Hashimoto thyroiditis (HT) is highly prevalent among reproductive-aged women and has a substantial negative impact on fertility. Currently, there is no specific treatment for Hashimoto thyroiditis. We hypothesize that acupuncture can halt or delay the progression of HT and improve fertility in child-bearing period female. We therefore designed a randomized controlled trial to test this hypothesis by comparing the therapeutic effect of acupuncture vs sham acupuncture in patients with Hashimoto thyroiditis.In this randomized controlled study, a total of 284 eligible patients will be assigned to acupuncture group (n = 142) or sham acupuncture group (n = 142) in a 1:1 ratio. All patients will receive 36 sessions in total for 12 consecutive weeks with the same acupoint prescription . The primary assessment is the titers of thyroid peroxidase antibodies (TPOAb) and thyroglobulin antibody (TGAb). Secondary outcomes include the thyroid function, ovarian function, the rate of primary ovarian insufficiency, and pregnancy outcome. The thyroid function and thyroid antibodies tests will be measured at weeks 0, 4, 8, and 12 after randomization. The ovarian function will be examined on the 2nd to 4th day of the menstrual period in the 1st month, 2nd month and 3rd month compared with baseline. Both the pregnancy outcome and the rate of primary ovarian insufficiency will be evaluated 1 year after treatment.This will be the first large-scale trial specifically evaluating acupuncture therapy in child-bearing period female with Hashimoto thyroiditis. If the study confirms the effectiveness of acupuncture treatment, more consistent acupuncture therapy can be set up for clinical practice.Chinese Clinical Trials Register identifier, ChiCTR2000031320, registered on 27 March 2020. HT is characterized by increased levels of thyroid peroxidase antibodies (TPOAb) and thyroglobulin antibody (TGAb), diffuse lymphocytic infiltration of the thyroid gland, manifestations of goitrous or atrophic gland, and frequent thyroid dysfunction in varying degrees.,7 HT is the most common cause of hypothyroidism. The pathogenesis is thought to be an immunologic attack that the presence of TPOAb tends to correlate with thyroidal damage and lymphocytic inflammation. TPOAb is directly cytotoxic to thyrocytes. In patients who have circulating thyroid peroxidase antibodies, there is a greater risk of progression from subclinical to overt hypothyroidism. A previous study indicates that largely increased levels of TPOAb are associated with a moderately increased risk of developing hypothyroidism in the patients of Hashimoto thyroiditis remained euthyroid. An epidemiological survey with a 20-year follow-up showed that the lifetime risk of incident hypothyroidism was 4% among TPOAb negative women, 23% for women with low-level TPOAb, 33% for those with mid-level TPOAb, and 53% for those with high TPOAb. Most cases of Hashimoto thyroiditis are considered to be the early stage of hypothyroidism. According to studies, 4% to 8% of the reproductive-age population suffered from subclinical hypothyroidism (SCH) with elevated thyroid stimulating hormone (TSH) more than 4.5 to 5.0 mIU/L and normal free thyroxine (FT4) levels concentration.–15 The risk of progression from SCH to overt hypothyroidism (OH) ranges between 2% and 5% a year. It is well known that thyroid hormones play important roles during the development and maintenance of reproductive function in women. Numerous studies have suggested that thyroid autoimmunity and hypothyroidism associated with an adverse effect on fertility as well as pregnancy.,17–21Hashimoto thyroiditis (HT) known as autoimmune thyroiditis is a common autoimmune thyroid disease (AITD) and endocrine disorder.,22 Fertility is impaired in women with autoimmune thyroid disease. An extensive meta-analysis study reported that the presence of TPOAb combined with normal thyroid function was associated with an increased risk of unexplained subfertility, implantation failure, miscarriage, recurrent miscarriage, preterm birth, and maternal post-partum thyroiditis.,25 A prospective study confirmed that the prevalence of TPOAb was greater (18%) in infertile women than that in healthy and fertile controls (8%). Autoimmune thyroid disorder and hypothyroidism have a negative impact on ovarian function.,28 Women with AITD have lower serum anti-Müllerian hormone (AMH) levels compared with age-matched controls, which means women with AITD have prematurely aging ovaries. A prospective controlled trial revealed that 5% of patients of premature ovarian insufficiency (POI) presented highly elevated antithyroid antibodies. Besides, 24.1% of patients with premature ovarian failure (POF) had thyroid peroxidase autoantibodies. Idiopathic low ovarian reserve was associated with more frequent positive TPOAb in Chinese women. The human ovary is often the target of an autoimmune attack. In this context, HT is likely to be associated with ovarian dysfunction and diminished ovarian reserve. TPOAb has been detected in all samples of follicular fluid obtained from women with thyroid autoimmunity, while they were absent in women without thyroid autoimmunity. In these patients with HT, the levels of TPOAb in ovarian follicular fluid is positively correlated with serum antibody levels. Monteleone et al speculated that TPOAb might passed through the blood follicle barrier during follicular evolution and damage growing follicles and oocytes via thyroid hormone receptors on granulosa cells, which may result in decreasing the quality and development potential of oocyte. TPOAb targets TPO antigen on granulosa cells, creating a hostile local immune microenvironment around the oocyte. Subclinical or overt hypothyroidism is often caused by HT, which is associated with a diminished ovarian reserve in infertile patients of reproductive age.,36 Recently, a retrospective study showed that SCH was associated with decreased ovarian reserve during later reproductive age. Therefore, Hashimoto thyroiditis has a substantial negative impact on the fecundity of reproductive-aged women. Impaired fertility has become a major public health problem that is associated with an increasing burden on society.For two decades, the association between TAI and reproductive failure has gained attention. Current therapeutic strategies for HT with normal thyroid function in the early stages include Se supplementation, glucocorticoids, and vitamin D.–41 However, the available data on beneficial effects of Se on thyroid autoimmune parameters are limited. On the other hand, caution should be taken when long-term selenium supplementation, the increased risk of type 2 diabetes was observed in a randomized clinical trial. Only an ambiguous causal relationship and few interventional studies reported that Vitamin D is beneficial in the management of thyroid disease, so the therapeutic potential of vitamin D remains debated. Although some small trials appear promising of glucocorticoids, corticosteroid use has many negative effects on key aspects of early pregnancy.,45 Once overt hypothyroidism is present, levothyroxine (LT4) is the treatment of choice for Hashimoto thyroiditis. Patients with HT who have the mere presence of detectable TPO antibodies does not, however, advocate empiric treatment with thyroid hormone. According to one study, women that are considered euthyroid, before pregnancy, are reclassified as having subclinical hypothyroidism, in cases of TSH between 2.5 and 4 mIU/L. Most endocrinologists use a TSH value of 2.5 mIU/L as the threshold for diagnosing hypothyroidism and starting levothyroxine in pregnant women because of an evident association between mild thyroid impairment and adverse outcomes in pregnancy. LT4 supplementation can slow the progression of HT, control the early stage of hypothyroidism, and support follicular development. The American Thyroid Association's recommendations for LT4 treatment also be considered for TPOAb positive women with normal thyroid function, although the quality of evidence is low. However, LT4 treatment before pregnancy in women is lacking sufficient data. An ideal TSH value before pregnancy can initiate a typical starting dose LT4 therapy (25–50 ug/d) When considering the potential benefits and risks minimization.To date, there are no specific therapies to effectively reduce the autoantibody level of HT. Patients with HT who have normal thyroid function and no obvious symptoms of oppression should be followed up and monitored thyroid function regularly. MOK pharmacopuncture ( a new form of acupuncture treatment) can regulate the imbalance of Th1/Th2 cytokines and help to suppress autoimmune response. Acupuncture can regulate the indexes related to immune function and correct the immune dysfunction to some extent, which is possibly the action mechanism of acupuncture-moxibustion in treating HT. Electroacupuncture (EA) intervention can regulate the thyroid hormone. The connection between thyroid and fertility is mainly based on cross-talk between the hypothalamic–pituitary–thyroid (HPT) axis and the hypothalamic–pituitary–gonadal (HPG) axis. The electroacupuncture has a benign regulating effect on the key hormones in the pituitary-target gland axis, such as thyroid-stimulating hormone (TSH), triiodothyronine (T3), tetraiodothyronine (T4), estradiol (E2), luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Therefore, we can draw the preliminary conclusion that acupuncture can delay the progress of Hashimoto's thyroiditis and improve its fertility. Although acupuncture is increasingly used as a nonpharmacologic therapy in clinical practice, the level of available evidence is rated as “low”. We designed an RCT to evaluate the effects of acupuncture on halting or delaying the progression of HT and fertility improving in child-bearing period female.According to the World Health Organization (WHO), acupuncture can be used to treat thyroid diseases. As integrative therapy or alternative medicine, acupuncture is safe and economical. Acupuncture can reduce symptoms and improve relevant biomarkers of thyroid disease patients.22.1,57 The flowchart of the trial is shown in Figure A single-center, patient-blinded, randomized controlled trial (RCT) is currently being performed. Acupuncture will be compared with sham acupuncture to determine if acupuncture has an effect on the reduction of antibody (TPOAb or TGAb) and fertility improving. The trial will be conducted in the Department of gynecology, Teaching Hospital of Chengdu University of Traditional Chinese Medicine. The protocol will be reported following the Consolidated Standards of Reporting Trials (CONSORT) statement and the Revised Standards for Reporting Interventions in Clinical Trials of Acupuncture (STRICTA).2.2 and has been approved by the Sichuan Regional Ethics Review Committee on Traditional Chinese Medicine . We registered the study on the Chinese Clinical Trial Registry (Registration No. ChiCTR2000031320).The research protocol is performed in accordance with the principles of the Declaration of Helsinki2.3 If they meet the study criteria, they will be invited to the Department of gynecology, Teaching Hospital of Chengdu University of Traditional Chinese Medicine to undergo the study. All participants will be recruited through hospital social media (WeChat) or posters in the community and hospitals. If a patient is interested in participating, she will contact the researcher by WeChat or telephone. Prospective participants will be asked to talk face to face with the researcher for a baseline screening visit after diagnosis. Eligible