| Part 1:Professor Cui Xuejiao's prescription for the treatment of impotence prescriptionObjecive:Impotence is a common disease in men and can be caused by a variety of pathogenic factors.Type 2 diabetes is a category of "thirst quenching" and is a common cause of disease.Long-term diabetes can cause refractory diabetes-type impotence.Many doctors try to use various specific and empirical methods to try to improve the erectile function of diabetic impotence patients,but they have little effect.Professor Cui Xuejiao is a professor and chief physician of Guangzhou University of Traditional Chinese Medicine,a doctoral tutor of Chinese medicine and a doctoral tutor of clinical medicine,and a third batch of the sixth batch of nationally renowned Chinese medicine experts.He has been working hard for more than 50 years,and has been well versed in all kinds of male intractable diseases.He has a unique understanding and rich experience in type 2 diabetes type impotence.In this study,we collected and treated the doctors of Cui Xuejiao for the treatment of type 2 diabetes-type impotence medical records for the past 3 years.Taking the case as the core,we collected the patient's age,medical history,syndrome type and other information,applied data mining technology for retrospective analysis,and summarized Professor Cui Xuejiao' s treatment of impotence prescription.The medication and the prescription rules,through the complex entropy cluster analysis to obtain new prescriptions,provide a data basis for better clinical treatment programs,and provide a reference for the inheritance of old Chinese medicine experience and type 2 diabetes type impotence clinical application.Method:The use of landing outpatient medical order system search,telephone follow-up and outpatient follow-up,etc.,with "erectile dysfunction","type 2 diabetes","yangwei","thirst" as key search terms,collected 2014.01.01 to 2018.12.During the period of 31,Professor Cui Xuejiao accepted the prescription of type 2 diabetic impotence patients who met the inclusion criteria at the clinic of the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine.And record the detailed information of related prescriptions such as:syndrome type,prescription,usage and usage as data source.A total of 214 prescriptions for type 2 diabetes-induced impotence were screened out.According to the Pharmacopoeia of the People' s Republic of China,the names of the drugs of 214 prescriptions are standardized.For example,the wine cellar and the hawthorn are collectively referred to as "Hawthorn";the aconite and the aconite are collectively referred to as "Aconite".The standardized prescriptions are entered into the“Traditional Chinese Medicine Aided Platform" system(V2.5)developed by the Institute of Chinese Medicine of China Academy of Chinese Medical Sciences,and a second review is carried out to ensure the authenticity and reliability of the data.Result:1.General clinical dataA total of 214 cases were collected and screened in this study.The minimum age of the patients included was 25 years old,the maximum age was 80 years old,and the average age was 37.44+8.67 years old.2.TCM symptom distributionAfter screening 214 patients who met the inclusion criteria,the researchers browsed the outpatient case system,patient cases and other four diagnostic data and the prescriptions of the doctors to identify their syndrome types in combination with the diagnostic criteria of each syndrome type,and took the main syndrome type(first syndrome type).According to the distribution of syndromes in patients with diabetes-induced impotence in the past three years,the proportion of patients with evidence and deficiency syndrome was found to be equivalent,accounting for 44.9%and 55.1%,respectively.Among them,the evidence was mainly caused by blood stasis,accounting for all occurrences.21%of the syndromes,the false deaths of the fires accounted for the vast majority,the proportion was 49.5%.After grouping all 214 patients according to age group,the distribution of patients in all age groups and the distribution of syndromes in different age groups are shown in Figure 17.It is found that the patients are mainly young and middle-aged people aged 31-50 years;The age group syndrome showed a specific distribution,and the 21?40 age group was mainly based on evidence,while the 41-80 age group was mainly based on deficiency syndrome.3.Drug medicinal analysisAfter frequency analysis of the four gas properties of the Chinese medicine related to the treatment of type 2 diabetes impotence,Professor Cui Xuejiao found that the drugs used were mainly warm-filled,accounting for 61%,followed by flat drugs,accounting for 20.5%,and again cold drugs.Accounted for 16.9%;the frequency analysis of the five flavors found that the drug properties were mainly Xin and Gan,accounting for 24.0%and 37%respectively.4.Drug returnThe frequency analysis of Professor Cui Xuejiao' s treatment of type 2 diabetes-related impotence related Chinese medicines found that the most important drugs belonged to the heart,liver,spleen and kidney.5.Frequency of drugsThe descriptive statistics of 214 prescriptions for treating impotence were analyzed.According to the frequency statistics,214 prescriptions for impotence contained 96 flavors of Chinese medicine,including up to 17 flavors and at least 7 flavors.According to the frequency of drug appearance,the frequency of drugs is ? 