Emotional dysregulation (ED) is now considered as an important symptom of attention deficit hyperactivity disorder (ADHD). It is believed to have a considerable impact on the severity of the disorder, one’s global functioning, and the prognosis. Our research aimed to evaluate and compare ED and cognitive emotional regulation strategies between ADHD and borderline personality disorder (BPD) patients.N = 279 ADHD, N = 70 BPD, N = 60 BPD + ADHD) were assessed with the Emotion Reactivity Scale (ERS), the Cognitive Emotional Regulation Questionnaire (CERQ), The Basic Empathy Scale (BES-A), the Adult ADHD Self-Report Scale (ARSV-v1.1) and the Beck Depression Inventory II (BDI-II). ADHD, BPD and comorbid patients were compared with each other and with samples of controls extracted from already published data.Four hundred six French-speaking outpatients . However, ADHD patients showed a higher use of adaptive cognitive emotional strategies and a lower level of ED than BPD patients. There is increasing evidence of a close link between emotional symptoms and attention deficit hyperactivity disorder (ADHD) –3. SeverStudies have estimated that 34 to 70% of adults suffering from ADHD have difficulties regulating their emotions . These eBesides the usual description of ED in BPD, namely heightened emotional reactivity to environmental stimuli, inadequate cognitive emotion regulation strategies resulting form difficulty identifying, differentiating, and labeling emotions have also been suggested as important components of ED –22. BPD Although several hypotheses explaining ED in ADHD have been suggested, including executive function deficits, social cognitions deficit, impaired emotional perception/processing, lack of empathy and impairment in first and second order theory of mind and poor mentalizing capacities , 23–31, The aim of our study was therefore to compare emotional reactivity, cognitive emotional regulation strategies, and empathy in subjects suffering of ADHD to subjects with BPD, or comorbid ADHD and BPD. We also evaluated the relationship between ADHD symptoms and ED severity.N = 279 ADHD, N = 70 BPD and N = 60 BPD + ADHD) were recruited in a specialized centre for diagnosis and treatment of adults suffering from ADHD and BPD at the University Hospitals of Geneva.Four hundred and six French-speaking outpatients  [Borderline Symptoms Check-List (BSL-23) for BPD patients as previously described [Diagnostic Interview for ADHD in adults (DIVA 2.0), assessing DSM-IV ADHD criteria [Screening Interview for Axis II disorders (SCID-II), assessing DSM-IV BPD criteria [Patients underwent a clinical evaluation at their entry of the program conducted by trained psychiatrists to ascertain the diagnosis of BPD and/or ADHD according to DSM-5 criteria, and to exclude any organic condition and/or comorbid disorders that could better explain the symptoms. With the exception of ADHD and BPD (see below), other comorbidities were assessed clinically and using medical records only, and no structured interviews were used. In addition, subjects were administered the RS v1.1) ; and theescribed . Finallycriteria   when psychostimulants were not yet started.Beck Depression Inventory II (BDI-II) to assess the current level of depression as an indirect tool to evaluate the current distress associated with suffering from ADHD or BPD [All participants completed the D or BPD . BDI-II D or BPD , 38.The study was approved by the ethics committee of University Hospitals of Geneva and all subjects provided informed written consent.Several instruments were used to assess emotion regulation and reactivity, cognitive strategies to avoid worries, and empathy.Emotion Reactivity Scale (ERS) is a self-report questionnaire enquiring about emotional experience on regular basis. It consists of 21 items measuring emotion reactivity, based on three aspects: emotion sensitivity, intensity, and persistence. Each item is rated on a 5-point Likert scale from 0  to 4 (completely like me), with scores ranging from 0 to 40 for emotion sensitivity, 0 to 28 for emotion intensity and 0 to 16 for persistence, and total scores ranging from 0 to 84 [The  0 to 84 . The val 0 to 84 . The scaCognitive Emotional Regulation Questionnaire (CERQ) is a 36-item questionnaire consisting of 9 conceptually different subscales based on different cognitive emotion regulation strategies, overall divided in two main domains: the adaptive strategies domain entails putting into perspective, positive refocusing, positive reappraisal, acceptance, refocus on planning; the non-adaptive strategies domain comprises self-blame, other-blame, rumination, and catastrophizing. Each subscale contains 4 items referring to thoughts after the experience of a threatening or stressful life event. The items are measured on a 5-point Likert scale, ranging from 1  to 5  [The  always) . The val always) . Explora always) , 43.Basic Empathy Scale in Adults (BES-A) is a 20-item self-report scale, focusing on two components of empathy, the cognitive and affective components [The mponents , 45. Eacmponents . The scaADHD patients were compared to a sample of controls extracted from published data that have used the ERS, the CERQ or the BES-A , 47, 48.p < 0.05. Post-hoc tests were performed using pairwise comparisons (Bonferroni correction).All analyses were performed using Stata v14. Univariate comparisons between clinical groups were conducted using the chi square test for qualitative variables  or Fisher’s exact test when assumption of frequencies for chi square test were not met, and a one-way ANOVA for quantitative variables . T-tests were used to compare ADHD patients to a sample of controls extracted from published data that have used the ERS, the CERQ or the BES-A , 47, 48.Questionnaire scores were analyzed with linear regression models, using the diagnostic group as a fixed variable. Models were adjusted for age and gender in the event of significant differences between groups. For all continuous predictors, we examined the assumption of normality of the distribution of residuals with residual plots. These revealed no breach of model assumptions.Secondary analyses were performed to assess the relation between symptomatology and questionnaire scores. ASRS total score (for ADHD and ADHD+BPD groups) or BDI-II scores were added into the model as continuous predictors. Finally, association with ADHD presentation and current depressive episode were also assessed.F = 6.5; df = 2/405; p = 0.002). BPD and BPD + ADHD patients were more likely to be female than ADHD patients . ADHD patients were more likely to have a job than the two other groups  and to have 2 or more children  (Table ADHD patients were older than the other groups (N = 177/279) of ADHD patients did have at least one comorbidity compared to 97.14% (N = 68/70) of BPD patients and 93.33% (N = 56/60) of the ADHD+BPD patients. The use of medication differed among participants. A small fraction of patients took stimulants: 12.90% of ADHD patients, 11.66% of ADHD+BPD patients, and 1.43% of BPD patients. No patients took non-stimulant ADHD medication. The use of other medications  were lower in ADHD patients compared to the two other groups . BDI-II scores were higher in BPD and BPD + ADHD than in ADHD group  (Table Patients with ADHD were less likely to have other comorbid disorders than BPD and ADHD+BPD patients. Taken together 63.44% N = 77/279 ofp < 0.001)  Table . BPD andp < 0.001; Mean: 36.66 standard deviation (SD): 17.52 vs Mean: 47.38 SD: 18.39) [p < 0.001; Mean: 35.02 SD: 17.14) [Our ADHD patients displayed higher ED as measured by the ERS total score compared to a patient population recruited from community and local psychiatric clinics   , and lower scores for non-adaptive strategies  than BPD and ADHD+BPD patients respectively. BPD and BPD + ADHD groups did not differ. The subscale scores showed an overall similar pattern, with exception of the ‘acceptance’ (adaptive) and ‘rumination’ (non-adaptive) subscales where all three groups showed comparable scores. ADHD patients had similar scores as BPD patients in the ‘blaming others’ subscale but were still significantly lower than BPD + ADHD patients  [Our ADHD patients showed poorer cognitive emotions regulation strategies as indicated by higher levels of all non-adaptive cognitive strategies than 611 adults from the general population control group (o 14.19) . Resultsp < 0.05)  Table .p = 0.03 and 32.39 (SD = 3.86) vs 37.62 (SD = 3.46) t = − 10.52; p < 0.001 respectively) but higher affective empathy (39.81 (SD = 7.37) vs. 37.49 (SD = 3.39) t = 2.61; p = 0.01)  and all the subscales . This association was also true when looking at attentional or hyperactive/impulsive symptoms (data not shown).We constructed a model adding ASRSv1.1 score as predictor of the ERS, CERQ, and BES scores, analyzing only ADHD and ADHD+BPD patients. We found that the total ASRSv1.1 score was positively associated with ERS total score  and with each of the non-adaptive subscales (data not shown). It was not associated with adaptive strategies . The significant association was observed for attentional as well as hyperactive/impulsive symptoms.ASRSv1.1 total score was also significantly associated with higher score on non-adaptive cognitive strategies total score , affective subscale score , but not cognitive subscale score . The association was found only with attentional symptoms .ASRSv1.1 was significantly associated with higher empathy total score . The difference between ADHD and BPD patients was no longer significant , however the difference between ADHD and ADHD+BPD remained significant .When adding BDI-II total score as predictor in the model, we found that current level of distress was associated with higher ERS total scores  and higher non-adaptive strategies . When adjusting for BDI-II, the difference between groups was no longer significant .BDI-II total score was also predictive of lower CERQ adaptive strategies .BDI-II was not associated with empathy total score  than those with the attentional presentation. There were no differences between the two presentations of ADHD on CERQ adaptive and non-adaptive subscales  or on BES total score .ADHD patients with the combined presentation had higher ERS total scores  and ADHD+BPD patients . This was true for all the ERS subscales.Thirty-three (47.13%) BPD, 44 (15.77%) ADHD and 25 (41.67%) BPD + ADHD patients had a current major depressive episode. When adding current major depressive episode as a predictor in the model, we found that the difference between groups was still significant, with ADHD patients showing lower ERS total score than BPD patients  and with higher CERQ non-adaptive strategies . When adjusting for current major depressive episode, ADHD patients still showed higher CERQ adaptive strategies than BPD patients , but the difference with ADHD+BPD was no longer significant . Furthermore, with this adjustment the difference between ADHD and BPD for CERQ non-adaptive strategies was no longer significant , but was still significant for the comparison between ADHD and ADHD+BPD  with lower scores in ADHD patients.Current major depressive episode was also associated with lower CERQ adaptive strategies .No association between BDI-II and empathy total score was found .We found that patients with ADHD had higher scores on scales measuring ED compared to community-based psychiatric patients. Previous studies suggested a slow return to emotional baseline, increased intensity and instability of negative emotions, and a predominance of negative emotions in ADHD , 4, 10. We found that ADHD as well as BPD displayed lower empathy than a sample of adolescents not suffering from psychiatric disorder. This slight deficit in empathy might contribute, as in BPD, to poorer emotion regulation capacities , 55. PreConcerning ADHD presentation, the combined type was associated with higher emotional reactivity as previously shown in other studies , 4. No dThe hypothesis of a possible additive effect of both disorders (ADHD + BPD) was not really corroborated by our results since no significant difference between BPD and ADHD+BDP was found. However, this observation might be due to a ceiling effect as the scales we used were designed to assess emotions present in the general population and not in highly emotionally dysregulated samples such as BPD.Part of the differences between groups was related to the level of distress as measured by the BDI-II . Our resOur study has several limitations. Firstly, we did not have a control population and used BPD patients as a comparison to assess ED, cognitive emotion regulation strategies and empathy in ADHD patients. We nevertheless compared our patients to samples from the general population, although not matched on age and gender, derived from others studies and we are quite confident that the assessment of emotion components in our study is a true reflection of the difficulties endured by patients suffering from BPD and ADHD. Secondarily, there was a substantial difference in the size of our three groups, the ADHD group being the largest one. This may have slightly biased our results keeping in mind that ED in ADHD subjects was the main focus of our study, the two other groups being here considered as comparison samples. Thirdly we used self-report measures to assess the different aspects of emotions. However it has previously been shown that self-report measures are reliable if used in combination with interviews, which was the case in our study . FinallyOur findings support the importance of exploring facets of ED as well as cognitive emotion regulation strategies in ADHD. These may, as in BPD, be an important feature of the disorder and be associated with its severity. In general, our results showed that maladaptive cognitive emotion strategies for management of emotion, such as ‘self-blame’, ‘catastrophizing’, ‘other-blame’ and ‘rumination’ are often used by ADHD patients and should be considered in individual and group psychotherapeutic approaches offered to these patients. Furthermore, ADHD patients showed a better use of cognitive emotional regulation strategies and less emotional reactivity than BPD patients. Similarities were also found between these populations, such as the tendency of using ‘blaming others’ and the absence of a difference in cognitive empathy, possibly suggesting a common deficit in perception of self and others in emotionally triggering situations.