participants will be randomized to acupuncture group or sham acupuncture group. There will be one year follow-up period after treatment.A total of 284 women with HT in child-bearing period will be enrolled in this study after their informed consents are obtained. The diagnosis of HT based on circulating antibodies to thyroperoxidase (mainly thyroperoxidase and thyroglobulin) and reduced echogenicity on thyroid sonogram.2.4Patients who meet all of the following conditions will be considered for enrollment. The inclusion criteria are as follows:1.females, aged between 20 and 45 years;2.meet the diagnosis of HT with normal thyroid function(TSH<5.0 mIU/L) and is trying to conceive;3.willing to join this research and sign an informed consent form.2.5The exclusion criteria are as follows:1.infertility caused by organic lesions of the reproductive system or male infertility;2.pregnant women or women in their lactation period;3.complicated with bleeding disorders , mental diseases , malignant tumor, or serious organic diseases;4.history of radioiodine therapy or surgical intervention of the thyroid;5.use of immunosuppressants, immunostimulants, or drugs that interfere with the production, transport, and metabolism of thyroid hormones ;6.participated in other clinical trials in the past 3 months.2.6Sample size will be decided by the primary outcome. The primary outcome of this study will be the mean change of TPOAb and TGAb from baseline to week 12. According to the results of our previous pilot study, titers of TPOAb was 253.86 ± 178.23 IU/ml in the acupuncture group and 318.98 ± 201.37IU/ml in the sham acupuncture group (TPOAb higher than 34IU/ml was the positive threshold of this research center). Considering participants recruited in the two groups are in a 1:1 ratio, the following formula was used to estimate sample size: α = 0.05, two-sided) and 80% power (β = 0.2), at least 134 patients should be enrolled in each group and 284 total participants will be recruited to allow for a 10% dropout rate.With a 5% significance level . Once consent has been obtained and baseline data collected, participants will be randomized to the acupuncture group (n = 142) or the the sham acupuncture group (n = 142). Randomization will be performed according to a random list of numbers generated with SPSS21.0 software . An independent researcher will prepare the assignments in opaque envelopes containing an allocation sequence number and take charge of the allocation sequence concealment.2.8Due to the specific nature of the intervention, blinding will be maintained for participants, the data collection researcher, and the statistician except for the acupuncturist. The acupuncturist, the data collection researcher, and data analysts are not allowed to exchange information.3There will be 4 weeks for baseline, 12 weeks for treatment, and 1 year for follow-up in this study. Each participant will receive 36 sessions of 30-minute duration over 12 weeks . Participants whose TSH is greater than 2.5mIU/L should be managed with a typical starting dose LT4 (25 ug/d). TSH levels should be optimized to <2.5 mIU/l, before conception. No additional treatment is allowed during treatment. Only licensed acupuncturists who have more than 5 years of experience will perform the treatment following the standardized operating procedures. The two groups of participants will be treated separately in different rooms to avoid communication between the participants. Each participant will take a supine position on the treatment couch which is separated by a curtain. Stick adhesive pads and disposable, sterile needles ) will be used for acupuncture.4Participants in acupuncture group will receive therapy at the Lianquan (RN23), Renying (ST9), Danzhong (RN17), Guanyuan (RN4), Qihai (RN6), Zusanli (ST36), Sanyinjiao (SP6) and Zhaohai (KI6) points. The acupuncture points are identified according to the method of point location issued by the WHO. After local area disinfection with alcohol wipes, RN4, RN6, ST36, KI6 and SP6 will be inserted perpendicularly 10 to 15 mm deep with a 0.25 mm × 40 mm acupuncture needle. RN17 (with the needle tip pointing towards the head) will be punctured obliquely 5 to 10 mm deep with a 0.25 mm × 25 mm acupuncture needle, while RN23 (with the needle tip pointing towards the root of tongue) will be punctured obliquely to the depth of 5 mm. ST9 will be punctured perpendicularly to a depth of 5 mm. De qi sensation  will be achieved through lifting, thrusting, and rotating.5 This non-invasive placebo needles can provoke a needling sensation as soon as it touches the skin. The acupoint selection and procedures will be the same as in the acupuncture group. The acupuncturists should pretend to manipulate the needle for 10 seconds on each point as well. No deqi sensation will be induced.Participants in the control group will undergo treatment with a special sham acupuncture device with a blunt-tip needle  which is similar to Streitberger needles device, without insertion.66.1The primary outcome in this trial is the titers of TPOAb and TGAb at baseline and at the 4th, 8th, and 12th week after randomization.6.2The secondary outcomes include:(1)thyroid function test including free triiodothyronine (fT3), free thyroxine (fT4), and thyroid stimulating hormone (TSH);(2)ovarian function including Anti Mullerian Hormone(AMH), follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2);(3)pregnancy outcome including pregnancy rate (%), pregnancy losses (%), and live birth rate (%);(4)primary ovarian insufficiency (%).The thyroid function test will be performed at the baseline, 4th, 8th, and 12th week after randomization. The ovarian function will be examined on the 2nd to 4th day of the menstrual period in the 1st month, 2nd month and 3rd month compared with baseline (-1st month). Both the pregnancy outcome and the rate of primary ovarian insufficiency will be evaluated at 6 months and 1 year after treatment.6.3Any adverse events, including acupoint hematoma, bleeding, fainting, serious pain, infection, stuck needles, and broken needles, will be recorded by the researcher. If adverse events occur during the intervention, management will be taken emergently. Serious adverse reactions will be reported to the ethical committee and rescue procedures will be initiated at once.6.4Before the trial, all researchers involved will receive specialized training to maintain the study quality. The training courses include how to select and exclude patients, how to stratified randomization, how to manipulate interventions correctly, how to fill the case report form, how to assess outcomes and manage data. When the clinical trial begins, the principle researcher will supervise the trial. The licensed acupuncturists who have more than 5 years of experience will perform intervention according to the pre-specified standard operating procedure. Raw data will be recorded in the case record form (CRF). Two data managers enter the data into the spreadsheet and check the electronic data respectively. To guarantee the objectivity of the data, effect assessment and statistics will be blinded during the study period. Measures are taken to improve compliance, such as health education, respect patients informed consent right sufficiently etc.6.5t test. For abnormally distributed variables, they will be expressed as Medians ± Interquartile Range (MIQR) and nonparametric tests will be used. Categorical variables will be presented by frequency and percentage and analyzed by χ2 test or Fisher exact test. A repeated-measures multifactorial analysis will be used to analyze value changes of TPOAb, TGAb, fT3, fT4, TSH, thyroxine, AMH, FSH, LH, and E2 across 4 testing time points  by professionals who will be blinded to the whole trial process. According to the principle of intention-to-treat (ITT), all randomized participants will be analyzed. Continuous variables with normal distribution will be expressed as Mean ± Standard Deviation (M ± SD) and compared by an independent sample Student 7 The presence of anti-thyroid antibodies has an increased risk of unexplained infertility, low fertilization rates, poor embryo quality in assisted reproductive technologies, miscarriage, preterm delivery, perinatal mortality, and maternal post-partum thyroiditis.,24 The relationship between reproductive failure and autoimmune conditions (including thyroid disease) has attracted worldwide attention in recent years. Consider that the early and middle stages of adulthood are periods of increased risk for many autoimmune diseases, particular emphasis was placed on the importance of reproductive problems in these groups. The association between TPOAb and subfertility has proposed several possible mechanisms:Thyroid dysfunction and autoimmune in women of child-bearing age are adverse risk factors for fertility and pregnancy.(1)The autoimmunity process may lead to subfertility or pregnancy loss;(2)Infertility or pregnancy abortion may be secondary to hypothyroidism. An increased risk of unexplained infertility for the euthyroid women who were positive for thyroid antibodies. There is no specific treatment modality to suppress autoimmune destruction by modern medicine, therefore, seeking complementary and alternative therapy such as acupuncture in the early stage is of great significance for the treatment and fertility preservation in the child-bearing period female with HT.Prolonged mild hypothyroidism may have a negative impact on the ovarian follicular reserve. Levothyroxine supplementation may improve fertility in infertile patients.,64 Given that there may be some benefit and negative influence may be avoided with adequate levothyroxine therapy aimed at keeping TSH <2.5mIU/L, we will manage the participants whose TSH is greater than 2.5mIU/L with a typical starting dose LT4 (25 ug/d).Acupuncture therapy has been used in clinical practice on thyroid disease for a long time. Currently, there is a lack of high-quality research and evidence on acupuncture for Hashimoto's thyroiditis. Therefore, we are conducting a randomized controlled study to evaluate the effect of acupuncture on halting or delaying the progression of HT and fertility improving in the child-bearing period female. To reduce the possible bias, we use the stratified randomization method to randomize the enrolled patients into acupuncture group and sham acupuncture group. Due to the particularity of this invasive operation of acupuncture, completely inert placebo acupuncture and blinding is difficult in acupuncture trials. For the placebo control and better blinding of the participants, we use tailor-made sham needles and adhesive pads to treat participants alone in a separate room which is separated by a curtain. There is no definitively recommendation or objection to treatment with levothyroxine in euthyroid women who are positive for thyroid antibodies before pregnancy. In women seeking pregnancy through assisted reproductive technologies, the guidelines recommend levothyroxine treatment of subclinical hypothyroidism, defined as a TSH > 2.5 mIU/L in many studies, with a goal for the TSH of <2.5 mIU/L. TPO is the key enzyme in thyroid hormone synthesis. The thyroid hormones T3 and T4 are synthesized in the thyroid gland in a process that crucially involves the iodoglycoprotein thyroglobulin (TG). Antibodies against the main thyroid antigens (such as TPOAb and TGAb) can induce a chronic lymphocytic thyroiditis, eventually leading to the destruction and loss of thyroid function.,68 TGAb may reflect a more initial type of immune response, while TPOAb can characterize later adaptive