous peony group was statistically significant(P<0.05), and there was no statistical significance in other Experimental group(P>0.05). Compared with the high concentrations of the HuaYuZhiTong Prescription group, the expression of ER mRNA in the low concentrations of Glycyrrhizin group was statistically significant(P<0.05), and there was no statistical significance in other Experimental group (P>0.05). Compared with the high concentrations of the SGD group, the expression of ER mRNA in the low concentrations of the Licorice group and the low concentrations of the Glycyrrhizin group was statistically significant (P<0.05), and there was no statistical significance in other Experimental group(P>0.05).Conclusion:1. The syndrome types of AD was relevance with the age and the main symptoms. The first syndrome of adenomyosis is qi stagnation and blood stasis type, and the incidence of dysmenorrhea was the highest, adenomyosis which is qi stagnation and blood stasis type and have the dysmenorrhea will be the focus of future research. 2. The results of the HuaYuZhiTong Prescription HPLC finger print suggested that Paeoniflorin and Glycyrrhizin were the main chemical components of HuaYuZhiTong Prescription, and this provided a basis for drug research.3. The results of the HuaYuZhiTong Prescription GC-MS finger print suggested that the main chemical components in the Volatile oil of HuaYuZhiTong Prescription maybe extract from Myrrh, Panax, Canton costas, Angelica, Cyperus rotundus, Licorice, and this provide a basis for Drug screening.4. Adenomyosis cells which was digested by Collagenase was cultured was stable, the 3th-6th cells were selected to be the model of drug intervention. The time was in 4-5 days when the cells was passaged, and 24h as a assay time.5. The influence of HuaYuZhiTong Prescription on the Adenomyosis cells was possible as following:①It indicates that it is one of the mechanisms for HuaYuZhiTong Prescription to induce apoptosis of the Adenomyosis cells, make it spontaneous regression, the effect of Volatile oil and Root of herbaceous peony were more, and paeoniflorin was the main chemical components of Root of herbaceous peony.②By decreasing the expression of PGF2αin adenomyosis cells, the intensity of uterine contractions was reducing, and thus alleviate dysmenorrhea:SGD and Volatile oil maybe the main effective components of HuaYuZhiTong Prescription in decreasing the expression of PGF2α, and Root of herbaceous peony and paeoniflorin maybe the main effective components of SGD; the other Experimental groups were inferior to HuaYuZhiTong Prescription in effect of decreasing the expression of PGE2, and the SGD group was the greatest impact group, the Volatile oil group was the scond.③One of the mechanisms for HuaYuZhiTong Prescription to induce the OTR mRNA expression in adenomyosis cells, decrease the binding of OT and OTR, which reduce the intensity of uterine smooth muscle contraction, effectively alleviate the symptoms of patients with dysmenorrhea:the Volatile oil and Remaining medicine and the paeoniflorin and glycyrrhizin maybe the main effective components.④One of the mechanisms for HuaYuZhiTong Prescription to induce the ER mRNA expression in adenomyosis cells, then the binging of E2 and ER was decreased, the intensity of uterine smooth muscle contraction was reduced, dysmenorrhea was minimized or disappeared:the SGD maybe the main effective components. And the uterus contraction decreased, the larger the contribution of peony.