The pathological characteristics, treatment strategies and prognosis of ovarian primary primitive neuroectodermal tumor (PNET) were unclear due to the rarity of PNET. All cases treated at Peking Union Medical College Hospital (PUMCH) between 1975 and 2016 and published in the English literature between 1980 to 2017 were reviewed.Finally four cases from PUMCH and 15 cases in the literature were included. The median age was 25 years (range 13–79), and the median diameter of the tumors was 13.4 cm (range 5.0–30.0). The most common initial symptoms were abdominal pain, bloating and a pelvic mass. Diagnosis primarily depended on immunohistochemical and fluorescence in situ hybridization data. Treatment consisted of surgery, various chemotherapy regimens and/or radiotherapy. The 5-year overall survival (OS) and progression-free survival (PFS) rates were 15 and 52%, respectively. For patients with OS and PFS > 12 months, the median ages were 21 years (range 13–35) and 17 years (range 13–35), respectively, while for patients with OS < 12 months and PFS < 12 months, the median ages were 48 years (range 14–79) and 25 years (range 18–79), respectively.No standard therapy for ovarian primary PNET exists, and an individualized strategy is recommended. Young patients seem to have better prognoses. Primitive neuroectodermal tumor (PNET) was first proposed by Hart and Earle  in 1973.All procedures performed in the study involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. All patients or their authorized deputies had presented consents. The Institutional Review Board of PUMCH had approved this study (ID HS-1390).Through an intensive search of the database of electronic medical records from PUMCH from 1975 to 2016, four cases of ovarian primary PNET were retrieved. The pathological results were verified by Dr. Bi. None of these cases has been published before.A 33-year-old woman presented with complaints of progressive backache and lower limb aching 7 months after laparoscopic right ovarian cystectomy, the pathology of which proved to be mature teratoma. Sonographic examination revealed a mass in the right iliac. Single-photon emission computed tomography showed a malignant metastatic lesion in the hip bone, and pathological review of the first surgical section suggested the possibility of an immature teratoma. Despite 11 courses of cisplatin-based combination chemotherapy and two courses of radiotherapy, the disease progressed during treatment, with multiple metastases in the right iliac fossa, iliac, sacrum and liver on imaging. Biopsy of the liver lesion proved it to be a metastasis. Subsequent palliative radiotherapy did not relieve her pain. Palliative surgery was performed with partial hepatectomy of the left lobe of the liver and resections of the omentum and metastatic lesions. The final pathological exam showed metastatic PNET from the ovaries. The patient declined further treatment and died 2 years after the bone metastasis.cluster of differentiation (CD)-99, neuron-specific enolase (NSE), vimentin and smooth muscle actin (SMA) but was negative for synaptophysin, alpha fetoprotein (AFP), epithelial membrane antigen (EMA), calretinin, a-inhibin, Melan A, and desmin. The patient refused further adjuvant chemotherapy and was lost to follow-up.A 59-year-old woman had undergone exploratory laparotomy and pelvic mass resection with elevated CA-125 (105 U/ml). Pathology suggested a lipoid cell tumor. The pelvic mass recurred 3 months after the primary surgery, and a second laparotomy revealed multiple solid tumors in the pelvic and abdominal cavities. Tumor cytoreductive surgery was performed with total hysterectomy, bilateral salpingo-oophorectomy, appendectomy, infracolic omental excision, and metastatic lesion resection, with a residual tumor less than 1 cm. The final pathological diagnosis was primary ovarian PNET. Immunohistochemical (IHC) staining showed a positive reaction for A 67-year-old woman presented with abdominal distention and changes in bowel habits that had occurred over the previous 4 months. She had undergone transabdominal hysterectomy and left salpingo-oophorectomy for some benign diseases 2 years prior. Colonoscopy and colonography indicated sigmoid colon adhesions and stenosis Fig. . The fin2) plus carboplatin (AUC 6). Contrast-enhanced computed tomography of the abdomen and pelvis revealed tumor recurrence during the course of chemotherapy. The patient died of disease progression; her overall survival (OS) time was 5 months.A 14-year-old girl presented with abdominal distention for 2 weeks, which worsened over 1 week. Sonographic examination revealed a 30 cm × 20 cm pelvic mass with the possibility of ovarian origin. Abdominal and pelvic magnetic resonance imaging (MRI) revealed a large mass in the abdominal and pelvic cavity, which showed a low and intermediate T1 signal intensity and a high, heterogeneous T2 signal intensity, with good enhancement  by private communication. SPSS 23.0  was applied for statistical analysis. Life tables were used to determine 1-, 3- and 5-year OS rates.The clinicopathological data and results of IHC and molecular genetics analysis are shown in Tables For all 19 cases, the median age of the patients was 25 years (range 13–79), and most of them  were premenopausal girls and women. The clinical manifestations were abdominal pain and/or distention , a pelvic/abdominal mass , irregular menstruation , and progressive backache and lower limb aching . The lesions were all unilateral with a median diameter of 13.4 cm (range 5.0–30.0). Of the 16 cases with exact FIGO stages, 3 (18.8%), 12 (75.0%) and 1 (6.3%) were FIGO stage I, III and IV, respectively. Except for one patient with bone metastasis, 18 underwent primary surgeries: 13 patients received tumor cytoreductive surgery or comprehensive staging, and 5 patients received non-comprehensive staging surgery. Overall, four patients accepted fertility-sparing surgeries.Eleven of the 19 cases (57.9%) were treated with different types of chemotherapy regimens: 4 patients received chemotherapy prescribed for germ cell tumors, such as PEB , VIP , VID  or VIA ; 3 accepted the TC regimen ; one underwent the EFT-2001 strategy prescribed for Ewing sarcoma family tumor (ESFT); one was prescribed melphalan, actinomycin and cyclophosphamide; and 2 were treated with unknown chemotherapy regimens. Three patients received a combination of chemotherapy and radiotherapy after surgery, and only one patient received simple adjuvant radiotherapy after surgery.IHC and/or molecular genetic analyses were performed for 13 of the 19 cases (68.4%). The most common positive IHC markers were CD99 , NSE  and FLI-1 (friend leukemia integration 1 transcription factor) . Molecular genetic analyses were conducted for 3 cases: two showed the chromosomal translocation t ; for the other case, EWS/FLI-1 chimeric mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR).After primary treatment, definite survival data were available for 18 of the patients. Eight of 18 (44.4%) experienced progressive disease and died, whereas 10 patients achieved complete remission (CR). However, 5 patients experienced tumor relapse , and 5 patients remained in CR. The median OS rate was 18.0 months , and the 1-, 3- and 5-year OS rates were 65, 31 and 15%, respectively. For 9 patients achieving CR after primary treatment, the median PFS was 18.0 months (range 3.0–84.0), and the 1-, 3- and 5-year PFS rates were 78, 52 and 52%, respectively. For patients with OS > 12 months and PFS > 12 months, the median ages were 21 years (range 13–35) and 17 years (range 13–35), respectively; for patients with OS < 12 months and PFS < 12 months, the median ages were 48 years (range 14–79) and 25 years (range 18–79), respectively.Of the four patients who selected fertility-sparing surgeries, one 25-year-old woman with stage IC1 disease had a recurrence 3 months after primary treatment but achieved CR after chemotherapy. She experienced two spontaneous pregnancies and delivered twice by cesarean section . AnotherPNET typically affects young people, and the most common age range is 10–19 years, followed by 20–29 years. Cases are rarely found in women over the age of 40. In our analysis, the median age of these patients was 25 years, which is far younger than the median age of epithelial ovarian tumors. Imaging studies suggest that these tumors are often solid, bulky pelvic masses, which are not specific for diagnosis. Although two of the four patients treated at PUMCH had elevated serum CA-125, there is no definitive tumor marker for PNET. Furthermore, the non-specific clinical presentations and imaging of ovarian primary PNET make early diagnosis and differential diagnosis very challenging.IHC analysis is critical for pathological diagnosis. Sen et al. suggested that the standard diagnostic methods depend on IHC staining of markers, such as CD99 and other neural markers including NSE, synaptophysin, vimentin, S-100, NF and PZ macroglobulin . AdditioSome authors have proposed that the diagnosis of primary ovarian PNET should be based on the presence of the EWSR1 fusion transcript and/or gene translocation detected by RT-PCR or FISH, especially when the tumor is located in an unusual site or when a diagnosis that is only based on morphology and CD99 is difficult . ApproxiNo standard therapy exists for ovarian primary PNET, and surgery is still the first choice of treatment. In our analysis, two of the four patients who accepted fertility-sparing surgeries experienced relapse, whereas three patients ultimately achieved CR. One patient had two full-term deliveries. Therefore, fertility-preserving comprehensive staging surgery may be appropriate for young stage I patients, even though the risk of recurrence is high.Due to the heterogeneity of treatment, achieving consensus regarding the regimens and courses of postoperative adjuvant chemotherapy is difficult. Initially, the chemotherapy regimen for ovarian primary PNET patients was based on the regimen for ovarian immature teratoma. Later, this tumor was regarded as a type of ovarian germ cell tumor. One 13-year-old girl diagnosed with primary ovarian PNET of at least FIGO stage IIIC received a high dose of chemotherapy and a peripheral blood progenitor cell transfusion, and there were no signs of tumor recurrence at 18 months after the completion of treatment . AccordiOverall, the effectiveness of postoperative adjuvant radiotherapy is not clear. Among the 19 cases evaluated, only one patient (5.3%) received simple adjuvant