immune response, a sort of immune escalation. Positive TPOAb and/or positive TGAb, have been used test for screening for HT. TPOAb (positive in 95% of HT patients) as the best serological marker of HT is more sensitive than TGAb (positive in only 60%–80% of HT patients).,70 Some surveys suggest that positive TPOAb are associated with an increased risk of developing hypothyroidism.,12 In a cohort study, a TPOAb level above the threshold of 500 IU/ml was associated with a moderately increased risk for developing hypothyroidism. The presence of TPOAb negatively influences folliculogenesis, spermatogenesis, fertilization rates, embryo quality and pregnancy rates. In Chinese women, idiopathic low ovarian reserve is associated with more frequent positive TPOAb. Therefore, TPOAb and TGAb will be used as the primary outcome to assess the effect of acupuncture on delaying or preventing disease progression and improving fertility in child-bearing period female with HT. Except for thyroid antibodies, evaluation of thyroid function in patients with HT is carried out by measuring the serum levels of thyrotropin (TSH), free thyroxine (FT4), free triiodothyronine (FT3). Anti-Mullerian hormone (AMH) is secreted by the granulosa cells of growing ovarian follicles. AMH is a suitable biomarker of ovarian age in women of reproductive age and the most widely used and reliable serum biomarker of ovarian reserve.,73 Follicle stimulating hormone (FSH) stimulates growth of ovarian follicles and aromatase expression in follicle granulosa cells. During reproductive aging in females, a rise in basal FSH is considered a sign of the reduction in the follicle reserve. Basal estradiol level is usually lower than 50pg/mL. It can indicate reproductive aging and hastened oocyte development if greater than 60 to 80 pg/mL in the early follicular phase. Luteinizing hormone (LH) plays an essential physiological role in follicle steroidogenesis and development and oocyte maturation. High basal LH levels are associated with significantly reduced fertilization rates and oocyte maturation, as well as impaired embryo quality, consequently resulting in an impaired pregnancy rate and higher miscarriage rate.,77 Collectively, ovarian function will be assessed using the basal endocrine in the early follicular phase.Thyroid peroxidase (TPO) and thyroglobulin(TG) are involved in thyroid autoimmunity as important antigens.,79 Electroacupuncture (EA) at the Guanyuan (RN4) or Sanyinjiao (SP6) can regulate hormone  levels in the HPO axis. Acupuncture from Renying (ST9) to Shuitu (ST10) can regulate neuro-immune-endocrine system to return thyroid hormone levels to normal and reduce the titer of antithyroid antibody. EA at Guanyuan (RN4) and Zusanli (ST36) has benign regulating effects on the key hormones of pituitary-target gland axis,such as FSH, LH, E2, TSH, T3, and T4. Lianquan (RN23), Renying (ST9), Danzhong (RN17), Guanyuan (RN4) and Qihai (RN6) points are located in Ren meridian which runs along the anterior median line of body and means dominating pregnancy. The thyroid gland is closely related to the Ren meridian because it is located in the anterior neck. According to the basic principles of acupoint indications, acupuncture the points on Ren meridian can regulate the qi of Ren meridian to treat thyroid diseases and improve fertility. Lianquan (RN23) and Renying (ST9) in the anterior neck near the thyroid gland can regulate thyroid function directly. Zhaohai (KI6) as one acupoint of Foot-Shaoyin connects with Ren meridian to treat thyroid disease.Acupoint selection has focused on Traditional Chinese medicine (TCM) theory or the treatment effects of acupuncture in this trial. SP6 and ST36 have been usually set for studies on endocrine disorder or reproductive syndrome.A limitation in this trial is that the acupuncturists cannot be blinded due to the nature of the intervention. We hope the results will provide reliable evidence and clarify the value of acupuncture as a treatment for child-bearing period female with HT.Conceptualization: Fangyuan Li, Zhang Qi, Lu Hua.Investigation: Xinxin Wang.Supervision: Mi Ling, Du Juan.Writing – original draft: Fangyuan Li, Zhang Qi.Writing – review & editing: Fangyuan Li, Lu Hua.


Hemiscorpius lepturus venom in rats. In this study, the neutralizing abilities of the equine and the recently introduced camelid antivenoms on the hemodynamic parameters  were assessed following envenomation by H. lepturus venom for 30 min at room temperature. Finally, equal amounts of the antivenoms were injected intravenously to observe the hemodynamic changes.At first, the electrophoretic profiles of both products were obtained by using the SDS-PAGE method (12.5%) and stained with Coomassie blue and silver nitrate. Secondly, different doses of the camelid antivenom  were given intravenously in 10 min before venom injection (400 µg/rat). The neutralizing potencies of camelid and equine antivenoms were measured by preincubation (100 µl) with th min).Based on the electrophoretic profile, it was evident that undesired proteins significantly decreased in equine antivenom, owing to impurities. Pretreatment with the camelid antivenom (100 µl), neutralized the elevation of the mean arterial pressure evoked with scorpion venom injection (88.15±4.56 versus 10.2±1.23 percent at the 8th min). The Incubation of the venom and the camelid antivenom counteracted the hemodynamic changes, but the equine product had no effect. The intravascular injection of the equine antivenom transiently increased the mean arterial pressure as compared to the control (108.67±8.63 mmHg versus 52.67±1.93 mmHg at the 10The most obvious finding emerging from this study was that the camelid antivenom neutralized the hemodynamic changes in rats significantly, but in comparison, the equine antivenom had just a minor ability. Scorpion sting as the oldest arthropod  . In thisHemiscorpius lepturus is one of the most dangerous scorpions in Iran 2 products reach the vital organs 5 to 9 times later following the venom exposure because of their large sizes . The precipitated camelid antivenom with saturated ammonium sulfate (SAS) was taken from Iranian Pasteur Institute following immunization of young camels with H. lepturus venom , were used in our study. In this case, raw venoms were milked by applying direct electrical shock (15 V) to their telsons. The collected venom was transferred to a microcentrifuge tube, lyophilized, and stored at -20 SDS-PAGEAt first, the protein concentrations of our samples were measured by the Bradford method with bovine serum albumin as the control . SecondlExperimental protocolMale Wistar rats (250–300 g) were housed in polycarbonate cages with free access to water and chow in the animal house of Bushehr University of Medical Sciences for one week before our study. They were anesthetized with ketamine  and xylazine  before the experiment.The anesthetized rats were placed supine under a heat lamp, and their temperature was monitored with a rectal tube connected to a thermometer . An incision was made in the right femoral area, and two cannulas were inserted in the femoral artery and vein, in order to administer the venom/antivenom and measure hemodynamic changes . Our data were analyzed using a pressure transducer  for a sustained recording of the arterial pressure by means of a Power Lab/4SP data acquisition system (AD Instruments). Final results were obtained 20 min after the beginning of the experiments following the administration of the venom or antivenoms. Mean arterial pressure was calculated using the following formula:  [(2 x diastolic) + systolic] divided by 3.Antivenom effectsThree groups of rats  were pretreated with different doses of the camelid antivenom  slowly ten min before the venom injection (400 µg/rat) . The lasThe effects of the camelid and equine antivenoms on hemodynamic parametersThe animals were divided into three groups (n=6), and the cardiac parameters were measured 20 min before the antivenin medications. Normal saline (200 µl) was injected in the first group as a negative control. The camelid and equine antivenoms with the same volume were infused via the femoral vein in two minutes. Inotropic, chronotropic, and arrhythmogenic parameters were evaluated in the last two groups and compared with the former. Statistical analysispost hoc test. Results were also analyzed by Student’s t-test. In all cases, differences were considered significant at P-value<0.05Results were expressed as mean±SD (standard deviation of the mean) and were evaluated using one- or two-way analysis of variance (ANOVA) followed by Tukey’s Gel electrophoresisThe electrophoretic profiles of the camelid and conventional antivenoms showed at least 7 and 3 bands in Coomassie blue staining distributed between 11-260 kDa . The majNeutralizing effects of the camelid antivenomThe scorpion venom evoked transient hypertensive effects considerably, and it reached the maximum level of 88.15% increase in mean arterial pressure (MAP) in eight minutes . The priHemodynamic potencies of the camelid and equine antivenoms Injection of the camelid and equine antivenoms  in separate experiments caused different inotropic, chronotropic, and arrhythmogenic properties among the anesthetized rats. There was no increase in MAP within 20 min after the camelid antivenom injection as compared to normal saline , while iNeutralizing efficiency of the incubated productsThe scorpion venom incubated with the equine antivenom (100 µl) increased the mean arterial pressure 53.8% at the fourth minute in the treated rats . AdditioScorpions are considered a threat to farmers, villagers, and passengers, especially in dry and hot environments. It is estimated that approximately 2.3 billion people are prone to scorpion bite . HoweverPrior studies have noted the importance of the careful evaluation of the hemodynamic changes, including myocardial damage, pulmonary edema, and occasional hypertension, due to scorpion envenomation, especially among infants . Cardiovascular deterioration caused by envenomation necessitates equine antivenom injection and symptomatic therapy as early as possible. Recently, camelid antivenom has been used due to its lower immunogenicity, more thermo-stability, and less potency to induce anaphylactic shock . H. lepturus, which belongs to the Scorpionidae family, has lower cardiogenic effects on rats compared to other venomous scorpions like Tityus serrulatus (1200 µg/kg versus 200 µg/kg) (It must be taken into account that 0 µg/kg) , 19-20. Furthermore, this study found that more attempts have been carried out by Razi Institute antivenom to eliminate the impurities of the equine antivenom .  H. lepturus venom  could significantly neutralize the transient arterial pressure elevation induced byus venom . Howeverus venom -25. Moreus venom . Unlike us venom , 27. Hottentota saulcyi envenomation (The present experiment was in line with the previous studies showing no neutralizing effects following animal pretreatment with the equine antivenom against cardiovascular changes caused by the venomous animals , 4, 28. nomation .There is no consensus among scientists regarding the neutralizing potency of equine antivenom on hemodynamic changes following envenomation in animals and humans. Returning to the hemodynamic results obtained in this study, it is evident that Razi Institute antivenom should be slowly infused in envenomed rats owing to its own tendency to raise mean arterial pressure. Furthermore, the camelid antivenom could be introduced as a novel therapy counteracting the hemodynamic dramatic changes.


