| Part I Experimental study on the treatment of secondary lymphoedema with Xiao Zhong Ru Mi Qing tabletsBackground and objectivesLymphedema is caused by lymphatic drainage disorder after lymphatic obstruction,and excessive protein-rich fluid accumulates in the interstitial tissue.It is divided into two major categories:primary and secondary Iymphedema.Most of the primary lymphedema is unexplained and caused by congenital dysplasia such as lymphatic expansion,valvular insufficiency or absence.Common causes of secondary lymphedema include filariasis,surgery,radiotherapy,trauma,repeated infections,and tumor invasion and metastasis.There are about 140 million patients with lymphedema around the world.10%of patients are primary lymphedema caused by congenital lymphatic system defects and the remaining 90%are secondary lymphedema,of which 45 million are filarial lymphedema and 20 million are upper extremity lymphedema after breast cancer surgery.Lymphedema is listed as the second most disabling disease by WHO,which seriously endangers the patients’physical and mental health.China was once one of the most serious countries with filarial lymphedema.China has basically eliminated filariasisby the beginning of this century and it is difficult to see filarial lymphedemanow.However,the number of patients with secondary lymphedema has not decreased.With the increasing number of patients with malignant tumor undergoing surgical radical surgery and/or radiotherapy,the incidence of secondary lymphedema has increased year by year.It has been reported that the incidence of postoperative lymphedema in breast cancer is 6%to 30%,the incidence of postoperative pelvic malignant tumors is 1%to 47%and the incidence of postoperative head and neck cancer is 22%to 56%.In the past,although countries in the world have carried out various treatment research including surgeryon these diseases,there is still no ideal cure.Therefore,it is still a key problem to explore effective treatment drugs for lymphedema and chyluriria.According to the treatment of traditional Chinese medicine,Wang Peiyi research group of the Shandong Institute of ParasiteControl and Prevention has developed the application of Xiao Zhong Ru Mi Qing tablets at 9g/d per person for one month which was a course to treat chyluria and limb lymphedema.This drug was respectively used to treat 315 and 341 cases of chyluria and limb lymphedema,and the total effective rate was 100%after 1-7 courses,among which the cure rate was 98.41%and 73.82%.In the past 20 years,the research group has treated more than 30,000 patients with lymphedema in 16 provinces and autonomous regions such as Anhui and Jiangsu,and all of them have achieved the same remarkable results as the above results.The research group found that the traditional Chinese medicine have significant anti-inflammatory and anti-swelling properties,reduce the permeability activation of capillaries and enhance the phagocytic function of reticular endothelial system,and have significant protective effects on lymphangial endothelial cells damaged by hypoxia.However,due to various conditions at that time,the mechanism of this drug could not be further studied.In this study,we will explore the efficacy and molecular mechanism of Xiao Zhong Ru Mi Qing tablets for the treatment of secondary lymphedema.Rodent models have been greatly welcomed due to their ease of access,cost-effectiveness,and stable lymphedema.The combination of radiation therapy and surgery has been shown to effectively promote stable lymphedema in the hind limbs of rats and mice.Compared with the hindlimb model,the rodent tail lymphedema model reported by Slavin et al.has the advantages of simple anatomy and surgical operation as well as good repeatability.It has been used for the surgical and gene therapy of lymphedema andthe study of the molecular mechanisms of lymphangiogenesis.Therefore,we will use the rat tail model to study the efficacy and molecular mechanism of Xiao Zhong Ru Mi Qing tablets in the treatment of postoperative lymphedema.The stratum spinosum of epidermidis is the site of lymph circulation and substance exchange in the skin.Laser Scanning Confocal Microscope(LSCM)combines laser and computer image processing technology on the basis of optical microscope to increase the resolution of optical microscope by 30-40%.The performance of optical sectioning can observe the three-dimensional structure of the tissue and cells.Theconfocal microscopy technique can non-invasively display the epidermal and dermal papilla layers with images that achieve histological resolution.In this part,we will construct a rat tail lymphedema model to simulate secondary lymphedema,and study the lymphedema with confocal microscope.Given the damage of secondary lymphedema,this paper studied the feasibility of laser scanning confocal microscopy in the diagnosis of secondary lymphedema by constructing a rat tail lymphedema model,and explored the effect and molecular mechanism of Xiao Zhong Ru Mi Qing tablets on secondary lymphedema.The study provide theoretical and experimental bases for the treatment of lymphedema by traditional Chinese medicine.Syudy Contents1.To establish a model of the rat