radiotherapy after surgery, and three (15.8%) received a combination of chemotherapy and radiotherapy after surgery. However, these patients had poor prognoses, with survival times ranging from 2 to 17 months. ESFT, however, is to some degree sensitive to radiotherapy, and adjuvant radiotherapy has been reported to help decrease the rate of local recurrence . For a tTargeted therapy of genetic mutations may provide new treatment options. A recent study has shown that EWS-FLI1 antagonists can inhibit the proliferation of ESFT cells in vitro ; consequThe risk factors that determine the prognoses of ovarian primary PNET patients are not clear. Due to the limited sample size, risk factors for survival could not be determined in the present study. Some authors have suggested that the prognosis is poor for those diagnosed with distant metastasis , 21. OurThe most important limitation of this study was the nature of the pooled analysis, which limited the sample size, caused treatment heterogeneity, and produced a bias regarding survival data. However, the rarity of ovarian primary PNET is associated with these shortcomings. In addition, identification of EWSR1 fusion transcripts and/or gene translocation detection by FISH are/is important for the differential diagnosis of ovarian PNET . NonethePrimary ovarian PNET is extremely rare, and the chief complaints of these patients are abdominal pain, abdominal distension and pelvic masses. The age of peak occurrence in this study was 10 to 19 years. Additionally, the gold standard diagnosis of this tumor was based on microscopic, IHC, and FISH data. No standard therapy exists to date, and most treatment options are empirical. Furthermore, the results of this study suggest that gynecological oncologists should perform comprehensive staging surgery or tumor cytoreductive surgery at the beginning, followed by postoperative adjuvant radiotherapy and combined chemotherapy . Although the prognosis of patients with early-stage treatment was acceptable, the therapeutic effect for patients with metastatic disease at diagnosis remains poor. Therefore, exploration of the optimal chemotherapy regimen for ovarian primary PNET continues to be necessary.


Morus multicaulis) branch bark powder have showed effective hypoglycemic activity in our previous research. This study aims to explore the mechanism of protect effect on diabetes mice of mulberry branch bark as food supplement based on non-targeted GC-MS metabolomics’ platform.Mulberry  for five weeks to study its therapeutic effect. The metabolite feature of diabetes model and treatment group mice were characterized using a gas chromatography-mass spectrometry-based metabolomics, complemented with the biochemical evaluation, histological inspection, immunohistochemistry observations and enzyme protein detection.A panel of endogenous metabolites were revealed that are relevant to disturbed metabolic processes among groups. The serum metabolic profiles were significantly different between the model group and treatment group. The manner of MBBP treatment showed to be significantly dose dependent and 20% MBBP treatment gain a relatively greater benefit than others. The metabolic disorders in model group include enhanced activation of the sorbitol pathway and galactose metabolite, increased activities of gluconeogenesis, fatty acid oxidation, proteins catabolism and attenuated activities of pentose phosphate pathway, glycolysis and aerobic oxidation pathways, internal synthesis of cholesterol, inositol production. MBBP treatment ameliorate these abnormal metabolize as revealed by differential metabolites comparing with that of model mice, such as decreasing the accumulation of ketone body, enhancing NADPH biosynthesis, partially reversing oxidative stress and energy metabolism disturbance.Mulberry branch bark had a re-balancing effect on the disturbed metabolic pathways in the diabetic mice. Based on the metabolic pathways network, oral administration of MBBP could ameliorate the hyperglycemia and hyperlipidemia symptoms in a global scale and restore the abnormal metabolic state to a near normal level in a dose dependent pattern.The online version of this article (10.1186/s12986-019-0335-x) contains supplementary material, which is available to authorized users. Diabetes mellitus, characterized by elevated blood glucose levels, is a common chromic metabolic disease affecting millions individuals worldwide . AccordiThere are two types of diabetes, namely type 1 and type 2. Type 1 diabetes happens when the pancreas does not produce enough insulin to function properly . Type 2 Morus alba) is a well-known medicinal plant and mulberry derived products in the form of mulberry fruit juice, mulberry leaves tea, root bark extracts and capsules are now commercially available as functional foods and dietary supplements [Morus multicaulis powder have showed effective hypoglycemic activity in mice which mainly contain prenylated flavonoids, Diels-Alder type adducts, stilbenes and polysaccharides [Mulberry  was purchased from Sigma-Aldrich Fine Chemicals, USA. All other chemicals and solvents were of analytical or HPLC grade. Methanol, acetonitrile, pyridine, n-hexane, methoxylamine hydrochloride(97%), BSTFA with 1% TMCS were purchased from CNW Technologies GmbH . L-2-chlorophenylalanine was from Shanghai Hengchuang Bio-technology Co., Ltd. .Morus multicaulis L.  Loud.) were collected from the mulberry garden of Soochow University, Suzhou, China, in November 2016. The bark, which was peeled from the mulberry branches, was dried at 100 °C for 2 h, pulverized into powder twice, and passed through a 100-mesh sieve. The powder were weighted and mixed with standard diet to get 5, 10, 20% MBBP diets for mice.The branches of the mulberry cultivar HuSang 32 from Male C57BL/6 mice (18 ± 2 g) were obtained and housed in SPF Animal laboratory of the Laboratory Animal Research Center, Soochow University . The mice were maintained under controlled conditions . All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Soochow University .n = 50): (1) normal diet supplied group , (2) high-fat diet and STZ injection group (Model), (3) 5% MBBP treated group (5%), (4) 10% MBBP treated group (10%) (5) 20% MBBP treated group (20%). The mice were then fed their individual diets for another five weeks.The mice were fed with a normal diet and a high fat diet containing 59% basic fodder, 20% sugar, 18% lard oil, and 3% egg yolk for five weeks. Then diabetes model was induced by intraperitoneal injection of freshly prepared citrate buffer (pH 4.5) solution of STZ (80 mg/kg) in high fat diet mice. Mice were randomly divided into five groups  injection (2 g of glucose/kg body mass). Subsequently, the blood glucose level is measured with a portable OneTouch glucometer  Ltd., China) at 15, 30, 60, 90 and 150 min after glucose injection.The pancreas was quickly removed from the mice and fixed with 10% formalin solution. Tissue dehydration was performed with increasing concentrations of acetone. The sample was then cleaned with xylene and embedded in paraffin, and 3-μm thick slices were cut on an HM340E microtome, stained with hematoxylin and eosin (H&E) and insulin immunohistochemical staining, and the histocyte structure and expression of insulin protein were imaged under optical microscope (200×).The serum insulin levels were determined by Insulin Assay Kit . The levels of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), malondialdehyde (MDA) in serum were measured using commercially available kits . The total cholesterol (CHOL), triglyceride (TG), high density lipoprotein cholesterol (HDLC) levels, low density lipoprotein cholesterol (LDLC), aspartate amino-transferase (AST), Uric acid (UA), Creatinine (Crea), Cholinesterase (CHE) levels in serum were assessed using a BS-800 Chemistry Analyzer .v/v) was added, and the mixtures were vortexed for 1 min, ultrasonicated at ambient temperature (25 °C to 28 °C) for 5 min, stored at − 20 °C for 10 min. The samples were centrifuged at 12000 rpm for 10 min at 4 °C. QC sample was prepared by mixing aliquots of the all samples to be a pooled sample. An aliquot of the 150 μL supernatant was transferred to a glass sampling vial for vacuum-dry at room temperature. And 80 μL of 15 mg/mL methoxylamine hydrochloride in pyridine was subsequently added. The resultant mixture was vortexed vigorously for 2 min and incubated at 37 °C for 90 min. 80 μL of BSTFA (with 1% TMCS) and 20 μL n-hexane was added into the mixture, which was vortexed vigorously for 2 min and then derivatized at 70 °C for 60 min. The samples were placed at ambient temperature for 30 min before GC-MS analysis.Serum samples stored at − 80 °C were thawed at room temperature. 50 μL of sample was added to a 1.5 mL eppendorf tube with 10 μL of 2-chloro-l-phenylalanine (0.3 mg/mL) dissolved in methanol as internal standard, and the tube was vortexed for 10 s. Subsequently, 150 μL of ice-cold mixture of methanol and acetonitrile  coupled with a mass selective detector . A DB-5MS fused-silica capillary column  was utilized to separate the derivatives. The analysis was performed under the conditions described in following: Helium (> 99.999%) was used as the carrier gas at a constant flow rate of 1 mL/min through the column. The injector temperature was maintained at 260 °C. Injection volume was 2 μL by splitless mode, and the solvent delay time was set to 5 min. The initial oven temperature was 50 °C, ramped to 125 °C at a rate of 15 °C/min, to 210 °C at a rate of 5 °C/min, to 270 °C at a rate of 10 °C/min, to 305 °C at a rate of 20 °C/min, and finally held at 305 °C for 5 min. The temperature of MS quadrupole and electron impact ion source was set to 150 °C and 230 °C, respectively. The collision energy was 70 eV. Mass spectrometric data was acquired in a full-scan mode (m/z 50–450).The QCs were injected at regular intervals (every 10 samples) throughout the analytical run to provide a set of data from which repeatability could be assessed.Raw GC-MS data were converted to. CDF format by Agilent Chem Station software and imported into ChromaTOF . The pretreatment process included the data baseline filtering and calibration of the baseline, peak alignment, deconvolution analysis, peak identification and integration of the peak area. Metabolites were qualitatived by the NIST and Fiehn database, which is lined to the ChromaTOF software. After internal standards and any known pseudo positive peaks, such as peaks caused by noise, column bleed and BSTFA derivatization