Salmonella and HIV infections, colorectal cancer or severe acute malnutrition, has been associated with anaerobic gut microbiome alteration, impaired butyrate production, mucosal immunity dysregulation and disruption of host-bacterial mutualism. However, the role of major antioxidant molecules in the human body, such as glutathione, ascorbic acid and uric acid, has been neglected in this context. Here, we performed an in vitro metabolomics study of the 3 most odorous anaerobic microbes isolated from the human gut in our laboratory  when grown in anaerobiosis or in aerobiosis with these 3 antioxidant molecules via gas and liquid chromatography-mass spectrometry (GC/MS and LC/MS). There was no growth or volatile organic compound production in aerobic cultures without the 3 antioxidant molecules. In anaerobiosis, the major metabolic products of the bacteria were thiols, alcohols and short-chain fatty acid esters. The production of alkanes, cycloheptatriene and, paradoxically, increased butyrate production, was observed in the cultures grown in aerobiosis with the 3 antioxidant molecules. The qualitative shift suggests specific molecular mechanisms that remain to be elucidated. The increased production of butyrate, but also isobutyrate and isovalerate in vitro suggests that these 3 antioxidant molecules contributed to the maintenance and active resilience of host-bacterial mutualism against mucosal oxygen and uncontrolled oxidative stress in vivo.Uncontrolled oxidative stress, reported in  Among the several gut microbial odorants, butyrate is of particular interest to human health as it is the preferred energy source for colonic cells3. In addition, it inhibits inflammation and carcinogenesis, reinforces the colonic defense barrier, modulates gut epithelium permeability, promotes satiety, increases sodium and water reabsorption  and reduces oxidative stress4. Production of butyrate by gut commensals contributes to immunity regulation of the host by inducing the differentiation of colonic and systemic regulatory T cells6 and has an important epigenetic effect on gut epithelial cells by inhibiting histone deacetylase (HDAC)8. More specifically, a lower butyrate-to-acetate ratio has been associated with nonalcoholic steatohepatitis (NASH), adenomatous polyps or colon cancer9.Odorants are volatile organic compounds (VOCs) detected by the human nose that generate an olfactive signal and mainly include sulfur compounds ), low-molecular-weight carboxylic acids, also known as short-chain fatty acids , aldehydes, amines and heterocyclic compounds. Odorants, according to their chemical class, combination and/or concentration, lead to good or unpleasant odors10. That is, there is a link between redox potential, oxidative stress and human microbiota according to the oxygen tolerance of each species and the abundance of antioxidants in the environment. For the first time, we found a statistically significant association between fecal redox potential and metagenomic relative aerotolerant predominance10. In severe malnutrition, the lack of antioxidants and nutrients necessary for endogenous antioxidants and major liver peroxisomal and mitochondrial dysfunction results in major and uncontrolled oxidative stress12. In children with severe acute malnutrition, we have shown a major depletion of anaerobic microbes (intolerant to reactive oxidative species)13, consistent with the collapse of fecal butyrate in those who die14. This led us to rediscover that antioxidants allow for most anaerobes, including methanogenic archaea, to thrive in an oxidative (aerobic) environment20.On the other hand, we have recently identified a link between redox state and the microbiome23 that produced the strongest smells and cultured them in our anaerobic culture laboratory: Clostridium sporogenes, Clostridium subterminale and Romboutsia lituseburensis. We compared the VOCs produced by these 3 strains grown under standard anaerobic conditions or in ambient air with the addition of antioxidants 17 using gas and liquid chromatography coupled with mass spectrometry. We were surprised to see that the profile of VOCs was quantitatively but also qualitatively different. Indeed, the aerobic metabolic profile (available only with the 3 antioxidant molecules) was different from the anaerobic metabolic profile, which was confirmed by analysis of polar metabolites. The anaerobic metabolic profiles allowed for us to distinguish each species with perfect accuracy. Unexpectedly, butyrate production by the bacteria was increased in aerobiosis with medium supplemented with uric acid, ascorbate, and glutathione, suggesting a critical role of these antioxidant molecules in the maintenance of butyrate production at the gut microbial-mucosal interface.Serendipitously, we observed an absence of unpleasant odor when anaerobes were grown aerobically with antioxidants. To test what might explain the mitigation of the adverse smell, we selected 3 strains of the human gut isolated by microbial culturomicsThe 3 bacterial strains did not survive, and there was no production of VOCs or SCFAs by the bacteria in aerobiosis without the 3 antioxidant molecules. Accordingly, we compared the amount and profile of VOCs, SCFAs and polar metabolites of cultures grown in anaerobiosis and in aerobiosis with ascorbate, glutathione and uric acid.Clostridium sporogenes .The number and total amount of VOCs were higher in the cultures grown in anaerobiosis than in those grown in aerobiosis for all 3 species, and the difference was significant only for odorous VOCs Fig. . Both thThe 37 different VOCs produced by the 3 strains corresponded to 6 chemical classes, including 15 short-chain fatty acid esters (SCFAEs), 7 sulfur compounds (including thiols), 7 alcohols, 11 alkanes, 2 aromatic compounds and 1 alkene Fig. . By analC. sporogenes and R. lituseburensis were grouped together, whereas C. subterminale was clearly different. This contrasts with the phylogenetic analysis , while all short-chain fatty acid esters, thiols and alcohols were associated with anaerobiosis methane, commonly called dimethyl disulfide) and C. sporogenes . For C. subterminale, 3-methylbutyl 3-methylbutanoate was the most anaerobically produced VOC  in the medium20, such as those containing an SH group18. This has been shown with alkali sulfides as early as 1898, pyruvic acid (1923), cysteine (1926), glutathione (1926), thioglycolic acid (1926) and many other thioacids (1928)18. As early as 1940, Brewer showed that the absence of trace oxygen was not sufficient for growing strict anaerobes and developed a medium based on sodium thioglycollate, D-glucose (dextrose) and agar18. Sodium thioglycollate lowers the redox potential, D-glucose is a carbon source for bacterial growth, and agar was critical for lessening convection (2). More recently, titanium(III) citrate (E°H = −480 mV) was also successfully used as a reducing agent for anaerobic growth, including the methanogenic Archaea Methanobrevibacter arboriphilus20. However, redox-lowering activity is not the only factor. Jones et al19. showed that titanium(III) citrate alone allowed for the slow growth of Prevotella brevis, Butyrivibrio fibrisolvens, and Eubacterium ruminantium. In contrast, the addition of cysteine hydrochloride, an antioxidant and sulfur source, increased bacterial growth by a factor of 10019.The 3 molecules  were critical for 34. The relative contribution to the antioxidant power of human plasma is 60% for uric acid, 15% for ascorbic acid, 10% for protein and 5% for alpha-tocopherol and bilirubin35. Glutathione is one of the major intracellular and mitochondrial antioxidants36. Uric acid is excreted in the gut and rapidly degraded by gut bacteria37. Further studies are needed to clarify the relative antioxidant power and role in microbiota maintenance of glutathione, ascorbic acid, uric acid, proteins, including glutathione-associated enzymes (glutathione-S-transferase (GST) and glutathione peroxidase (GPX)), alpha-tocopherol38, beta-carotene38 and gut bilirubin derivatives 39 in the gut.Thioglycollate and titanium(III) have not been described in humans, while glutathione, ascorbic acid and uric acid are present and major antioxidants in animal and human plasma, gut and colonic mucosaet al.40 reported that there is an inducible ula regulator capable of anaerobically catabolizing L-ascorbate in several bacterial species. An additional YiaK-S operon was required in aerobiosis. This use was associated with the production of short-chain fatty acids, such as acetate40. However, the fact that ascorbate or uric acid may serve as a carbon source for bacteria in the gastrointestinal tract does not preclude a potential antioxidant role. Moreover, the detection of oxidized glutathione confirms the antioxidant role of reduced glutathione in the medium. While butyryl-CoA:acetate CoA-transferase activity is dominant in butyrate-producing bacteria in the human digestive tract29, it is the butyrate kinase pathway that is present in R. lituseburensis29. Further studies should investigate the specific molecular mechanisms linking the presence of oxygen, glutathione, ascorbic acid, and uric acid in the stimulation of butyrate production.Beyond aerobic survival, we have shown the notable existence of an aerobic metabolic profile that is different from that of bacteria grown under anaerobic conditions. The mechanisms allowing for this change between the 2 metabolic profiles will have to be elucidated in future studies. Linares 2 and ATP, and this is associated with water and sodium absorption25. Our findings shed light on the beneficial role of antioxidants on gut microbiota and butyrate production, previously reported in vivo, particularly with oligomeric proanthocyanidins (OPCs)42, but the microbial mechanism was not understood. Here, we show that antioxidants unravel an unexpected and unexplored aerobic metabolic profile of human gut Clostridia, and this profile is possibly active in vivo near the human gut mucosa. The physiological effect of the possible microbial production of alkanes, alkenes, and