tail lymphedemaand evaluate the model with laser scanning confocal microscope.2.To explore the effect of Xiao Zhong Ru Mi Qing tablets on secondary lymphedema.3.To explore the molecular mechanism of Xiao Zhong Ru Mi Qing tablets on secondary lymphedema.MethodsTest one:Study on the evaluation of the rat tail lymphedema model by laser confocal microscopy1.Establishment and grouping of animal models:Wistar rats of 6-8 weeks old were selected to establish rat tail’s lymphedema models by surgical cutting and ligation of caudal lymph vessels.They weredivided into lymphedema group,sham group and normal control group(n=6).2.Evaluation of rat tail lymphedema1)The change of rat tail diameter:The diameter of the rat tail at 20mm from the root was measured with a vernier caliper,and changes in the skin of the rat tail were observedbefore and 1,3,5,7,9,11,14,21,28,35,42 days after surgery.2)The changes of lymphatic reflux in rat tail were detected by radionuclide imaging.3)The changes of rat tails’ skin were detected by laser confocal microscope.4)The pathological changes of rat tails’skin morphology and structure were observed by HE staining.5)The expression of Proxl in rat tails’ skin was observed by immunohistochemistry.Test two:Effect and molecular mechanism of Xiao Zhong Ru Mi Qing tablets on secondary lymphedema1.Establishment and grouping of animal models:Wistar rats aged 6-8 weeks were selected and the method of modeling was the same as in experiment one.They weredivided into treatment group andcontrol group.The treatment group included low,medium and high dose groups.The control group included lymphedema group,sham operation group and normal control group.There were 6 rats in each group.On the postoperative day,the treatment group was given different doses of Xiao Zhong Ru Mi Qing tablets for 28 days to observe its curative effect.2.Evaluation of rat tail lymphedema after intervention1)The change of rat tails’ diameter:The diameter of the rat tail at 2 cm from the root was measured with a vernier caliper,and changes in the skin of the rat tail were observedbefore and 1,3,5,7,9,11,14,21,28 days after surgery.2)The changes of lymphatic reflux in rat tail were detected by radionuclide imaging.3)The changes of rat tail skin were detected by laser confocal microscope.4)The pathological changes of rat tails’ skin were observed by HE staining.5)The expressions of Proxl and VEGF-C in rat tails’ skin were observed by immunohistochemistry.Results Test one1.The successful establishment of rat tail lymphedema modelsIn the lymphedema group,the edema of the tail began to swell on the 1st postoperative day and gradually aggravated.The edema peak reached on the 7th postoperative day andblisters could appear on the skin in severe cases.Then the tail edema gradually subsided.On 42 days after surgery,there were only mild swellingand the wound scar healed,but the skin of the tails were hard and fibrotic.In the sham operation group,the tails of the rat were slightly swollen,and the wound healed on the 7th postoperative day.2.Evaluation results of rat tail lymphedema1)The change rate of rat tails’ diameterOn the 1st postoperative day,the change rate of tails’ diameter in the lymphedema group increased by 0.188 compared with the normal control group,which was statistically significant(P<0.0001).On the 7th postoperative day,the change rate of tails,diameter in the lymphedema group increased by 0.595 compared with the normal control group,with the greatest statistical difference(P<0.0001).The change rate of rat tails’diameter in the sham group was 0.012 higher than that in the normal control group,with no statistical difference(P--0.78).2)Radionuclide imaging of rat tails’ lymphatic refluxOn the 7th postoperative day,2 rats in each group were subjected to rat tails’lymphocyte imaging.In the lymphedema group,a large amount of tracer retention occurred in the subcutaneous areas of the surgical site and distal end,and the proximal lymphatic chain and lymph nodes were not developed.The normal control group and the sham-operated group showed that the lymphatic chain of the tail was well developed,the pelvic bark was round or oval and the shape was clear.3)Laser confocal microscopeThe rat tails’ skin of the lymphedema group(n=5),the sham operation group(n=4)and the normal control group(n=4)were evaluated by confocal microscopy before and 7 days after surgery.Image obtained by using ImageJ software for grayscale measurement.There was no significant difference in the preoperative gray value between the lymphedema group and the normal control group(P=0.4315).On the 7th day after surgery,the gray value of the lymphedema group increased by 25.32 compared with the normal control group.There was a statistically significant difference between the two groups(P<0.0169).There was no significant difference between the sham operation group and the normal control group(P>0.5667).4)HE stainingThere was a marked acute inflammatory change in the skin of the lymphedema group:edema,hyperkeratosis and spongiformity in the epidermis,uneven connection between the epidermis and the dermis,extension of the dermal papilla,and 2-3 fold tissue expansion between the bone and epidermis.Significant