procedure, were removed from the data set, the CSV file was obtained. The resulting data matrix which consists of sample code, peaks’ name, retention time, quant mass and peak intensities was further processed using Microsoft Execl 2010. Total chromatographic area normalization was applied to reduce the deviation between each sample. Finally, the normalized dataset was imported into SIMCA 14  for multivariate statistically analysis.Protein was isolated from hepatic tissue using lysis buffer enrich with phosphatase and protease inhibitors  and quantitated using Bradford protein quantification kit . Equal amounts of protein were loaded and separated on sodium dodecyl sulphate polyacrylamide gels and then incubated with specific antibodies. The following primary antibodies were used: GLK (bs-1796R), PEPCK1 (bs-4972R), PFK1 (bs-3982R), CPT1A (bs-2047R) from Bioss Inc. ; G6P  from Sant Cruz biotecnology ; HMGCR (BM4908) from Boster biological technology . Images were captured with G: BOX chemiXR5 imaging System  and analyzed with Gel-Pro32 software.t-test or ANOVA, and results with a P-value < 0.05 were considered to be statistically significant.The data were analyzed using Origin 9.1 software. The data of model groups was compared with that of normal group and the data of MBBP treat group was compared with that of model groups. Statistical significance was performed using unpaired 2-tailed Student’s http://www.metaboanalyst.ca).Multivariable analysis including principal component analysis (PCA) and orthogonal projection to latent structures-discriminant analysis (OPLS-DA) analysis was performed by Simca 14. Pathway analysis was performed with Metaboanalyst 4.0  in the mice of model group. Uric acid (UA) and Creatinine (Crea), as biochemical index of kidney dysfunction [Aspartate amino-transferase (AST), which were biochemical markers for assessing hepatic injury, was significantly elevated  = 0.546, which points to a stable model. Q2 is low, 0.226, indicating that although patterns are found they are not conclusive. Each point represent one sample in the score plot. The quality control (QC) samples in the PCA score plot overlapped indicate samples behaved stable for the duration of the run. There are no outliers in the score and DModX plot shows no deviating observations. Normal group and Model group were completely separated and far away. The MBBP treat groups close to normal group with a dose dependent pattern. This demonstrate severe metabolic disturbance induced by high fat diet and STZ could be ameliorated after MBBP feed in mice.There is a lot of GCMS data and it is hard find any trends or outliers by analyzing the raw data. Therefore PCA analysis were done after unit variance scaling Fig. . The PCAn = 200) were presented in Fig. Orthogonal partial least squares discriminant analysis (OPLS-DA) was subsequently employed to explore intergroup difference in Fig. p values. In general, a threshold of VIP > 1 was consider as the relevant metabolites for interpreting the discrimination, fold change value ≥1.5 or ≤ 0.667 were supposed to be obvious up-regulated or down-regulated in concentration, and Student’s t test p value set to 0.05 (p < 0.05) was believed to be a significant difference. Based on the strategy, a total of 46 discriminating metabolites resulting from model group were selected when compared with normal group  value, fold change values of metabolites and the corresponding The significant metabolites include sugars , sugar alcohols , organic acids , fatty acid , amino acids  and others . Xenobiotics originated from plant and syntheitic drugs such as trehalose, guaiacol, quinic acid, phloroglucinol, beta-Sitosterol, Atrazine-2-hydroxy, 1-Aminocyclopropanecarboxylic acid, albendazole were excluded from all metabolite list.The SUS plot is used when there is a three group investigation. Two OPLS-DA models construct a SUS plot Fig. . Each poThe details of this trend were improvably visualized in heat map by 30 metabolites in Fig. http://www.metaboanalyst.ca) to explore biologically meaningful metabolic patterns and the most impacted pathways. Compared to normal mice, the significant perturbed metabolic pathways in serum of high fat diet and STZ injection were determined  and SMPDB (http://smpdb.ca/), five meaningful metabolic pathways were summarized, including carbohydrate metabolism, lipid metabolism, protein metabolism, energy metabolism, oxidative stress. Based on the above pathway analysis, a map of the diabetes related metabolic pathways was constructed  the formation of NADPH for synthesis of fatty acids and steroids and counter the damaging effects of oxygen radicals. (2) the synthesis of ribose for nucleotide and nucleic acid formation . Inhibit+ and increased NADH/NAD+ result in redox imbalance and oxidative stress through reduced level of glutathione and increased reactive oxygen species concentrations [Hyperglycemia increases glucose metabolism via the sorbitol pathway. Intracellular glucose is predominantly metabolized by phosphorylation and subsequent glycolysis, but when increased, some glucose is converted to sorbitol . Sorbitotrations . The incIn the molecular pathway of inositol, after insulin links with the cell, inositol-second messengers are produced . In modeIncreased concentration of palmitoleic acid, lauric acid and glycerol phosphate in model group indicate rapid mobilization of triglycerides from adipose tissue leading to increased levels of plasma free fatty acids. The free fatty acids are taken up by numerous tissues and metabolized to provide energy . The actThe amount of cholesterol carried by LDL-cholesterol (LDL-C) were observed to be high in model group mice by biochemistry assay. The high level of cholesterol in serum was mainly from the high fat diet feed in model mice. The declined level of squalene and zymosterol, as cholesterol synthesis intermediates , showed Insulin deficiency or tolerance has lead complex biochemical changes in glucose metabolism and lipid metabolism, it still be the key for protein metabolism. Insulin has a global effect on protein metabolism, increasing the rate of protein synthesis and decreasing the rate of protein degradation . Thus, t2 and ATP. This may be due to ATP deficit via depletion of TCA cycle in mitochondrion [During the process of catabolism of protein, ammonia was transferred in muscle and other various tissues, the level of glutamine and alanine increased in model group for they carry ammonia to liver in circulatory system . In livehondrion  or activhondrion .In MBBP treatment group, we found the process of protein degradation has been reversed and urea cycle defects disappeared based on the backward changing data in MBBP group.+ and high NADH/NAD+ ratio in mitochondria [The most notable change in the metabolic profile of diabetes model is reduced glucose oxidative metabolism and increased fatty acids β-oxidation metabolism. The main fuel of the body inclined to be fatty acid instead of glucose as fuel to produce energy in model mice. Oxidation of fatty acid lead to excess production of NADH and Hchondria . This slchondria . And redchondria . In addiThe increase of creatinine in serum were observed in model group which was degraded from phosphocreatine. This suggested more high-energy phosphate bond in phosphocreatine has been transferred to form ATP for energy demand and confirmed energy deficit state in mode group mice.In MBBP treatment group, the TCA cycle come to rescue, the balance of energy supply inclined to be normal judging by the level of malic acid, succinic acid, fumaric acid, citric acid and creatinine in MBBP treatment groups.2.- is generated from the NADPH oxidase reaction or mitochondrial electron transport chain (ETC) are dismutated by the mitochondrial Mn-SOD or cytosolic Cu-Zn-denpendent SOD [2O2, if the latter is not reduced by catalase (CAT) [With the reduction of NADPH supplied in pentose phosphate pathway, organism would fail to meet their anabolic demands and combat oxidative stress. Besides, the sorbitol production in polyol pathway intensified the insufficiency of NADPH and up-regulation of the NADH oxidase complex in model group. NADPH is crucial for the regeneration of GSH via the glutathione disulphide (GSSG) reductase, following Odent SOD . NADPH ise (CAT) , 51. By se (CAT) .Several biochemical parameters which were measured to reflect the status of oxidative stress confirmed the oxidative imbalance in model group mice. Comparing with the normal group, the level of malonaldehyde (MDA), as a lipid peroxidation product, increased in model group; the activity of antioxidant enzyme Glutathione peroxidase (GSH-Px) and catalase (CAT) also declined in model group; the activity of total-SOD were found a little higher in model group. These demonstrated reactive oxygen species (ROS) generation and oxidative stress occurring in the model mice. MBBP treatment could ameliorate these abnormal parameters as revealed by decreased concentration of MDA and increased activity of GSH-Px and CAT, when compared with model mice.Diabetes is a chronic disease with serious metabolic disturbances in carbohydrate, protein  and fat In addition, there are sufficient data to correlate the magnitude of the abnormalities in glucose metabolism with those of fat (or protein) metabolism. In model group, the ketone bodies increase to compensate for impaired glucose metabolism as energy providers for tissues; increased fatty acid oxidation compensates glucose oxidation defects to produce ATP. However, there is no alternative pathway for pentose phosphate pathway to produce NADPH, an important reducing power in cytoplasm, and this results in an imbalance in redox state. These abnormal metabolism is substantially reversed in MBBP treatment group. This suggested the mulberry branch bark play a therapeutic role through multiple targets or key targets. The hypoglycemic effect of MBBP were related to their regulation of oxidative, increase of inositol, amelioration of disordered metabolism which were influenced by insulin or not. It open up avenue of investigation of signaling pathways of these process for more detailed studies of the inherent molecular mechanism and targets.The observed beneficial effects of mulberry branch bark are postulated to be brought about by the activity of various identified specific constituents  and some nonspecific compounds. Its nutrient composition  may also play a role and be indispensably as therapeutic nutrition. Because this is in line with the principle of balanced diet in the treatment