lysophospholipids near the human gut mucosa will need to be clarified in future studies. Nevertheless, the effect of butyrate on human gut mucosa and immunity is well known and is key to human health43.The conservation of butyrate production in aerobiosis with antioxidants observed here is particularly important because butyrate itself limits the oxygenation of the digestive lumen; in colonocytes, oxygen consumption with oxidation of butyrate by colonocytes gives CO44. Our study was an observational study and was designed to observe microbial metabolism according to the environment (atmosphere) and the presence of 3 major antioxidant molecules in the human body but not to clarify the molecular mechanisms. After linking gut redox and the microbiome10, this is the first study linking major antioxidants in the human body, gut microbiome and health through butyrate production. Future studies should investigate total microbial mass and metabolic pathways using biochemical analysis, proteomics, transcriptomics and genomics45 and include a control group grown in anaerobiosis with these 3 antioxidant molecules. It may be considered that the difference in the concentration of volatile organic compounds may also be related only to the difference in growth. However, this would only quantitatively and not qualitatively explain the differences in the metabolic profile.Gut microbes are key and instrumental elements in the complex mechanisms linking environment, behavior, diet and human healthClostridia to thrive and maintain microbial production of the key molecule butyrate in the presence of oxygen (in aerobiosis). This work provides new arguments for understanding the critical role of antioxidants in the maintenance of gut homeostasis and host-archaeal-bacteria mutualism and their resilience to uncontrolled oxidative stress and radical attack. This key role of antioxidants on microbiota probably extends beyond the digestive tract, as the beneficial effect of butyrate has been suggested for other human microbial ecosystems, such as the skin46, the gut-brain axis47 and atopic diseases48.Here, we demonstrated that a combination of 3 major antioxidants in the human body  allowed for human gut 23 and obtained from the Collection de Souches de l′Unité des Rickettsies , were used for this study: Clostridium sporogenes CSURP3957, Clostridium subterminale CSURP3759 and Romboutsia lituseburensis CSURP3909. These bacterial strains were previously cultured anaerobically for 24 hours at 37 °C on Columbia agar supplemented with 5% sheep blood . Then, the bacterial inoculum was calibrated to 106 bacteria/mL in PBS using a densitometer , and 0.1 mL of this solution (105) was inoculated into different culture bottles using a syringe. Classical anaerobic culture conditions in standard anaerobic blood culture  under a 100% nitrogen atmosphere were compared with aerobic culture conditions supplemented with an antioxidant cocktail in aerobic blood culture bottles (Becton Dickinson). Negative culture controls were performed under classical aerobic conditions in aerobic blood culture bottles (Becton Dickinson) without antioxidants. Sheep blood (5%) was previously added to each culture condition. The antioxidant solution was prepared as follows: a stock solution containing 4% ascorbic acid , 0.4% glutathione and 2% uric acid  was prepared in distilled water and filtered using a 0.2 μm filter after adjusting the pH to 7.5 with 10 M KOH. Then, 1 mL of this solution was injected into the aerobic blood culture bottle (Becton Dickinson) containing 40 mL of culture medium supplied by the manufacturer  using a sterile syringe and needle to avoid contamination. All blood culture bottles were incubated at 37 °C for different times  before measurement of VOCs, SCFAs and polar metabolites.Three strains of anaerobic bacteria, isolated by culturomics studies49. No viable bacteria were isolated after aerobic culture, while the viability and growth of all strains were confirmed in aerobic blood culture bottles supplemented with antioxidants and in anaerobic blood culture bottles.To confirm the viability of the bacterial strains at the end of the experiment, the blood culture content was subcultured anaerobically for 48 hours on Columbia Agar medium supplemented with 5% sheep blood (BioMérieux). Then, identification of the three bacterial strains was performed with MALDI-TOF-MS as previously describedTo measure the temporal production of VOCs in the bottles, different incubation times were tested for each culture condition: 48, 72 and 96 hours at 37 °C. After selecting the most suitable incubation time of 72 hours, VOCs were measured in triplicate for each condition. VOCs were collected for 10 minutes using a PDMS/DVB 65 μm SPME fiber  inserted in the headspace of each culture bottle. Gas and liquid chromatography-mass spectrometry (GC/MS and LC/MS) analyses were carried out with a Clarus 500 gas chromatograph equipped with a SQ8S MS detector  and with a liquid chromatograph. All data were collected and processed using Turbomass 6.1 (Perkin Elmer). A spectral database search was performed using MS Search 2.0 operated with the Standard Reference Database 1 A . Chemical identifications were validated with both reverse and forward scores above 800. The measurement of a blank sample was carried out in parallel, consisting of a noninoculated blood culture bottle containing the same culture medium as the inoculated bottles. Consequently, the results only considered VOCs measured in culture samples after subtracting the corresponding blank values.et al50.After finding that nonesterified short-chain fatty acids (SCFAs) were not detected by our first approach, we measured acetic, propanoic, isobutanoic (isobutyric), butanoic (butyric), isopentanoic , pentanoic , hexanoic (caproic) and heptanoic acids with a Clarus 500 chromatography system connected to a SQ8s mass spectrometer (Perkin Elmer) using the method described by Togo Clostridium sporogenes was cultivated under the same conditions as described above. Five culture replicates for the anaerobic and aerobic antioxidant cocktail culture conditions were prepared, as well as their five respective culture controls (without bacteria). Collected cell pellets from 1 mL cultures were resuspended in 400 µL of methanol at -80 °C to quench the sample. After centrifugation, the supernatants were dried and reconstituted in 50 µL of 10% acetonitrile, 90% water and 0.1% formic acid. Pooled quality control (QC) samples were prepared by pooling all bacterial samples with equivalent quantities (culture controls were not pooled). Metabolites were first separated by reversed-phase chromatography using an ACQUITY UPLC I-Class System chromatograph  and then monitored using a hybrid ion mobility QTOF mass spectrometer (Waters). Compounds were ionized in a Zspray ESI source using a default “Labile Tune” to reduce compound fragmentation before QTOF monitoring. Ionization parameters were set as follows: 0.8/2.0 kV for positive/negative modes, source/desolvation temperatures = 120/600 °C, and cone/desolvation gas = 100/600 L/h. Ions were monitored using a Sensitivity HDMSe data independent analysis method between 50 and 1000 m/z as follows: scan time, 0.15 s; collision energy ramp, 20–40 eV; and automatic mass correction during survey . Mass and CCS calibrations were run before analysis batches using the Major Mix solution (Waters). Raw data were then processed by UNIFI software to collect “components” described by retention time, drift time and precursor/fragment masses. Automatic parameters were set to detect 4D peaks above 150 and 75 counts for low and high energy scans. The marker table was sent to the EZinfo multivariate statistics software  for data exploration by PCA and then data classification according to both anaerobic and aerobic groups using an OPLS-DA model. The selected markers were then labeled on the marker table in UNIFI and searched using the elucidate tool against the following structure libraries: metabolite structures  and the KEGG + HMDB databases (Chemspider access). Potential identities were then added to an inhouse structure library including.mol files from Chemspider  and then targeted on the initial raw data according to the following parameters: a 5 ppm mass tolerance, a 2 mDa mass tolerance for predicted structure fragments, a 5% CCS deviation, and multiple adducts  within 5 ppm and 0.1 minutes. Identifications were then filtered with the following parameters to validate metabolites: mass error <2 ppm for precursor ions, <2% CCS deviation (if available), at least one predicted structure fragment, <10% error on CCS estimates, <5 ppm error on RMS isotope match and <15% RMS percent isotope intensity51. The consistency of metabolites between positive and negative experiments were also checked . The fold change was defined as the ratio of response values from the mean response for each condition.C. sporogenes, R. lituseburensis and C. subterminale) and atmosphere  were considered qualitative variables. Unless noted otherwise, three replicate values were measured. Comparing aerobiosis with antioxidants and anaerobiosis, three t-tests (one by species) were performed, and differences were determined using the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 1% (VOCs) or 5% (SCFAs). Each row was individually analyzed without assuming a consistent SD. When 3 groups were compared , two-way ANOVA was also performed with correction for multiple comparisons using Tukey’s test. Principal component analysis with Pearson metrics was performed, and biplots were shown to explore the association between strains, atmosphere and metabolites. Statistical tests were two-sided. A p-value ≤ 0.05 was considered significant. These statistical analyses were performed using GraphPad v8.0  and XLSTAT v19.1 software .The peak areas of each detected VOC (arbitrary units) and the concentration of each SCFA (mM) were assessed as quantitative variables. Species (The data that support the findings of this study are available from the corresponding author.Supplementary information.