changes in cell structure were observed,and a large number of fibroblasts and histiocytes were observed,while a large number of neutrophil infiltrations were observed,and granulation tissue was observed in the vicinity of the center.5)PROX1 immunohistochemistryLymphedema was quantitatively assessed by lymphatic administration of PROX1 immunohistochemical staining.The mean number of lymphatic vessels in the lymphedema group,the normal control group,and the sham operation group were respectively 4.333 ± 0.816,0.833 ± 0.408,and 1.000 ± 1.095.The lymphedema group increased by 3.5 compared with the normal control group,and there was a statistically significant difference between the two groups(P<0.0001).There was no significant difference between the sham operation group and the normal control group(P=0.7).The lymphatic area was measured by ImagJ software.The mean lymphatic area in the lymphedema group,the normal control group,and the sham operation group were respectively0.037 ± 0.010mm2,0.006 ± 0.004mm2 and 0.007 ± 0.006mm2。The lymphedema group increased by 0.031 compared with the normal control group,and there was a statistically significant difference between the two groups(P<0.00001).There was no significant difference between the sham operation group and the normal control group(P=0.70279).Test two1.Changes in appearance of the rat tail after interventionExcept for the normal control group,the tails’ edema began to swell on the first day after surgery and gradually increased in the remaining groups.The lymphedema group was the most swollen,and the high-dose inflammatory sputum group was the lightest.The edema peak reached the 7th day after surgery,and the tails’ edema gradually subsided.There was only mild swelling at 28 days postoperatively and the wound scar healed.2.Evaluation results of rat tails lymphedema after intervention1)The change rate of rat tails’ diameterOn the 1st postoperative day,the change rate of tails’ diameter in the normal control group decreased by 0.21 compared with thelymphedema group,which was statistically significant(P<0.0001).On the 7th postoperative day,the change rate of rat tails’diameter was lower than that of lymphedema group,and the high dose group decreased by 0.17(P<0.0001),which was statistically different.Compared with the lymphedema group,the change rate of the tails’ diameter of the low-dose group decreased by 0.02 On the 28th postoperative day(P=0.1657).With the increase of drug concentration,lymphedema decreased gradually.The change rate of rat tails’diameter decreased by 0.04(P=0.01)in the middle dose group,and it decreased by 0.12(P<0.0001)in the high dose group,which was statistically different.No threshold saturation effect was found.2)Radionuclide imaging of rat tails’ lymphatic refluxOn the 28th postoperative day,more tracer retention occurred in the skin and subcutaneous tissue at the distal end of the rat tail,the rat tails’ lymphatic chain was incomplete and the pelvic lymph nodes were not developed in the lymphedema and low-dose groups.The development of the tails’lymphatic chain was not clear,and the pelvic lymph nodes were partially developed in the middle dose,high dose and diosmin groups.The lymphatic chain of the rat tail was clearly developed,the pelvic lymph nodes were round or oval,and the shape was clear in the normal control group.3)Laser confocal microscopeThe gray value of the low-dose group decreased by 6.5(P=0.2869)compared with that of the lymphedema group,showing no statistical difference.With the increase of drug concentration,the degree of lymphedema was gradually reduced.The gray value of high dose group was decreased by 21.85(P=0.0008)compared with that of the lymphedema group,showing statistical difference.4)HE stainingIn the lymphedema,middle dose and low dose groups,the epidermis had varying degrees of edema,uneven connection between the epidermis and the dermis,dermal papilla extension,and tissue expansion between the bone and epidermis.The cell structure was changed,fibroblasts and histiocytes were observed,neutrophil infiltration was observed,and granulation tissue was observed in the area near the center.In the normal control,diosmin and high dose groups,there was a boundary between the dermis and the epidermis.There was no increased cell between the epidermis and the dermal cells,no enlarged nuclei and hyperkeratosis.5)PROX1 immunohistochemistryThe mean lymphatic area of the low-dose group decreased by 0.06(P=0.0036)compared with that of the lymphedema group,showing statistical difference.With the increase of drug concentration,the degree of lymphedema was gradually reduced.The lymphatic area of high dose group was decreased by 0.1(P<0.0001)compared with that of the lymphedema group,showing statistical difference.6)VEGF-C immunohistochemistryThe gray value of lymphatic vessels stained by VEGF-C in low dose group decreased by 25.3(P<0.0001)compared with that of the lymphedema group,showing statistical difference.With the increase of drug concentration,the degree of lymphedema was gradually reduced.The lymphatic area of high dose group was decreased by 33.9(P<0.0001)compared with that of the lymphedema group,showing statistical