21 times,that is,the Chinese medicine with frequency of drug?10%has 28 flavors.7.Analysis of association rulesAccording to the association rule Apriori algorithm,the support degree is set to 10%,20%,and 30%respectively(indicating that the frequency of occurrence of the drug combination accounts for at least 10%,20%,and 30%of the total number of prescriptions),and the confidence is 0.9.The core drug combinations for the treatment of sputum were extracted at different levels.According to the "group rule" analysis,19440 core drug combinations with a support degree of 10%were obtained;8237 core drug combinations with a support degree of 20%;the support degree was 30.%of the core drug combinations are 8051.8.Complex system entropy clustering analysisApplying the method of complex system entropy,further correlation analysis is carried out on the possible drug combinations of 214 prescriptions.According to the data mining method of complex system entropy clustering,the total number of prescriptions is used to pre-read the data extracted by different parameters.Set the correlation degree to 5 and the penalty degree to 2,and perforim hierarchical cluster analysis to obtain 6 core drug combinations.Conclusion:During the period from 2014.01.01 to 2011.12.31,the majority of patients admitted by Cui Xuejiao were mainly kidney-yang deficiency,blood stasis,damp-heat bet,and the patient population gradually became younger.According to the application of descriptive statistics,association rules algorithm,complex system entropy cluster analysis,Wenyang Huoxue Recipe is the main prescription for the treatment of impotence,and the combination of drugs extracted in the new prescription suggests that it is also necessary to pay attention to both spleen and dampness.Lishui Tonglin products to help kidney yang.Part 2:Based on NO/cGMP pathway to explore the effect and mechanism of Gross Saponins of Tribulus Terrestris on erectile dysfunction in type 2 diabetesObjective:1.Construct a rat model of type 2 Diabetes Mellitus Erectile Dysfunction(T2DMED);2.To confirm that type 2 diabetes damages endothelial function by mediating oxidative stress,promotes smooth muscle fibrosis and apoptosis of corpus cavernosum and leads to erectile dysfunction;3.It was confirmed that saponins can improve the expression of NO/cGMP pathway by improving the endothelial function of penis in T2DMED rats,improve smooth muscle fibrosis,and inhibit apoptosis to improve erectile function.Method:1.Establishment of T2DMED rat model50 body masses of about 180-220 g(6-7 weeks old)were injected with Apomorphine(APO)(APO 100 ? g/kg was injected subcutaneously into the posterior neck of the rat,and placed in a transparent experiment to observe the cage.Observed in a dark and quiet environment for 30 minutes,recorded the presence or absence of erectile function:the standard of penile erection in rats was:penis enlargement,foreskin retreat,glans exposed to congestion),and adult male SPF SD rats with erectile function were randomly assigned.Rats were randomly divided into normal control group(6 rats)and intervention group(44 rats).Both groups were raised in SPF animal laboratory of the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine.The experimental environment was set as follows:light environment:7:00-19:00 light,19:00-7:00 dark;temperature setting:18 24?;noise control within 60dB;relative humidity control range is 40%?70%;After 3 days of adaptive feeding,the normal control group was fed with normal feed and common reference water,and was given a double-distilled water at a dose of 15 ml/Kg at a frequency of 1 time/day;the intervention group was pre-emulsified high-sugar high-fat emulsion.Gastric administration(about 10%cholesterol,35%lard,2%sodium cholate and 40%sucrose),the intragastric dose is 15ml/Kg,the frequency is 1 time/day,supplemented with normal feed and free to eat and saturated sucrose Water and ordinary drinking water were free to eat:after 8 weeks,the normal control group was injected with citrate-sodium citrate buffer(30 mg/kg),and all the rats in the intervention group were intraperitoneally injected with streptozotocin(STZ)(30 mg/kg)to specifically destroy islet ? cells and replicate the T2DM model.On the 3rd and 7th day,the blood glucose was measured by the tail-cut method,and the random blood glucose was>16.67 mmol/L.The blood glucose was measured once every 2 weeks and continued to feed according to the previous protocol.On the 16th week,Fasting blood glucose was measured and APO was injected again.Rats without erectile function were screened.T2DMED rats with random blood glucose>16.67 rmmol/L and no erectile function were divided into 4 groups,which were divided into 5 groups:normal control group.