Medicago and its relatives, Trigonella and Melilotus comprise the most important forage resources globally. The alfalfa selected from the wild relatives has been cultivated worldwide as the forage queen. In the Flora of China, 15 Medicago, eight Trigonella, and four Melilotus species are recorded, of which six Medicago and two Trigonella species are introduced. Although several studies have been conducted to investigate the phylogenetic relationship within the three genera, many Chinese naturally distributed or endemic species are not included in those studies. Therefore, the taxonomic identity and phylogenetic relationship of these species remains unclear. In this study, we collected samples representing 18 out of 19 Chinese naturally distributed species of these three genera and three introduced Medicago species, and applied an integrative approach by combining evidences from population-based morphological clusters and molecular data to investigate species boundaries. A total of 186 individuals selected from 156 populations and 454 individuals from 124 populations were collected for genetic and morphological analyses, respectively. We sequenced three commonly used DNA barcodes  and one nuclear marker (GA3ox1) for phylogenetic analyses. We found that 16 out of 21 species could be well delimited based on phylogenetic analyses and morphological clusters. Two Trigonella species may be merged as one species or treated as two subspecies, and Medicago falcata should be treated as a subspecies of the M. sativa complex. We further found that major incongruences between the chloroplast and nuclear trees mainly occurred among the deep diverging lineages, which may be resulted from hybridization, incomplete lineage sorting and/or sampling errors. Further studies involving a finer sampling of species associated with large scale genomic data should be employed to better understand the species delimitation of these three genera. The three genera are closely related and the latter two genera were included in Medicago sensu lato by some authors (Medicago sativa L.), which is known as “the queen of forage crops”  and alfalfa are widely used in the treatment of diabetes, menstrual cramps and high cholesterol  were found to be informative for phylogenetic resolution at species level within Medicago (trnK-matK and psbA-trnH) and the nuclear DNA (nrDNA) marker (GA3ox1), and combined morphological variation with molecular evidence to examine phylogenetic relationships and species delimitation of Medicago, Trigonella, and Melilotus, using species from China as a case study.DNA barcodes, such as families . Howeverfamilies . Previouelilotus , while aelilotus . FurtherMedicago . Therefo1, 15 Medicago species , eight Trigonella species and four Melilotus species are recorded  are introduced and widely cultivated in China. Some of them  have been escaped to roadsides, fields and stream banks and adapted to variable environments. The remaining species are naturally distributed in China and adjacent countries, and only Medicago archiducis-nicolai is endemic in China. This endemic species together with other three Chinese species  belong to the sect. Platycarpae. Phylogenetic analysis based on three DNA markers  indicated that M. archiducis-nicolai is clustered with M. ruthenica and M. platycarpos, but M. edgeworthii is clearly not nested within the sect. Platycarpae clade (Trigonella arcuata and Trigonella cancellata) and other four Trigonella  remains unclear. To examine the delimitation and relationships among these Chinese species, we sequenced three DNA barcode regions  and one nuclear marker (GA3ox1), and remeasured 19 morphological traits at the population level. Specifically, we aimed to address the following questions: (1) Which marker is the most effective for phylogenetic resolution? (2) Do the two “medicogoid” species (T. arcuata and T. cancellata) also cluster with the Medicago clade? (3) How many species should be recognized in the three genera in China based on the integrated results of molecular and morphological variation? (4) Are the phylogenies reconstructed by nrDNA regions and chloroplast DNA (cpDNA) fragments congruent?According to the Flora of Chinarecorded . Among tae clade . In addiT. pamirica. In addition, we also collected three introduced species  because of their significant values as legume forage and included the well-known model species M. truncatula in our analyses, resulting in a total number of 22 species  in the western or southwestern borders of China, we could only collect one or two populations for each species (T. emodi and three samples of T. fimbriata from the specimens in Kunming Institute of Botany Herbarium (KUN). We followed standard protocol and collected the individual samples that were at least 20 meters apart in naturally distributed populations. In total, we collected fresh leaves and specimens for all the 21 species from 156 localities in China. We used silica gel to dry the fresh materials, and deposited all voucher specimens in Lanzhou University. The elevation, latitude, and longitude of locations were recorded using the mobile phone application “GPSkit v.2.3.9.” We selected 1–6 individuals from each of the 156 populations for molecular analysis, which resulted in a total of 186 individuals. The detailed information for the sampling localities and individuals used for molecular analysis was listed in For the naturally distributed species of the three genera in China, we sampled 18 out of 19 species (94.7%) but failed to find the species  species . For the species  in the f species . We alsopsbA-trnH and trnK-matK) and two nrDNA regions (GA3ox1 and ITS) to amplify and sequence 166–186 individuals of the 21 species.The whole genomic DNA of all samples was isolated from nearly 30 mg dried leaves according to the TIANGEN plant genomic DNA Kit  following the manufacturer’s instructions. We used previously published primers  of two c® Master Mix  containing dNTPs, rTaq polymerase and 10 × PCR buffer, 10 μM of two primers and ddH2O added to 25 μL. For trnK-matK with nucleotide length of more than 2500 bp, multiple primer pairs were used for PCR amplification and sequencing (MW241635-MW242342).We performed the PCR amplification in 25 μL volume containing 1 μL of plant DNA with 50–150 ng/μL, 12.5 μL 2 × TSINGKEquencing . The PCRquencing , 45 s atM. truncatula and four species of genus Trifolium  that were selected as outgroup , 186 trnK-matK , 177 GA3ox1 , and 178 ITS . The length of the four aligned markers ranged from 497 bp for psbA-trnH to 2565 bp for trnK-matK ; Clade 2 contained four species Medicago lupulina (sect. Lupularia), Medicago minima, M. polymorpha, and M. truncatula (sect. Sphaerocarpos), and M. sativa complex comprising three species, M. sativa, Medicago falcata, and Medicago × varia (sect. Medicago); Clade 3 contained four “medicagoid” species ; while other three Trigonella species  were grouped as Clade 5. Clade 4 included all the four Melilotus species. Clades 4 and 5 were basal to all other groups and the four “medicagoid” species (Clade 3) were reciprocally monophyletic to all Medicago species (Clades 1 and 2). All the five clades were strongly supported in both ML and BI analyses, except for Clade 2, which had a bootstrap value of 78 , both at the independent and concatenated marker levels . The physequence  had the sequence . The nucinferred . Clade 1ue of 78 .M. edgeworthii was grouped with the four “medicagoid” species, which was nested within Medicago clade. Clade 2 in the cpDNA tree was split into two clades in the nrDNAS tree both with high support. Clade 1 was moved to a basal position in the nrDNA tree. In general, phylogenetic relationships among species within each clade received higher support in the nrDNA tree compared to those in the cpDNA tree. Moreover, the three species of M. sativa complex or the two Trigonella species (T. emodi and T. fimbriata) were always grouped together and could not be separated from each other in both phylogenetic trees.In contrast, phylogenetic relationships among and within the Clades 1–3 in the nrDNA tree were incongruent with those in the cpDNA tree . For exaM. ruthenica and M. archiducis-nicolai. In Clade 2, the three species of M. sativa complex were clustered together as indicated by the phylogenetic tree, whereas other species formed distinct clusters. Clade 4 had similar clustering pattern as Clade 1, where overlap was present between Melilotus indicus and Melilotus officinalis, which was not highly supported as two distinct species although samples from each species seemed to group together in the phylogenetic trees. The four “medicagoid” species (Clade 3) formed generally distinct clusters, while the two Trigonella species (T. emodi and T. fimbriata) could not be distinguished based on morphological characters as shown for Clade 5. Finally, the four “medicagoid” species were morphologically closer to M. edgeworthii than the other Trigonella species  as well as trnL-F, rbcL, and matK are commonly used as core barcodes for plants  may be useful when the commonly used barcodes exhibit relatively low resolution in species delimitation.DNA barcodes are standardized fragments of DNA that were developed with the aim of distinguishing between both closely and distantly related species of different genera and families . Multiplr plants . The nucr plants , and thu studies . The disM. polymorpha) out of the 21 Chinese species can be delimited with high support values in the phylogenetic trees and distinct morphological clusters  have already been revised as Medicago species (M. monantha and M. orthoceras). Our results indicated that the four “Medicagoid” species formed a monophyletic group and nested within Medicago. Therefore, the other two species  should be also transferred to Medicago.