of diabetes mellitus. Together with the results in this study, mulberry branch bark have shown the potential of treating diabetes mellitus as a supplementary food or raw material in functional food. However, when oral administration of mulberry branch bark powder are applied in people with diabetes, the dosage of other chemotherapeutic medicine may be decreased. The exact dosage of mulberry branch bark when combined with other medicine or not in diabetic people needs to be further studied.As a research based on untargeted metabolomics which provide insights into fundamental biological processes of diabetic mice fed with mulberry branch bark in this study, the relevant information have been extracted from the large-scale data in a holistic manner. However, untargeted metabolomics casts a much more indiscriminate net, considering all metabolite information in an experiment to examine multiple sets of profiles without bias . For eacAdditional file 1:Figure S1. The effect of high fat diet, STZ injection and MBBP treatment on the body weight of mice. Figure S2. Total ion current (TIC) chromatogram of serum of mice of Normal group (a), Model group (b), 5% MBBP treat group (c), 10% MBBP treat group (d), 20% MBBP treat group (e) obtained from GC-MS analysis. (DOCX 589 kb)Additional file 2:Table S1. Peaks identified in TIC chromatogram of serum from five groups after peak alignment. Table S2. Differential metabolites in response to 5% MBBP treat group vs. model group. Table S3. Differential metabolites in response to 10% MBBP treat group vs. model group. Table S4. Differential metabolites in response to 20% MBBP treat group vs. model group. (DOCX 57 kb)


Idiopathic membranous nephropathy (iMN) is the major cause of end-stage renal disease (ESRD). Recent guidelines suggest limiting immunosuppressants only to high risk patients for ESRD. The present study is aimed at identifying new predictors for the renal outcome of iMN patients. We conducted a retrospective cohort study covering a period from January 2003 to December 2013. We enrolled participants who had received their first renal biopsy at our medical center in Taiwan with the diagnosis of iMN. Clinical, pathological and laboratory data were collected from medical records. Analyses with Mann–Whitney U test was used for continuous variables and Chi-square test for categorical variables. The Kaplan-Meier curve was used for the analyses of patient survival and renal survival. Youden index was used for evaluating the performance of a dichotomous diagnostic test for renal and patient outcomes. Cox proportional hazard regression was used to determine factors affecting renal survival.A total of 99 patients with renal biopsy-confirmed idiopathic iMNs were enrolled. C3 level ≤114 mg/dl predicted patient outcome (p < 0.001) with good predictive power (AUC = 0.736). The univariate analysis showed that risk factors for poor renal outcome were older age , high BUN , poor baseline renal function , C3 ≤ 93.4 mg/dl , NLR > 3.34  and PLR > 14.48 . Stage of iMN did not fully account for the risk of ESRD. This is the first evidence that serum levels of C3 ≤ 93.4 mg/dl predicted poor renal outcomes with good predictive power. Easily obtained markers, NLR > 3.34 also predicted poor renal outcomes. Its common pathological finding is the presence of subepithelial deposition over glomerular basement membrane (GBM). The outcome is heterogeneous, spreading from a self-limited status to an end-stage renal disease (ESRD). In addition, the mainstream treatment for iMN is immunosuppressants. However, there is no consensus among medical professionals regarding the timing, types and doses of immunosuppressants. Recently, published in Kidney Disease Improving Global Outcomes (KDIGO) have suggested alkylating agents to be used only in patients with the highest risk for kidney failure in order to avoid unnecessary complications, like infections2. Therefore, the identification of the risk factors for renal outcome of iMN is needed.Idiopathic membranous nephropathy (iMN) is the major cause of nephrotic syndrome in adults3. It is based on three parameters: (a) time-averaged proteinuria (highest sustained 6-month period of proteinuria), (b) creatinine clearance (CCr) at diagnosis, and (c) the slope of CCr over 6 months. There is however no long-term follow-up study published for this model. In addition, no validation of this model has been made to include other other non-western races. Increasingly more markers 6 have been developed to strengthen the predictive power. Furthermore, the knowledge of molecular pathogenesis of iMN is actually rare. Recently, differentially expressed microRNAs and their downstream network may be involved in iMN pathogenesis and could be considered as potential diagnostic biomarkers of MGN7. Until now, none of the above approaches is perfect, and the outcome of iMN remains poorly predicted. Therefore, other factors responsible for predicting the outcome of iMN needed to be discovered. Here, we conducted a long-term retrospective study on such patients and analyzed the risk factors for their renal outcomes.Toronto Risk Score was proposed in 1997 to predict the renal outcome in patients of iMNWe conducted a retrospective cohort study covering the period from January 2003 to December 2013. We enrolled participants of ages >20 years and who had received a first renal biopsy for their diagnoses of iMN in our medical center. Graft renal biopsies were excluded. In this medical center, we had the largest population of renal biopsies (>8000 times over 30 years) in Taiwan. We collected patients with the diagnosis of iMN, instead of secondary MN. All recruited patients also did not have other autoimmune disease and infection disease. This study was approved by Ethics Committee of Taichung Veterans General Hospital, IRB number: CE15125B. All methods were carried out in accordance with relevant guidelines and regulations and informed consent was obtained from all subjects.8) (ml/min.1.732 m2), bloodwhite blood cell (WBC) (/cumm), blood red blood cell (RBC) (/cumm), hemoglobin (g/dl), neutral and lymphocyte ratio (%), platelet count (/cumm), uric acid (mg/dl), sodium (meq/L), potassium (meq/l), calcium (mg/dl), phosphaste (mg/dl), magnesium (mg/dl), albumin (g/dl), total protein (g/dl), glutamate oxaloacetate transaminase (GOT) (U/L), glutamate-pyruvate transaminase (GPT) (U/L), total cholesterol (mg/dl), triglyceride (mg/dl), low-density lipoprotein (LDL) (mg/dl), high-density lipoprotein (HDL) (mg/dl), fasting and postprandial blood sugar (mg/dl), glycated hemoglobin (%). We calculated NLR as the ratio of neutrophil to lymphocyte counts, and PLT as the platelet to lymphocele counts. Markers for chronic infection or inflammation are as follows: hepatitis B status, hepatitis C status, Anti-Nuclear Ab (ANA), anti-double stranded DNA (anti-dsDNA), Anti-neutrophil cytoplasmic antibodies (ANCAs), proteinase 3 (PR3) and myeloperoxidase (MPO). Urinary samples were tested for spot urine protein (mg/dl), spot urine creatinine (mg/dl), spot urine albumin (mg/dl) and 24-hour proteinuria (g/day).All data were obtained through our reviewing of medical records. At the time of renal biopsy, baseline data were collected. They included the following: gender, age, body height (cm), body weight (kg), and systolic or diastolic blood pressure (SBP and DBP). From blood samples the following data were also collected: blood urea nitrogen (BUN) (mg/dl), serum creatinine (mg/dl), estimated glomerular filtration rate . In the latter case, dialysis or kidney transplantation was performed according to our management guidelines.All pathological samples were reviewed by the same pathologist. Participants had the diagnosis of iMN based on stages of iMN (Ehrenreich- Churg)Chi-square test for the categorical variables. Kaplan-Meier curve was used to represent patient survival and renal survival. Youden index was used to evaluate the performance of a dichotomous diagnostic test for renal and patient outcomes. Cox proportional hazard regression was used to determine factors affecting renal survivals (univariate and multivariate cox models). Values of p < 0.05 was considered statistically significant. All statistical procedures were performed using the SPSS statistical software package, version 17.0 .For continuous variables, data were expressed as mean ± SD, and for categorical variables, as numbers (percentages). Mann–Whitney U test was used to analyze the continuous variables and nd or 3rd renal biopsies, as well as secondary iMN . Finally, 99 patients with renal biopsy-confirmed idiopathic iMNs were analyzed in this study. The whole duration of follow-up was 11 years (January 2003 to December 2013). The mean duration of follow-up was 6.2 ± 3.2 years. Totally, 79 patients reached endpoint (20 loss of followed or dead). Based on stages of iMN (Ehrenreich- Churg)9, we separated 99 participants into two subgroups: subgroup 1 (stage 1–2)10 and subgroup 2 (stage 3–4)10, as shown in Table 2). Metabolic syndrome was less frequent: i.e. 132.0 mmHg of SBP, 171.18 mg/dl of LDL, and 5.90% of HbA1c. Their proteinuria was severe, 7.99 g/day of daily urine protein. The participants in the more severe stages of iMN had lower percentages of neutrophils  and lower levels of IgM . Patients in stage 3–4 iMN had higher serum creatinine levels (1.10 ± 0.73 vs. 2.05 ± 2.30) but the difference with stage 1–2 was not significant (p = 0.086).Initially, 187 times of iMN confirmed by renal biopsy patients were screened. Three of them were excluded due to graft kidney biopsy. We further excluded within the remaining 184 native kidney biopsies, the 2As shown in Fig. Table 10, including Toroto Risk Score with 85–90% of the accuracy3. However, we believed more new parameters are needed. Firstly, this score was based on clinical and laboratory variables in Western population of Italy, Finland and Toronto. The application to other races is not clear. Besides, parameters in this model are late markers. This model was developed 20 years ago and the outcome prediction of iMN had not improved since. Thirdly, most clinical skepticism of this model is the lack of long-term studies. Finally, they only collected common parameters for analyzed . Therefore, more and newer biomarkers to predict outcomes of iMN are needed.That baseline poor renal function  were risk factors for poor long term renal function, is a finding of our study in consistence with other studies11. Staging of iMN (Ehrenreich-Churg) was reported more than 40 years ago9, and