(n=6),T2DMED group(n=6),GSTT group(n=6),Sildenafil group(n=6),mixed group(GSTT+sildenafil)(n=6).After grouping,the diets of each group were changed to normal diet and ordinary drinking water,and they were given to GSTT group saponin solution(dissolved in double distilled water)40mg/kg/d,Sildenafil group to citrate The non-tablet suspension(dissolved into powdered powder and dissolved in double distilled water)was intragastrically administered at a dose of 5 mg/kg/d.The mixed group was given a solution of 40 mg/kg/d+citric acid in a solution of Gross Saponins of Tribulus Terrestris(GSTT).The sildenafil suspension was intragastrically administered with 5 mg/kg/d;the normal control group and the T2DMED group were given the same amount of double distilled water;the rats in each group were intragastrically administered once a day for 4 weeks.2.Evaluation of erectile functionAfter 4 weeks of treatment,10%chloral hydrate was anesthetized by intraperitoneal injection at a dose of 100 mg/kg,routinely fixed,prepared,sterilized,and the midline incision was made in the abdomen and neck,respectively,and the bilateral cavernous nerve and common carotid artery were exposed;The bipolar silver electrode was placed on the right cavernous nerve,and two 23-G infusion needles connected to the pressure transducer were inserted into the right penis root and the right common carotid artery(the infusion tube was filled with 250 U/ml).Heparin saline)and connected to a pressure transducer to record I CP and MAP,respectively(ICP,Intracavernous Pressure:MAP,Mean Arterial Pressure;mean arterial pressure;both for quantitative assessment of erectile function),stimulation The parameters are set to voltage 5V,frequency 20HZ,stimulation duration 30s,interval 5min.The actual pressure of the ICP and MAP is converted to a digital signal by a pressure transducer,and the ICP value and the ICP/MAP value are recorded by a Delphis electrophysiometer recorder.3.eNOS expression level detection3.1 ImmunohistochemistryTissue samples were fixed,embedded in paraffin,and then sectioned at a thickness of 5 pm.After dewaxing and rehydration,the antigen was repaired and blocked,and incubated with an antibody against endothelium-derived nitric oxide synthase(eNOS)overnight at 4 ?.The sections were then incubated with biotin-conjugated secondary antibody for 30 minutes at 37 ?,followed by incubation with avidin-4 horseradish horseradish peroxidase complex for 30 minutes at 37°C,then DAB color The original body is processed to carry out the reaction and visualize.Sections were counterstained with diluted hematoxylin and fixed with neutral glue.Five high power fields(200X)were randomly selected for each slice.The image of eNOS iumunohistochemistry in each group of penis tissues was analyzed using Image-Pro Plus 6.0 image analysis software.3.2 Real-time PCRThe rat corpus cavernosum was taken and the expression of eNOS mRN was detected by polymerase chain reaction(PCR).3.3 Iumunoblotting(Western blot)The rat corpus cavernosum was taken and the expression of eNOS protein was detected by iumunoblotting.4.Determination of NO and cGMP expression levelsThe kit was removed and allowed to equilibrate to room temperature for about 30 minutes at room temperature.Add 100 U1 of the standard to the wells of the standard coated plate,add 20 p 1 of the sample to be tested,and add the sample dilution to 100 U1.Mix and incubate in a 37 ? incubator for 20 min.Wash the plate:discard the liquid,use dry,add the washing solution,remove the washing solution,dry,and repeat the plate washing 4 times.100 ?1 of the enzyme standard working solution was added to the standard well and the sample well,mixed,and incubated at 37 ? in an incubator for 30 min.Wash the plate:Same as above.100 ? 1 of 1×HRP was added to the standard well and the sample well,mixed,and incubated at 37 ? for 30 min.Wash the plate:Same as above.100 ? 1 of 1TM color developing solution was added to the standard well and the sample well,mixed,and incubated at 37 ? for 30 min.100 ?1 of the stop solution was added to the standard well and the sample well,respectively,and mixed,and the absorbance(OD value)of each well was measured with a wavelength of 450 nm in 15 minutes.Using the concentration and OD value of the standard,a linear regression equation is calculated,and the concentration of the measured sample is calculated based on the OD value measured by the sample.5.Determination of ROS expression levelThe expression of Reactive Oxygen Species(ROS)was measured by DHE superoxide anion fluorescent probe according to the method of Davidson et al.The tissue of each slice was taken in five regions under a 200 x optical microscope.Analysis using Image-Pro Plus 6.0 image analysis software.The ROS content and the crude protein extract of the homogenate of the tissue were determined using a kit developed by the Nanjing Institute of Bioengineering.The ROS content is defined as the concentration of hydrogen peroxide in the reaction