“Trigonella/Melilotus clade), deep relationships among the early diverging lineages within Medicago  recovered in the two trees are inconsistent. Such discordance of gene trees derived from nuclear and cytoplasmic markers has been found in numerous studies and usually explained by two non-exclusive factors, i.e., hybridization and introgression, and/or incomplete lineage sorting . However, incomplete lineage sorting that causes the retention of ancestral polymorphism in different species and/or sampling error cannot be completely excluded because of the relatively small sampling size. We, therefore, recognized that population genomic level data and more species sampling would be necessary to clearly discriminate among these possible scenarios.Although the clades inferred in the cpDNA and nrDNA phylogenetic trees all received strong statistical supports, the relationships among them are incongruent in the two trees. Except for the two basal clades 4 and 5 , accession numbers: MW241635–MW242342.The data presented in the study are deposited in the NCBI repository (GR and JL conceived and designed the project. JC, WG, and MY collected the materials. JC and GW carried out the lab work and measured the morphological traits. JC, SW, and AL performed the analysis. JC and GR wrote the manuscript with the help from NS and JL. All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Moreover, statistical differences were observed between Apatos and both Putty (p = 0.014) and Gel 40 (p = 0.007) groups, at 30 days, in regard to particle percentage. Within each group, regarding new bone formation, mp3 showed significant differences (p = 0.028) between 15 (40.93 ± 3.49%) and 30 (52.49 ± 11.04%) days. Additionally, intragroup analysis concerning the percentage of particles revealed a significant reduction in particle occupied area from 15 to 30 days in mp3 and Gen-Os groups (p = 0.009). All mp3, Gen-Os and Apatos exhibited promising results in terms of new bone formation, thus presenting suitable alternatives to be used in bone regeneration.Collagenated porcine-derived bone graft materials exhibit osteoconductive properties and the development of different formulations intends to enhance bone regeneration. This study aims to evaluate bone healing in a rabbit cancellous bone defect in response to grafting with different physicochemical forms of heterologous porcine bone. Twenty-six adult male New Zealand White rabbits received two critical size femoral bone defects per animal (n = 52), each randomly assigned to one of the five tested materials . Animals were sacrificed at 15- and 30-days post-surgery. Qualitative and quantitative  histological analyses were performed. Histomorphometry showed statistically significant differences in all evaluated parameters between mp3 and both Putty and Gel 40 groups, regardless of the timepoint ( Nowadays one of the most pressing subjects in modern oral rehabilitation is the recovery of lost or resorbed bone architecture, aiming at a functional and aesthetic recovery ,2. SeverBone substitutes consist of any biomaterial, biologic or synthetic, intended for implantation in humans with the prospect of rebuilding bone mass, strengthening bone structure or filling bone loss ,13. BoneCurrently, material selection for a given intervention is based on several factors such as the characteristics of the material itself, the type of bone defect to be treated, the operator’s preferences, the associated costs, and the patient’s acceptance. The range of clinical situations is wide, and a single material may not be the universal solution, but it is rather imperative to observe its characteristics, as well as its formulation and presentation, that may be best suited for each specific clinical condition. Although autologous bone is still considered the gold standard when it comes to bone substitutes ,17, clinUndecalcified bone tissue samples and histomorphometric techniques revolutionized the understanding of bone structure and physiology ,30. In fCurrently, the material selection for a given intervention is based on several factors such as characteristics of the material itself, type of bone defect to be corrected, operator’s preferences, ease of handling, associated costs and patient’s acceptance. The present study focuses on novel formulations of porcine-derived xenografts, with some offering new presentations that may render an easier handling and application with the possibility of maximizing technical performance if histological outcomes reveal promising.The main purpose of the current study is to evaluate bone healing in a rabbit cancellous bone critical size defect in the lateral aspect of the distal femur in response to filling with five different physicochemical forms of heterologous porcine bone .The null hypothesis of this experimental work states that the five different porcine-derived bone graft materials exhibit similar histological and histomorphometric results.This experimental protocol was approved by the national regulatory authority in animal research, as well as by the University of Trás-os-Montes and Alto Douro Ethics Commission (N° CE 29/2015). Animal housing and manipulation, as well as experimental procedures and data reporting followed Portuguese legislation related to the use of animals for experimental purposes  and European Legislation Directives on the protection of animals used for scientific purposes (Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010).Sample size calculation was performed using G*Power software considering bone defect as the unit, sustained on previous study results obtained in the scientific work of Palma et al. . In thisThree possible levels of significance were considered, α = 0.01, α = 0.05 or α = 0.10. Three different power levels were also considered, 0.80 (1−β = 0.80), 0.90 and 0.95. A bilateral Student’s t-test of independent samples and an allocation ratio between groups of 1:1 was also used to calculate the sample size.Oryctolagus cuniculus), weighing around 5.2 ± 0.56 kg, were included. This sample was divided into two series—15 and 30 days—of 13 animals each. In each animal, two test sites were surgically created in the lateral aspect of the distal femur of opposite limbs, thus a total of 52 test sites constitute the sample for research.Based on the obtained data, a sample consisting of 26 adult male New Zealand White rabbits  conditions.Surgical procedures were performed at the Veterinary Hospital of the University of Trás-os-Montes and Alto Douro and before undergoing surgery, animals were moved to specific presurgical rooms to maintain their stress levels low, as well as those from all housed animals. After surgical procedures, animals were transferred to specific recovery cages until full awareness and locomotion capabilities were demonstrated and then moved to their respective standard cages for unrestricted locomotion. Commercially dry food with controlled contaminant concentrations, according to manufacturer, was provided once a day and water was supplied ad libitum. Tested materials were randomly distributed using a computer algorithm in relation to animal, anatomical location and placement sequence, thus keeping the animal number to a minimum necessary by decreasing the effects of individual variation.-®, Tecnoss, Torino, Italy)Apatos group—100% porcine cortical bone granules (600–1000 µm particles) without preservation of collagen Gen-Os group—100% porcine cortico-cancellous bone granulated mix (250–1000 µm particles) with preserved collagen mp3 group—90% porcine cortico-cancellous bone granulated mix (600–1000µm prehydrated particles) with preserved collagen with 10% collagen gel Putty group—80% porcine cortico-cancellous bone granulated mix (<300 µm micronized particles) with preserved collagen and 20% collagen gel Gel 40 group—60% porcine cortico-cancellous bone granulated mix with preserved collagen (<300 µm micronized particles) and 40% type I and III collagen gel , a capnograph and cardiac auscultation.Animals went through a twelve-hour fasting period, maintaining only water supply, before anesthesia which was initially induced using medetomidine (0.15 mg/kg SC) and butorphanol (0.1 mg/kg IM). Fifteen minutes after this initial procedure, the marginal ear vein was located, a gentle local trichotomy was made, and a slow injection of ketamine (5 mg/kg IM or EV) was administered. Endotracheal intubation was then performed with a small endotracheal tube in order to maintain a controlled anesthetic state by continuous inhalation of isoflurane (0.25–2%) in an oxygen flow of 0.5–2 L/min/kg. Monitoring throughout the surgical procedure was done using a noninvasive oximeter  and ensuring adequate sterilization conditions for the surgery. During surgical procedures, only the intervention zone was exposed.The area of interest was trichotomized, the animal was placed on a heated operating table in lateral decubitus and the limb was immobilized and suspended without excessive pressure to expedite disinfection procedures and allow for an easier establishment and maintenance of the sterile surgical field. Both surgeons then started preparing the surgical table, disinfecting the surgical field with a povidone-iodine solution  was created in each test site (n = 52), in the lateral aspect of the distal femur in both limbs, through the distolateral cortex of the stifle joint without compromising the medial cortical bone and joint biomechanics. The volume of material placed on each bone defect was approximately 0.2 cc (V = πr2h).Only one negative control per group was used, as critical size bone defects are already sufficiently established and referenced in the literature, requiring no model validation and, thus, reducing the number of animals ,39.®, Tecnoss, Torino, Italy) was shaped, hydrated and applied over the filled bone defect, overlapping its margins  was administered for pain control. A single long-term amoxicillin dose  was also administered. Animals were placed in individual recovery cages at a dark, quiet, comfortable, clean location with controlled temperature and free of objects or materials that potentially could cause any harm when animals began attempts to move. When necessary, body temperature was maintained with the aid of thermally insulating wrapping materials. When full capacity for consciousness and locomotion was observed and anesthetic recovery was achieved, animals were then transferred to their standard cages with unrestricted movement and free access to food and water. Daily monitoring was conducted to evaluate any changes in food or water intake, body weight, typical ethological patterns of the species and for the presence of abnormal signs or adverse reactions, as well as pain.At the end of each experimental period (15 and 30 days postoperatively), animals were euthanized individually, in an isolated room with no sensory access to other animals, by administering a lethal dose of ketamine hydrochloride. Animal necropsy was performed to assess the systemic impact of the biomaterials under study. After macroscopic analysis, fragments of the main organs  were collected and processed for studies. The corpses and waste from euthanasia were subsequently incinerated in accordance with current legislation.