it was focused only on the status of GBM. The predictive value for renal outcome has not been discussed except by Marx et al. in 199910. The HR for ESRD of stage III/IV vs. I/II was found to be high at 5.3 (p = 0.002). After that, no study has validated the predictive power of such staging system. In our study, we found no statistical significance between different disease stages . We believe the background histological severity is not reflected only GBM status, but also by glomerular segmental sclerosis and tubulointerstitial injury both of which can affect the long-term renal outcome. According to Chen et al.11, over a median follow-up of 34 months, segmental sclerosis and tubulointerstitial injury are independent risk factors respectively for 20 and 50% of renal function decline and the progression to ESRD. In brief, GBM status (Ehrenreich-Churg) alone failed to fully predict long-term renal outcome.The morphologic severity of histologic sample with the diagnosed of iMN is related to long-term renal outcomes15. In 1984, C3 deposits were first detected in half of those patients with idiopathic iMN17. Furthermore, C3d deposits were also detected in all patients associated with IgG deposits17. Patients with glomerular C3 deposits exhibited more severe proteinuria, which supported the association of complement activation with disease severity in human iMN17. Recently, using more sensitive immunohistological stainings, C3 deposits can be detected in almost all cases of iMN19. The greater the deposition of C3, the lower the serum C3 level, which predicted poor long-term renal function. In addition, low C3 level is a risk factor for poor patient survival as found in our study. Patients with poor renal function may tend to receive more immunosuppressants in treatment and consequently wound develop moreimmunosuppressant related complications, such as infections.This present study is the first of its kind to show that serum C3 ≤ 93.4 mg/dl predicted poor renal outcome  with good predictive power (AUC = 0.643). The complement system is important for the pathogenesis of iMN. Subepithelial immune deposits activate the complement system which starts activation of the C3 component, conversion of C5, and subsequent formation of the C5b-9 complex in the podocyte membrane20, cardiovascular disease21, malignancy23, community-acquired pneumonia24, carotid atherosclerosis25 and stroke-associated pneumonia26. Its normal levels are between 0.78 and 3.5327. NLR predicts renal prognosis in lupus nephritis28 and granulomatosis with polyangiitis29. In addition, NLR is a risk factor for community-acquired acute kidney injury and NLR is associated with poor recovery of renal function30. Neutrophilia and lymphocytopenia are both indicative of disease severity. Also, an increased NLR level may be related to a low lymphocyte count. It has been proposed that under physiologic stress, the induced increase in cortisol secretion causes lymphopenia31. Similarly, PLR is also considered an inflammatory marker in thrombotic events, inflammatory diseases, and malignancies33. Recently, PLR has been associated with all-cause of mortality in the elderly with chronic kidney disease34, and it is associated with higher C-reactive protein in ESRD patients35. Platelets are viewed as immune cells and can interact with other types of immune cells, including endothelial cells, T-cells, neutrophils, and mononuclear phagocytes. These interactions might initiate and exacerbate the inflammation in the arterial wall36. Although without definite association, we still recommend routine checks for iMN patients, of such easily obtainable markers (like NLR and PLR), to improve the predictive power of renal outcome,especially NLR. NLR > 3.34 may have higher predictive power than PLR > 14.48. Firstly, both multivariable and univariate Cox regression models showed that NLR is a significant predictor of renal survival. NLR had predictive power only in univariate Cox regression. Besides, the predictive power (AUC) of NLR > 3.34 is higher than PLR > 14.48 (0.684 vs. 0.600)Both mean value of NLR > 3.34 (p < 0.01) and PLR > 14.48 (p = 0.002) predicted poor renal outcome. This is the first study to report such findings. We are also the first to demonstrate that NLR levels predicted renal outcome in iMN patients. NLR is a simple and inexpensive laboratory marker of the inflammatory status, including cirrhosis37. However, in clinical practice, we did not measure this marker. Therefore, our study reflected results in the real world. Finally, we did not record complications of immunosuppressants, which could cause more infection-related mortality.There are several limitations of this study. Firstly, the subjects were chosen from a single medical center even though the samples of renal biopsies were relatively large in number (>8000 over 30 years). Secondly, there was insufficient data on treatments. The standard treatment for iMN was consistent according to current guidelines in this medical center. Angiotensin-converting Enzyme inhibitors (ACEis) or angiotensin II Receptor Blockers (ARBs) were prescribed to all patients with iMN. This immunosuppressive therapy was given for those experiencing a progressively declining GFR, or refractory and persistent proteinuria above 4 g/day after maximal ACEis or ARBs. Thirdly, we did not measure anti-PLA2R antibodyStages of iMN did not fully explain the risks for ESRD. This is the first study to identify serum levels of C3 ≤ 93.4 mg/dl predicted poor renal outcome with good predictive power. Easily obtained markers, NLR > 3.34 predicted poor renal outcomes.


Sepsis and other infectious complications are major causes of mortality and morbidity in patients after cardiac surgery. Whereas conventional blood culture (BC) suffers from low sensitivity as well as a reporting delay of approximately 48–72 h, real-time multiplex polymerase chain reaction (PCR) based technologies like “SeptiFast” (SF) might offer a fast and reliable alternative for detection of bloodstream infections (BSI). The aim of this study was to compare the performance of SF with BC testing in patients suspected of having BSI after cardiac surgery.Two hundred seventy-nine blood samples from 169 individuals with suspected BSI were analyzed by SF and BC. After excluding results attributable to contaminants, a comparison between the two groups were carried out. Receiver operating characteristic (ROC) curves were generated to determine the accuracy of clinical and laboratory values for the prediction of positive SF results.n = 41) of blood samples were positive using SF and 17.2% (n = 49) using BC (n.s. [p > 0.05]). In six samples SF detected more than one pathogen. Among the 47 microorganisms identified by SF, only 11 (23.4%) could be confirmed by BC. SF identified a higher number of Gram-negative bacteria than BC did . The combination of BC and SF increased the number of detected microorganisms, including fungi, compared to BC alone . C-reactive protein (CRP) , procalcitonin , and interleukin 6 (IL 6)  plasma concentrations were higher in patients with a positive SF result. Using ROC analysis, IL-6 (AUC 0.836) and CRP (AUC 0.804) showed the best predictive values for positive SF results.14.7% :A884, 2015). Nosocomial infections represent the main non-cardiac complication after cardiovascular surgery and are associated with substantial morbidity, increased mortality, prolonged hospitalization and, eventually, economic burden , 3. RespThe LightCycler® SeptiFast (SF) is a multi-pathogen probe-based real-time PCR system targeting DNA sequences of 25 commonly observed bacteria and fungi present in blood samples within a few hours. However, data about the impact of PCR-based diagnostics on clinical decision-making process and modification of empirical antimicrobial therapy are very limited. A recently published prospective randomized trial demonstrated that in addition to a reduction in the time required for initial pathogen identification, the use of PCR was clearly able to reduce the time required for therapy modification from 38 to 19 h, however without reaching statistical significance . CurrentIn this retrospective observational study, data were collected between January 2009 and February 2013 on all consecutive patients with SF at our Intensive Care Unit (ICU), Department of Thoracic and Cardiovascular Surgery, West German Heart Centre Essen, Germany, in our institutional database. The diagnosis of the suspected BSI was made by clinical judgment by the treating physicians on basis of the occurrence of systemic inflammatory response syndrome (SIRS)/Sepsis criteria . The dec(I)Suspected bacterial or fungal BSI(II)Collection of paired blood samples for SF and at least two sets of BCs (two aerobic and two anaerobic bottles) from a peripheral vein or a central venous line at the same time point (within two hours)Patients were considered for inclusion in the study only if the met the following criteria:The BC and SF results were compared separately by positivity of samples and by detected species of microorganisms/isolates.2 produced by growing microorganisms. BC bottles were incubated up to seven days. In case of a positive signal on the Bactec instrument, 10 μL blood from aerobic blood culture was plated onto chocolate agar, blood agar, MacConkey agar, chromogenic yeast medium, and, if anaerobic bottle were positive, additionally onto two solid anaerobic media . Identification and susceptibility testing was performed according to the EUCAST  standard using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry VITEK MS, the VITEK2  and WalkAway MicroScan  [Blood samples  were collected by sterile venipuncture or from a central venous catheter (CVC) after disinfection of the connector and inserted into aerobic and anaerobic bottles and were sent to the laboratory. Samples then were incubated into the Bactec 9240 Plus , an automated microbial detection platform based on the colorimetric detection of COGermany) .The LightCycler® SeptiFast test M Grade  is an in vitro nucleic acid amplification test for the detection of bacterial as well as fungal DNA in human blood. It allows the identification of 25 bacterial and fungal species , material . Positivmaterial .Isolates identified by PCR were considered to be pathogens or contaminants using a modified algorithm, combining microorganism pathogenicity, interpretation of blood culture results, and clinical, laboratory, and microbiological data . The thr2 test of Fisher’s exact test.Statistical analyses were performed with