difference.Conclusions1.The model of rat tail lymphedema can be successfully constructed by cutting and ligating the rat tail lymphatic vessels.Laser confocal microscopy can qualitatively and quantitatively evaluate the state and extent of lymphedema in animal models.2.Low-dose Xiao Zhong Ru Mi Qingtablets have therapeutic effects on rat tails’lymphedema after surgery.With the increase of drug concentration,the therapeutic effect on secondary lymphedema is gradually increased.High-dose Xiao Zhong Ru Mi Qingtabletshave the most obvious therapeutic effect on secondary lymphedema.3.Xiao Zhong Ru Mi Qingtablets promote the formation and regeneration of lymphatic vessels by increasing the expression of VEGF-C in lymphatic endothelial cells.Part Ⅱ The retrospective study of May-Thurner Syndrome with lower extremity DVTBackground and objectiveMay-Thurner syndrome(MTS),also known as iliac vein compression syndrome(IVCS)or Cockett syndrome,refers to the condition where the left common iliac vein is compressed by the posterior aspect of the right common iliac artery and the anterior aspect of the lumbar vertebra.This condition leads to gradual luminal stenosis or occlusion,reflux in the iliac vein and its distal veins,and a series of clinical symptoms.The anatomical characteristics of MTS increase the risk of deep venous thrombosis(DVT).Embryology and special anatomical structure are the main causes of MTS Both the iliac and umbilical vascular systems are important vascular bundles during fetal development.The venous system of the embryo is developed from the primitive venous system.The primitive venous system includes yolk,umbilical cord and main venous system.With the continuous development of the main venous system,the inferior vena cava and the common iliac veins in the sacral tail during the 6th to 10th pregnancy were successively formed.Unlike iliac vein development,the iliac artery system originated from the 5th lumbar vertebral artery.At the 5th week of fetal development,the bilateral umbilical artery and the internal iliac artery establish a pathway through which the fetus carries the hypoxic blood back to the mother.As the main source of fetal oxygen-rich blood,umbilical cord system is vital to life.Given these characteristics,evolutionary pressure has allowed the umbilical system to take precedence over the development of the iliac vascular system.To ensure that the umbilical vascular system is first connected to the placenta,the iliac vascular system has to make concessions in its morphological and functional development.This may be the underlying cause of the iliac vein being crossed by the iliac artery.Although the right common iliac artery oppresses the left common iliac vein is the direct cause of the formation of the venous spurs,the positional relationship between the right common iliac artery and the lumbar spine may be the real reason for MTS.Other parts of the body also have arteriovenous parallel,such as femoral artery and femoral vein.In the femoral sheath,the pressure of the femoral artery is transferred evenly to the femoral vein.In MTS,however,any pressure from the right common iliac artery may be converted to compression of the left common iliac vein due to its proximity to the lumbar spine.Common clinical manifestations of MTS are deep vein thrombosis(DVT)and chronic venous insufficiency(CVT).Deep vein thrombosis of the lower extremities usually manifests as sudden swelling of the lower extremities,pain in the groin area,thighs,and gastrocnemius muscles,elevated skin temperatureand severe cases of femoral green swelling or femoral white swelling.18-49%of left lower extremity deep vein thrombosis is caused by compression of the left iliac vein.If DVT is not promptly and effectively diagnosed,20%-40%of patients will develop post-thrombotic syndrome(PTS).Chronic venous insufficiency of the lower extremities is caused by long-term lower extremity venous hypertension,and PTS is one of the main causes of lower extremity venous hypertension.Common clinical manifestations are as follows:superficial varicose veins,swelling,pain,hyperpigmentation and ulceration.In patients with clinically symptomatic lower extremity chronic venous insufficiency,up to 55%of patients have stenosis or obstruction of proximal vessels,such as iliac vein compression,of which MTS accounts for approximately 2%to 5%of all causes.Treatment of MTS includes surgical reconstruction and endovascular surgery.Before endovascular surgery was not extensive,surgical reconstruction surgery had made some achievements.The indications are mainly for patients with venous occlusion and failure due to conservative or endovascular treatment.Related surgical procedures include:(1)palma-dale surgery;(2)artificial vascular bypass transplantation;(3)posterior right iliac artery anastomosis;(4)iliac artery suspension;(5)iliac vein loosening and pad decompression.Jost et al.