® system . Regarding staining methods, toluidine blue was used.The material was harvested en bloc after meticulous soft tissue dissection. Collected samples were processed with an undecalcified technique, providing high-quality histological images, without morphological distortions of relevant structures or meaningful artefacts with the high-precision Exakt® SMZ 1500, Tokyo, Japan) and an optical microscope  in order to produce photographic records of the observations. Qualitative analysis focused mainly on the observation and recording of histomorphological tissue characteristics, bone/biomaterial interface, and the presence or absence of inflammatory response. The quantitative histological analysis, histomorphometry, was performed based on the image analysis software Bioquant® Nova  which calculates the area of different tissues based on color variation. The same operator performed all calibration procedures, as suggested in literature [A qualitative and quantitative analysis of the processed study material was performed. For both analyses, slides obtained by the histological processing were observed using a stereomicroscope , according to both biomaterial and period of evaluation. Additionally, descriptive statistics included the construction of dispersion diagrams for each evaluated parameter.Statistical analysis was performed using the commercially available IBMKruskal–Wallis and Mann–Whitney nonparametric tests were used to detect significant differences in the median across the groups. Subsequently to Kruskal–Wallis testing, Dunn–Sidak post-hoc testing with correction for multiple comparisons was carried out. It should be noted that, although there were measures taken in two different times, the animals were different and, therefore, only independent tests were used.This report follows the Animal Research: Reporting of In Vivo Experiments Guidelines (ARRIVE Guidelines) .The postoperative period was uneventful, with no systemic or local complications resulting from the surgical intervention being detected. Rapid recovery with healthy ethological behaviors revealed a reduced impact of the experimental protocol on animal health and welfare. Macroscopic observations of the en bloc collected femurs, showed no morphological changes or the existence of inflammatory processes beyond those expected and resulting from the surgical procedure. The histopathological analysis of collected organs showed no anatomopathological abnormalities.The set of analyzed bone defect cross-sections showed a sequential and centripetal process in which trabeculae of new bone coated, integrated and bound the particles among themselves and to the defect walls. At 30 days, large areas of immature bone tissue can be found, along with some lamellar bone and intense bone formation activity, thus indicating an ongoing remodeling process .All analyzed bone defect cross-sections displayed a remarkable and intense osteoclastic activity, often adjacent to also significant amounts of osteoblast cells and osteoid matrix related to osteogenic phenomena.Between 15 and 30 days, there is a clear difference in terms of bone remodeling phenomena shown by the notable areas of immature and some lamellar bone tissue trabeculae that colonize interparticle spaces but also replace the particles themselves .All bone defect transversal sections displayed intense bone neoformation with extensive bone trabeculae networks. At 15 days the central region of the defect already presented bone formation. At both timepoints, bridging phenomena are observed, with newly formed bone tissue integrating and connecting graft particles both on the peripheral and central areas of the defects. At 30 days lamellar bone tissue is more prevalent. Intense osteogenic activity is recorded at both timepoints with numerous active osteoblasts and osteoclasts. This activity is inferred by the presence of osteoblasts characterized as large mononucleated cells with an ovoid profile and abundant basophil cytoplasm, reflecting a high rate of protein and proteoglycan synthesis, often arranged in single cell lines along the immature bone surfaces, thus believed to synthesize organic components of bone matrix (osteoid) prior to their mineralization. The cell-mediated process of particle resorption was visible .The results shown for both groups in both timepoints have almost no variation in terms of tissue quality, with particle dispersion and migration being visible in both materials. Few to no bone formation is observed beyond the peripheral zones of the defect at both 15 and 30 days of evolution. Histomorphometric analysis was based on cross sections showing the entire defect, as shown in p = 0.028), with a percentage of new bone at 30 days (52.49 ± 11.04%) being statistically higher than the percentage of new bone observed at 15 days (40.93 ± 3.49%). Regarding new bone formation , the comp = 0.009) and Gen-Os group (p = 0.009), with a reduction in particle occupied area percentage from 15 to 30 days. Concerning the percentage of particles  in the dp < 0.05).At 15 days of evolution, comparing the groups among each other, the mp3 group presented statistically significant differences with the Putty and Gel 40 groups, independently of the analyzed parameter (p = 0.035) and Gel 40 (p = 0.015) groups. Regarding the percentage of particles (p = 0.022) and Gel 40 (p = 0.011) groups, as well as between the Apatos group and the Putty (p = 0.014) and Gel 40 (p = 0.007) groups.At 30 days, considering the percentage of new bone , statistarticles , statistTaking into consideration these results, the null hypothesis stating that the five different porcine-derived bone graft materials exhibit similar histological and histomorphometric results, is then rejected.This experimental study was designed with the aim of understanding and evaluation of the bone regeneration potential of different bone graft materials in a physically contained bone defect. Formulations with isolated particles needing hydration and new formulations containing different carriers and particle concentrations were evaluated.Although the cellular response to different biomaterials is based on in vitro studies, these tests do not accurately mimic in vivo realities. On the other hand, human models for bone regeneration studies have some major inconveniences such as the impracticality of performing adequate histological evaluations, even in cases where a biopsy can be performed, as well as challenges designing a study with satisfactory internal validity ,33. TherRegarding the chosen intervention location, although skullcap healing model would embryologically be considered as the gold standard in bone graft material evaluation for dentistry, research shows a tendency for abnormal particle migration when different formulations are tested in skull models. For that reason, a model of critical defect, defined as the smallest bone defect that does not heal spontaneously, with less than 10% new bone formation during the life of the animal, in rabbit’s femoral condyle was chosen for this study ,42,43,44Concerning the tested materials origin, Corbella et al.  publisheConsidering the addition of collagen to bone substitutes, as presented by some of the tested materials, Nannmark et al.  reportedComparing Apatos group with other tested materials, this cortical biomaterial presented the lowest amount of osteoclastic cells throughout the observed samples. When found, osteoclasts are adjacent to newly formed bone and very rarely around bone graft particles. Iezzi et al. . describAt Gen-Os group, either at 15 or 30 days, all defect transversal sections displayed a remarkable and intense osteoclastic activity adjacent to significant amounts of osteoblast cells and osteoid matrix related to osteogenic phenomena, demineralizing, resorbing and disintegrating bone graft particles. Hence, it might be discussed that as a result, resorbed particle matrix may release various growth factors responsible for the proliferation and differentiation of pre-osteoblasts that migrate to this area from perivascular zones or bloodstream and create conditions for an effective colonization by cells of the osteoblastic line. Following these processes of osteoclastic degradation, several osteoinductive molecules  contribute to the synthesis and mineralization of osteoid matrix ,59,60. Ip = 0.009) may potentially be explained by the intense osteoclastic activity which leads to particle fragmentation and resorption. Regarding new bone formation, the mp3 group presented the highest percentages of all tested materials in both time periods with 40.93 ± 3.49% at 15 days and 52.49 ± 11.04% at 30 days. Barone et al. [Concerning 90% porcine cortico-cancellous bone granulated mix with preserved collagen with 10% collagen gel (mp3), while performing the surgical part of this experimental study, the syringe presentation allowed for an easier handling of the material. This presentation appears to be an improvement over classical particulate materials and confers a safe and seemingly stress-free way of applying the material without wasting. The fact that this formulation is prehydrated also presents a major advantage for the clinician. Comparing mp3 group with other tested materials, in a strictly histological evaluation, this biomaterial seems to perform better than any other tested material at any timepoint. At 15 days the bridging phenomena with woven bone tissue even at the centre of the defect was evident and at 30 days a trabeculae network creates a continuity between opposite margins and also along the defect’s entire perimeter. Extensive areas of osteoblasts and osteoid in particle and trabeculae periphery reflect still ongoing osteogenic processes at both timeframes. Heterogeneous shapes, dimensions and tinctorial characteristics are also found in some granules, portraying particle resorption and demineralization process instigated by numerous osteoclasts broadly found adjacent to the biomaterial. These findings are in agreement with literature descriptions ,48,65,66e et al.  studied e et al.  used rabe et al.  comparedAlthough the images from Putty and Gel 40 groups displayed in Regarding the discrepancies between our findings and some of the literature, we conjecture these differences may occur due to the demanding of our critical size defect or the different methodologies of experimental study design and evaluation, even though this experimental work attempted to mimic clinical conditions by using an heterologous pericardium membrane to cover the defect and prevent material displacement, which seems to have been effective in the Apatos, Gen-Os and mp3 groups. The tested porcine-derived bone graft materials presented favorable biocompatibility and appeared to undergo extensive resorption, demineralization and particle disintegration processes that may lead to biomaterial replacement by newly formed bone. All mp3, Gen-Os and Apatos exhibited promising results in terms of new bone formation, thus presenting suitable alternatives to be used in bone regeneration. Putty and Gel 40 show unfavorable histological outcomes when compared to the remaining biomaterials, with statistically significant histomorphometric differences when compared to mp3, regardless of the parameter and timepoint considered.