SPSS Statistics 19 . Continuous data were expressed as median ± 95% confidence interval (CI); categorical data were expressed as percentage. Comparisons between two groups were carried out using unpaired Student’s t-test for normally or the Mann-Whitney Rank Sum Test for non-normally distributed data. Multiple groups were compared with ANOVA. Univariate analysis was performed on the quantitative variables using the Student t-test or Mann-Whitney test and on the qualitative variables using the Chip-value of less than 0.1 between the two groups using Student t-test, Mann-Whitney test, Chi2 test of Fisher’s exact test were selected for ROC analyses. The optimal cut-off concentration was defined by the highest Jouden index (J = sensitivity + specificity – 1). Statistical significance was assumed for a p-value < 0.05.To measure the sensitivity and specificity of laboratory and clinical data at different cut-off values, a conventional receiver operating characteristic (ROC) curve was generated. All variables showing a p < 0.001).During the study period, 279 matched blood samples from 169 patients suspected of having BSI were analyzed with conventional BC and SF. Out of these, 78% (132/169) were under antibiotic treatment at this time. Contaminants were significantly more frequent among blood cultures than SeptiFast . As illustrated in  Fig. 2 = 10.86, p < 0.001 for SF and χ2 = 12.35, p < 0.001 for BC). BC analyses resulted in 51% sensitivity, 83% specificity, 46.7% positive predictive value (PPV), and 54.1% negative predictive value (NPV) whereas SF resulted in 51% sensitivity, 84% specificity, 46.4% PPV, and 54.4% NPV. s.After excluding contaminants, SF identified 47, while BC identified 49 episodes of BSI  and Candida spp. . In contrast, the most frequently observed pathogen in SF was Candida spp.  followed by Enterobacter spp. (8/47) and Klebsiella spp. .With BC, CoNS was the most frequently detected agent  followed by SF identified 7/33 21%) Gram-positive bacteria, 28/35 (8%) Gram-negative, and 12/67 (67%) fungi, while BC identified 28/33 (85%), 12/35 (34%), and 9/18 (50%), respectively , but for fungi, the difference to BC was not relevant . In contrast, detection rate of Gram-positive bacteria was significantly higher with BC compared to SF .Gram-negative detection rate was significantly higher with SF than with BC (χE. coli (n = 3), Klebsiella spp. (n = 6), E. faecium (n = 4), Enterobacter spp. (n = 7), E. faecalis (n = 1), P. aeruginosa (n = 3), A. fumigatus (n = 3), C. spp. (n = 6), S. marcescens (n = 3), S. maltophilia (n = 1). BC detected the following pathogens in SF negative samples: CoNS (n = 13), E. faecium (n = 10), C. albicans (n = 6), S. marcesens (n = 3), Klebsiella spp. (n = 2), S. aureus (n = 1), E. coli (n = 1), other Streptococcus spp. (n = 1), E. faecalis (n = 1), M. morganii (n = 1). Polymicrobial infections were observed in seven patients. Five episodes were detected by SF; while BC identified multiple agents in only four specimens.SF identified 36 pathogens that were not found in BC, while BC detected 39 pathogens in SF negative specimens. Microbial strains exclusively identified by SF were: p = 0.01). In addition, prevalence of acute kidney Injury (AKI) with need for renal replacement therapy (RRT) was higher in SF positive patients .Several variables of the patients with and without pathogen identification in SF and BC were compared, respectively Table . Whereasp = 0.009), procalcitonin (PCT)  as well as interleukin 6 (IL-6)  were significantly higher in patients with positive SF result. In contrast, patients with negative PCR had a significantly higher WBC than patients with positive PCR .Laboratory markers of inflammation differed significantly between groups: C-reactive protein (CRP) . Comparing patients with positive and negative BC, demographics, inflammatory markers and organ function did not differ whereas ICU-stay was longer in individuals with positive blood culture (16 days [p = 0.044).Patients with proven BSI in SF suffered from a more complicated postoperative course with prolonged ICU stay compared to SF-negative patients  as well as CRP  showed the best predictive values for positive SF results  microbiological diagnostic information led to therapy adaptations Table . Only onOur data demonstrate that the PCR-based SF test might represent a rational adjunct tool to the traditional BC method for rapid etiologic diagnosis of BSI in patients after cardiothoracic surgery. SF detects significantly more Gram-negative microorganisms than BC, whereas BC was superior regarding Gram-positive pathogens.Early and reliable diagnosis of BSI and identification of bacteria and fungi is essential to initiate appropriate therapy in septic patients within one hour after sepsis as recommended by current guidelines , 16. Forn = 148) rapid identification of involved pathogens by multiplex-PCR led to adjustment of therapy [A meta-analysis including a total of 34 studies enrolling 6012 patients with suspected sepsis reported a high specificity with a modest and highly variable sensitivity . Recent  therapy . A rando therapy . Even in therapy . Taken tPatients after cardiothoracic surgery significantly differ from other cohorts: The use of cardiopulmonary bypass leads to a damage of the gastrointestinal mucosa, subsequent increased permeability, possible bacteremia, and the activation of a self-limited inflammatory response. The incidence of fungal infections especially in transplant recipients is, due to immunosuppression, higher than in the general ICU population. Commonly used biomarkers for bacterial infection might not work properly in the cardiothoracic population , 23.In accordance with previous studies, the results of the present study demonstrate that SF, compared to BC, provided a better management of contaminants and a lower contamination rate . In respIn accordance with recently published data, we observed a clear superiority of SF in detecting Gram-negative organisms compared to conventional BC . The reaS. aureus was only associated with a 2.2-fold increased risk of mortality [BSI caused by Gram-negative bacteria is associated with a 7-fold increased risk of early mortality after cardiac surgery, compared with no BSI . In contortality . TherefoAspergillus amplicons were detected by PCR as compared with BC. SF could improve patient outcome as a result of rapid and accurate fungi detection and the consecutive timely initiation of appropriate therapy [Since invasive fungal infections with Aspergillus are frequently associated with high morbidity and mortality, in particular immunocompromised patients benefit from prompt initiation of anti-fungal therapy . However therapy . Hence, S. aureus, representing an important pathogen associated with high mortality. CoNS are a major constituent of human skin commensal flora, which were once considered relatively apathogen and a likely contaminant. But in patients with foreign materials  these organisms, due to their propensity to form a biofilm and to display resistance to multiple antibiotics, have increasingly been recognized as a cause of clinically significant infections. Thus, due to the significant number of infections that would be missed, SF could not replace blood culture for the identification of bloodstream infections. In addition, in SF pathogen identification is restricted to the 25 tested microorganisms and, moreover, susceptibility testing is not possible. In respect of the ongoing problem of multi drug resistance susceptibility testing is of increasing importance [However, SF was inferior to BC in detecting Gram-positive bacteria including portance . TherefoEven though in our study antimicrobial therapy was escalated due to the results of SF in eight patients, no de-escalation was done. As most of our patients were already on broad spectrum antibiotics and several blood cultures were drawn before choosing SF as diagnostic tool, empirical antibiotic therapy was considered to be adequate for most of the pathogens detected in SF and de-escalation was not done due to the lack of susceptibility testing.Recent studies could demonstrate that use of new PCR based technologies in the management of septic patients lead to a significant reduction in treatment costs with a an average net saving of 9970 € per patient . This ecMencacci investigated the predictive role of procalcitonin in patients with suspected sepsis for positive test results in BC and PCR and revealed an area under the curve of 0.927 for SF positivity . When apThere are several potential limitations to this study. First, our study suffers from the general limitation of a single-center, retrospective investigation: the results may not be applicable to other clinical settings with different patient characteristics, resources, and laboratory procedures. In addition, due to the small number of specific pathogens, the power to detect a difference between the groups is limited.In interpreting the results of this study heterogeneity in the methods of drawing blood samples for BC must be considered as a limitation. It could not be ensured that all collected samples complied with the guidelines for drawing blood samples for BC, what can affect both for sensitivity and specificity .A major limitation is the fact that there were no predefined criteria for performing PCR e.g. presence of more than two SIRS criteria. The algorithms used in this study to differentiate between contamination and infection of BC and SF were not evaluated in the cardiothoracic population. Therefore, the reliability of this algorithm in this setting is uncertain. However, as there is no published algorithm for cardiothoracic patients, we modified the originally published algorithm to incorporate specific characteristics of our patient’s cohort e.g. the presence of prosthetic heart valves or other extracorporeal devices.Due to the retrospective nature of our study we could not ensure that the same blood sample was used for SF and BC.It has to be mentioned that the SF test is not available in the United States yet.The PCR-based SF test might represent a valuable addition to the BC method for rapid etiologic diagnosis of bloodstream infections in patients after cardiothoracic surgery. This applies in particular for individuals with Gram-negative bacteremia. Since SF missed a certain number of Gram-positive pathogens, can only detect a limited number of pathogens and is unable to determine antibiotic susceptibility, it should always be used in conjunction with traditional blood culture methods.