system is reduced by 1 Umol per milligram of tissue protein per minute at 37 ?as an anti-reactive oxygen unit(U/mg).6.Determination of cavernous smooth muscle fibrosis levelThe middle portion of the penis was sectioned and stained according to the Masson kit using the Masson method.The collagen tissues of the penis sponge(stained in blue or green)and smooth muscle tissues(stained iri red)were observed under light microscope,and the smooth muscle/collagen ratio of the cavernosus was analyzed by Image-Pro plus 6.0 software.7.Determination of apoptosis level of cavernosal cellsThe cells of each group of penile tissues were detected by TUNEL apoptosis detection kit using the deoxyribonucleotide terminal transferase-mediated dUTP nick end labeling(TUNEL)staining method.Apoptosis rate(this procedure was performed according to the kit test instructions).TUNEL images(200X)of each group of penile tissues were analyzed using the analytical measurement image software Image-pro plus 6.0.Four samples were randomly taken from each sample,and the cells stained with brown dark staining were apoptotie cells,and the ratio of the cells to the total number of cells in the visual field was the apoptotic rate.Result:1.Progress of the experimentDuring the whole experiment,the normal control group was in good condition and no accidental death;the intervention group showed obvious symptoms of polydipsia,polyphagia,and polyuria,and there was no significant change in body weight after modeling.During the experiment,15 of the intervention group did not meet the standard of modeling,6 of which did not meet the criteria for type 2 diabetes,9 did not meet the standard of erectile dysfunction;5 rats died during the intervention of high-fat and high-glucose emulsifier.2.Evaluation of erectile functionThe ICP and ICP/MAP values of GSTT group,Sildenafil and mixed group were significantly higher than those of T2DMED group(P<0.05),but still lower than the normal control group(P<0.05).3.eNOS expression level3.1 ImmunohistochemistryImmunohistochemistry showed that the expression of eNOS in GSTT group and mixed group was significantly higher than that in Sildenafil group and T2DMED group(P<0.05),and there was no significant difference between them(P>0.05).3.2 Real-time PCRFluorescence quantitative PCR showed that the expression of eNOS nRNA in GSTT group and mixed group was significantly higher than that in Sildenafil group and T2DMED group(P<0.05),and there was no significant difference between them(P>0.05).3.3 ImmunoblottingWestern blotting showed that the expression of eNOS mRNA in GSTT group and mixed group was significantly higher than that in Sildenafil group and T2DMED group(P<0.05),and there was no significant difference between them(P>0.05).4.NO expression levelCompared with the T2DMED group and the Sildenafil group,the GSTT group and the mixed group significantly increased the NO level(P<0.05).5.eGMP expression levelThere was no significant difference between the GSTT group and the Sildenafil group in improving cGMP levels(P>0.05),which was significantly higher than that in the T2DMED group(P<0.05),and the mixed group was significantly better than the two(P<0.05),but still low.In the normal control group(P<0.05).6.ROS expression levelCompared with T2DMED group and Sildenafil group,GSTT group and mixed group can significantly reduce ROS content(P<0.05).7.Penile cavernous smooth muscle/collagen ratioMasson staining showed that the smooth muscle/collagen ratio of GSTT group and mixed group was higher than that of T2DMED group and Sildenafil group,the difference was statistically significant(P<0.05),but still lower than the normal control group,the difference was statistically significant(P<0.05).).8.Penile cavernosal cell apoptosis indexTUNEL staining apoptotic rate test showed that the apoptotic index of GSTT group and mixed group decreased compared with T2DMED group and Sildenafil group,the difference was statistically significant(P<0.05),but still higher than the normal control group,the difference was statistically significant.(P<0.05).Conclusion:1.It is confirmed that the T2DMED model can be successfully established by the modified high-fat and high-sugar emulsion by intragastric induction+intraperitoneal injection of STZ.2.It was confirmed that T2DM can cause erectile dysfunction by mediating oxidative stress,apoptosis of cavernous endothelial cells and smooth muscle fibrosis of corpus cavernosum;3.Confirm that GSTT has a mild regulation of blood glucose and can improve oxidative stress in rats by reducing ROS content;It was confirmed that GSTT can inhibit the apoptosis of rat corpus cavernosum in T2DMED rats,improve the endothelial function of rat corpus cavernosum,increase the expression of eNOS,NO and cGMP,and improve the erectile function.5.Confirm that GSTT can improve smooth muscle fibrosis and improve erectile function;6.Confirm that GSTT has a synergistic effect with Sildenafil in improving the erectile function of T2DMED rats;... |
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