(2001)analyzed the medical records of patients who received ilio-cava or inferior vena cava reconstruction due to benign stenosis or obstruction of the iliac vein from January 1985 to June 1999,indicating that the primary and three-year patency rates of surgical reconstruction were 54%and 62%.Due to the low long-term patency rate and large trauma of surgical reconstruction surgery,endovascular therapy is preferred by patients with MTS.For these patients,percutaneous transluminal angioplasty(PTA)is usually performed for temporary expansion,followed by stent placement.Studies have shown that stent placement has a good effect on the treatment of primary iliac vein occlusion.The primary,assisted primary and secondary patency rates were respectively 79,100and 100%at 6 years.Patients with MTS are usually associated with iliac vein and its distal vein thrombosis.Currently,it is mainly treated by catheter-directed thrombosis(CDT)or percutaneous mechanical thrombectomy(PMT).Some researchers integrated these different endovascular treatment techniques,such as CDT combined with stent placement or PMT combined with stent placement,and achieved good results.Two majorseries from different groups on stenting in postthromboticiliac vein obstruction reportedthat the primary,assisted primary andsecondary patency rates were respectively 57,80 and 86%at 6 years,and they were respectively 83,89 and 93%at 10 years,.The main purposes of this study were to investigate the incidence of MTS in patients with acute lower extremity DVT,the association between MTS and the risk of PE in patients with acute lower-extremity DVT,and the effectiveness of endovascular treatment to MTS.MethodsThis study was approved by the Ethics Committee of the Affiliated Hospital of Jining Medical University.As this study was a retrospective analysis,the patienst informed consent was waived.This study collected clinical data from 146 patients with acute lower extremity DVT diagnosed and treated in the Department of Vascular Surgery,Affiliated Hospital of Jining Medical University from June 2014 to September 2016,and conducted retrospective analysis.The effect indicators include:gender,age,high risk factors,site of disease,D-dimer,lower extremity venous color Doppler,venography,pulmonary artery CTA,iliac vessels CTA,catheter direct thrombolysis,iliac vein stent placement and follow-up results.All of the analyses were performed with the statistical software packages R(http://www.R-proj ect.org,The R Foundation)and EmpowerStats(http://www.empowerstats.com,X&Y Solutions,Inc.,Boston,MA).Results1.The gender difference between DVT with MTS group and DVT without MTS group was statistically significant(P<0.001),while the age difference was not statistically significant(P=0.759).2.There was no statistically significant difference between DVT with MTS group and DVT without MTS group in risk factors(P=0.179).3.Thelocation difference between DVT with MTS group and DVT without MTS group was statistically significant(P<0.001),while the onsettime difference was not statistically significant(P=0.460).4.There was a statistically difference between DVT with MTS group and DVT without MTS group in D-dimer(P=0.042).5.There was a statistically significant difference between DVT with MTS group and DVT without MTS group in thrombosis type(P<0.001).6.There was a statistically significant difference between DVT with MTS group and DVT without MTS group in the diagnosis of MTS with iliac vessels CTA(P<0.001).7.There was a statistically significant difference between DVT with MTS group and DVT without MTS group in pulmonary embolism cases(P=0.006).8.112 patients with DVT who underwent pulmonary CTA were divided into MTS group and non-MTS group,and comparative analysiswas conducted.Univariate regression analysis found thatMTS(OR 0.3,95%CI 0.1-0.7;P=0.008),right lower extremity DVT(OR 3.93,95%CI 1.1-14.6;P=0.0406),D-dimer tertile(OR 2.8,95%CI1.1-7.5;P=0.0343)were common risk factors for pulmonary embolism.Multivariate regression analysis showed that MTS was negatively correlated with PE in the unadjusted model(OR 0.28,95%CI 0.11-0.71;P=0.0075);MTS was negatively correlated with PE in model 1(OR 0.17,95%CI 0.04-0.68;P=0.0125);MTS was negatively correlated with PE in Model 2(OR 0.15,95%CI 0.03-0.71;P=0.016).9.78 patients underwent catheter directed thrombolysis(CDT)in the DVT with MTS group.The follow-up time was 1-24 months.69 cases were followed up and 9 cases were lost to follow-up.The follow-up rate was 88.46%.25 patients who underwent CDT and iliac venous stent placement were followed up for 1-24 months after surgery.25 patients were followed up,0 patients were lost to follow-up,and the follow-up rate was 100%.The stent patency rate of this group was 100%after 1 month,3 months,6 months,1 year and 2 years.There was no significant difference in the effect of CDT group and CDT+Stent group on VDS at different time points using random effects model.ConclusionsIn this study,MTS occurs in female patients and is prone to secondary deep vein thrombosis in the left lower extremity.It is more common in both central and mixed types.The accuracy of iliac vessels CTA in the diagnosis of MTS is high.The incidence of pulmonary embolism in patients with acute lower extremity DVT combined with MTS is low,and MTS is an independent risk factor for pulmonary embolism.Endovascular treatment with MTS lower extremity DVT has a good mid-term effect and high patency rate. |
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