This study aimed to evaluate patterns of posttransplant malignancies among renal transplant recipients (RTRs) in South Korea using nationwide data.The nationwide cohort assessed in this study included RTRs from January 1, 2010, to December 31, 2014. We analyzed cancer incidence during the time course after renal transplantation. Additionally, we calculated standardized incidence ratios (SIRs) to evaluate the risk of malignancies in RTRs.A total of 1343 RTRs  were assessed. Among them, 104 (7.7%) developed malignancies after transplantation, most commonly in the thyroid cancer (23.1%). The SIR for all cancers was 3.54; particularly, the SIRs for renal cancer, myeloma, and non-Hodgkin lymphoma were 16.31, 24.02, and 28.64, respectively. Females showed a higher risk of malignancy than males (SIRs: 4.04 for women and 3.26 for men). The median interval between transplantation and malignancy diagnosis was 27.2 months (range 12.3–54.8 months).RTRs in South Korea demonstrated a high risk of malignancy after transplantation compared with the general population. This indicates that close surveillance and routine screening for cancer in RTRs are needed. Renal transplantation (RT) is considered as the most desired treatment option for patients with irreversible chronic kidney disease . There hIn Western countries, nonmelanoma skin cancer is the most common malignancy after RT , 9. MeanIn South Korea, a single-center study reported malignant lymphoma and stomach cancer as the two most common types of malignancies . HoweverThe National Health Insurance (NHI) system is a public medical insurance system in South Korea. The HIRA database includes demographic data on sex, age, and residential areas and clinical data on diagnosis, procedures, and prescriptions. The nationwide cohort assessed in this study was based on HIRA claims data and included patients who received RT from January 1, 2010, to December 31, 2014. This study was approved by the institutional review board (IRB) of Ajou University Hospital (IRB No. AJIRB-MED-EXP-16-493).Patients were identified by procedure codes indicating RT (R3280). Moreover, we confirmed use of immunosuppressants such as azathioprine, cyclosporine, prednisolone, mycophenolate mofetil, tacrolimus, and sirolimus. Malignancies after RT were confirmed using the main disease codes in claims data from inpatient and outpatient first visits. Malignancies were diagnosed based on pathological findings and identified by principal diagnoses using codes based on the International Classification of Diseases, 10th Revision. We excluded malignancies diagnosed before RT as well as in situ malignancies.We analyzed the patterns of malignancies during the time course after RT. Descriptive statistics were calculated to estimate the frequency of the cancer site using the diagnostic code based on the first hospital visit. We also analyzed standardized incidence ratios (SIRs) and corresponding 95% confidence intervals (95% CIs) to estimate the relative risk of malignancies between RTRs and the general population . All staA total of 1343 patients who received RT between 2010 and 2014 were identified in South Korea. The median age of these patients was 50 years (range 1–75 years); 871 patients (64.9%) were men and 472 (35.1%) were women , stomach cancer (n = 7), pancreatic cancer (n = 2), and larynx cancer (n = 1) were confirmed only in males. The cancers that were found only in females were breast cancer (n = 7), gynecologic cancer (n = 3), colon cancer (n = 1), anal cancer (n = 1), and ureteral cancer (n = 1).The mean follow-up time for RTRs was 25.4 ± 16.7 months. The most common malignancies after RT were thyroid cancer (23.1%), renal cancer (8.7%), stomach cancer (6.7%), liver cancer (5.8%), lung cancer (5.8%), and breast cancer (5.8%) . Patients with leukemia had a median age of 32 years, whereas those with prostate cancer had a median age of 67 years . Among all cancer types, brain tumor (SIR 26.16), leukemia (SIR 19.26), renal cancer (SIR 16.68), and cervical cancer (SIR 16.73) represented the highest risk of malignancy in females. Meanwhile, males had a high risk for developing myeloma (SIR 25.90), non-Hodgkin lymphoma (SIR 30.21), and Kaposi’s sarcoma (SIR 37.85).The long-term use of immunosuppressants and dialysis therapy before RT has been shown to increase the risk of malignancies in RTRs compared with the general population –17. The Another study in South Korea reported a 4.3% rate of malignant development during a 37-year follow-up after RT at a single center . Kim et In our study, the distribution of sites of posttransplant malignancies differed from that of studies in Western populations. Skin cancer is the most common malignancy in Western countries. However, the incidence of skin cancer is low in Asian countries. In Japan, stomach cancer, renal cancer, and lymphoma are more common , 11, wheThe age at diagnosis of malignancy was different between cancer types Table . ThyroidIn our study, thyroid cancer, gynecologic cancer, prostate cancer, and lymphoma were confirmed in 36 patients. These cancer types are difficult to detect using only abdominal and chest computed tomography (CT). Particularly, cervical cancer detection requires Pap smears annually. Our study also confirmed three patients who were diagnosed with colorectal cancer after RT. Although fecal occult blood tests for colorectal screening have a low false-negative rate, flexible sigmoidoscopy for pathological confirmation is ultimately needed. Therefore, a screening test is needed for identifying these difficult-to-detect cancers. Promoting health examinations among RTRs and using 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET)-CT in evaluating status in a systematic manner could be considered. FDG PET-CT has not been useful in detecting primary prostate cancer. However, it can be useful for detecting posttransplant malignancies as a systematic screening test, although its cost-effectiveness may be a concern. A previous study has confirmed that appropriate screening and early detection of cancer could reduce mortality and improve prognosis of RTRs .Besides screening, lifestyle modifications are also important. Lung cancer was confirmed in six patients  in this study. Since lung cancer is associated with smoking, RTRs should be strongly encouraged to quit smoking, and screening with low-dose CT is recommended for smokers. On the other hand, six patients were diagnosed with liver cancer (SIR 2.74). South Korea is within the epidemic region of hepatitis B, which is an important risk factor of liver cancer. Therefore, active attention is necessary in the management of viral infections through the use of vaccination among RTRs. Before transplantation, patients should receive vaccination for hepatitis B virus if they do not already have the specific antibody.This study has several limitations. The insurance claims registry was limited in terms of accuracy and validity and due to absence of universal screening methods. Clinical data such as type of pathology, laboratory data, and imaging results were underreported. Moreover, the data lacked social factors associated with cancer risk, such as lifestyle  and family history. Since the period of legal use of HIRA claims data is limited to 5 years, the shorter follow-up period in this study relative to that in previous studies was not adequate to detect malignancies with a late onset of ≥5 years. Therefore, further studies with a longer follow-up period should be conducted.This study confirmed that cancer risk was significantly increased in RTRs compared with the general population. The types and risk of cancer after RT in South Korea were different from those in other countries. This geographical variation would be helpful in improving cancer screening and follow-up recommendations for RTRs in Asian countries, specifically in South Korea.


Female patients are more likely to have tendon injuries than males, especially those who has a higher concentration of relaxin. Previous studies have demonstrated that relaxin attenuates extracellular matrix (ECM) formation. However, the mechanism of relaxin on tendon repair remains unclear. We hypothesize that relaxin inhibits tendon healing by disrupting collagen synthesis.A patellar tendon window defect model was established using Sprague-Dawley rats. The center of the patellar tendon was removed from the patella distal apex and inserted to the tibia tuberosity in width of 1 mm. Then, the rats were injected with saline (0.2 μg/kg/day) or relaxin (0.2 μg/kg/day) for two and four weeks, which was followed by biomechanical analysis and histological and histochemical examination.Mechanical results indicated that relaxin induces a significant decrease in tear resistance, stiffness, and Young’s modulus compared to those rats without relaxin treatment. In addition, it was shown that relaxin activates relaxin family peptide receptor 1(RXFP1), disturbs the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteases (TIMPs), and reduces the deposition of collagen in injury areas.Relaxin impairs tendon healing in rats. Also, relaxin might lead to tendon injury more commonly for females than males.The online version of this article (10.1186/s12891-019-2729-3) contains supplementary material, which is available to authorized users. Tendons, which are dense fibrous connective tissues composed of tenocytes and abundant ECM, connect muscles to bones and facilitate in the transmission of muscle-generated forces to the bones, resulting in joint motion . With thResearch investigations on sex hormones have recently increased. There is a quiet difference between women and men for the risk of tendon and ligament injuries. Sex hormonal differences may one of the major reasons . For exaWhen mice are pregnant, relaxin transforms the pubic articular cartilage to a pliable interligament in adapt to the enlarged uterus. Meanwhile, relaxin itself or combined with estrogen reduce public collagen content, which is antagonized by progesterone, in non-pregnant rats .It has been shown that relaxin is antifibrotic as it downregulate fibroblast activity, increase collagenase synthesis, and inhibits collagen-1 lattice contraction in rat kidneys, which is stimulated by transforming growth factor-β . AccordiDragoo et al.  showed tHowever, the effect of relaxin on tendon and ligament healing remains unclear. We hypothesized that relaxin inhibits tendon healing, and carried out this study to test it in a rat patellar tendon window defect model.All animal procedures were approved by the Ethics Committee of the First Affiliated Hospital of Soochow University .A total of 36 female Sprague-Dawley rats, 6–8 weeks old and weighing from 250 to 300 g, were used in this study. The rats were purchased from the Experimental Animal Center of Soochow University. Also, they were kept in a ventilated environment with a 12 h:12 h light-dark cycle at a constant temperature of 21 °C.To induce tendon defects, two stacked sharp blades, described in a previous study, was used to remove the center of the patellar tendon (1 mm in width) from the patella distal apex and insert to the tibia tuberosity without causing any damage to the fibrocartilage zone  The right patellar tendons of rats, sacrificed 2 and 4 weeks post-operatively, were kept at room temperature in PBS-soaked gauze pads until testing within 1 h after collection. To carry out tensile testing of the tendons, the patella and the proximal tibia were fixed at each clamp (5 mm in between two clamps) of a testing machine . The samples were tested at a speed of 1 mm/s until failure.For histological analysis, the left patellar tendon was fixed in 4% paraformaldehyde for one day. After embedding in paraffin, the samples were sectioned longitudinally at a thickness of 6 μm, then deparaffinized in xylene, washed, and hydrated with washing ethanol. The sections were processed for histological examination using hematoxylin-eosin (H&E) and Masson trichrome staining. To carry out the regenerating tissue quality evaluation, we adopted a histological scoring system . The hisThe sections were de-waxed, hydrated and inactivated. Then the tissue were blocked with 5% normal goat serum after antigens repairing and incubated at 4 °C overnight using Col I (1:500), Col III (1:500) , RXFP1 (1:200) , MMP1 (1:500), MMP9 (1:500)  and MMP13 (1:500) (Biorbyt) monoclonal antibodies. Finally, they were incubated with HRP-conjugated secondary antibody  for 1 h. Sections were colored by incubating with the chromogen 3.3′-diaminobenzidine (DAB) tetrahydrochloride and counterstained with hematoxylin.p values less than 0.05.One-way ANOVA and post-hoc Bonferroni tests with SPSS 20.0 were adopted to carry out statistical analyses of histological score and biomechanical testing. Statistical significance was assigned at p < 0.05). This observation suggests that surgical operations change the mechanical properties of tendons. The results demonstrate that the vehicle and relaxin groups have similar mechanical properties during the earlier period of repair. However, the data obtained four weeks postoperatively indicated that the control group exhibited significantly higher maximum load and Young’s modulus (p < 0.05) than the relaxin group. These findings suggest that relaxin disrupts tendon healing, whereas the control group exhibited regeneration. No statistical difference in tendon length and cross-sectional areas between the three groups after two and four weeks was observed.There was no loss of specimens during the tests. The results of mechanical testing showed that tissue repair after tendon removal at each postoperative time point in the vehicle and relaxin groups was worse than in the control group Table . At two p < 0.05). However, the relaxin group presented lower histological scores (p < 0.05) than the vehicle group, indicating that the relaxin group exhibited poor tendon structural recovery .At week 2 and 4 after surgery, immunohistochemistry staining of Col I and Col III were applied in the new tendon tissues. The expressions of Col I and Col III in the neo-tendon tissues were stronger in the vehicle group and more uniform at both weeks 2 and 4. Compared to normal tendons, the expressions of Col I and III were more intense, suggesting that more ECM was deposited into the wound areas during tendon healing   Fig. b. These p < 0.05). However, no difference in MMP1 and TIMP1 expression between the two groups was observed. Relaxin treatment effectively increased the expression of MMP9 and MMP13. These findings indicate an alteration in the balance between MMPs and TIMP expression