To evaluate three Tai Chi (TC) exercise programs as intervention measures to compare their effects on improving rate of change in bone mineral density (BMD) in elderly individuals with osteoporosis (OP) and to propose the optimal exercise duration.n = 13, 7 males/6 females), TC Kung Fu Fan group (TCKFFG) , TC Softball group (TCSBG) , and a control group (CG) . Except for the control group, each group received different TC exercise programs four times a week for 60 min per session, lasting for 16 weeks. BMD was measured using dual-energy X-ray absorptiometry (DXA) at the L2-L4 lumbar vertebrae, Ward's triangle, femoral neck, and greater trochanter. The rate of change of BMD was calculated using the formula.A randomized controlled trial (RCT) was conducted to identify study participants based on inclusion and exclusion criteria. Due to subject attrition, the number of participants analyzed decreased from 60 to 49. These participants were divided into four groups: 24-style TC Chuan group (24TCCG) (P < 0.05), but their effects on the improvement of femoral neck and greater tuberosity BMD change rates were similar (P > 0.05). In addition, compared to the other exercise regimens, 24TCCG demonstrated more significant improvements in BMD at the L2-L4 lumbar vertebrae region and exhibited a more pronounced improvement in Ward's triangle BMD after only 8 weeks (P < 0.05). Short-term (≤ 4 weeks) TCKFFG was more effective than TCSBG in enhancing femoral neck BMD (P < 0.05). However, statistical significance was not found (P > 0.05) in all other cases.Compared with CG, all three TC groups showed significant improvements in BMD changes (These three TC exercise programs have similar positive effects on the BMD of the femoral neck and greater trochanter. However, compared with other exercise schemes, 24TCC showed a more significant improvement in BMD of the L2-L4 lumbar vertebrae region after just 8 weeks, as well as a more pronounced improvement in BMD of Ward's triangle. In terms of improving femoral neck BMD, TCKFF was found to be more effective than TCSB in less than 4 weeks. This study provides evidence for the effectiveness of TC exercise in improving BMD and preventing OP in the middle-aged and elderly high-risk population.The online version contains supplementary material available at 10.1186/s13018-023-04324-0. Aging is associated with decreased bone health and an increased risk of osteoporosis (OP) , 2. OP iIn recent years, there has been growing interest in the potential benefits of Tai Chi (TC) exercise in improving BMD in this population. TC is a unique Chinese exercise method that combines gentle, flowing movements with rhythmic breathing and meditation . It is cTherefore, this study addresses the aforementioned issues and innovatively evaluates the effects of three types of TC exercise on improving the rate of change in BMD and reducing the risk of OP in middle-aged and older individuals at risk. It aims to provide evidence for the potential optimal exercise duration in TC exercise for the prevention and treatment of OP in the middle-aged and older population.This investigation was structured as a randomized, double-blind controlled trial, executed with strict adherence to the ethical tenets delineated in the Declaration of Helsinki of the World Medical Association , and sanThis study recruited middle-aged and older individuals engaged in teaching activities in TC societies through random selection. Inclusion and exclusion criteria were established to limit variability and maintain subject baseline characteristics. Inclusion criteria included being between 55 and 65 years of age, voluntarily participating in TC club instruction, not being a manual laborer, being able to effectively perform the technical demands and loads of the movements during TC instruction, and having been assessed as "positive" by the ONE-MINUTE OSTEOPOROSIS RISK TEST (OMOST) . ExclusiWe conducted a power analysis and estimated the sample size using G*Power software (version 3.1.9.7) . Based on = 15, 8 males/7 females), TCKFFG , TCSBG , and CG , but at a later stage, due to subject attrition the number of people in each group ended up being 24TCCG , TCKFFG , TCSBG , and CG . The intervention's purpose and design protocol were kept confidential from TC teachers and BMD measure participants to maintain blinding. However, blinding was not extended to the study subjects due to the instructional requirements of the TC exercise programs.This study employed randomization for coding subjects who met inclusion criteria, utilizing stratified random grouping with SPSS 26.0 software. Personnel not involved in the study were responsible for coding subjects to four different groups, including 24TCCG  but involve different exercise methods:(i) 24TCC: It consists of 24 movements, including the basic actions and postures of TC, focusing primarily on the smoothness and coherence of movements, while emphasizing the fundamental principles of TC.(ii) TCKFF: In TCKFF, the movements of TC are combined with the use of a fan, requiring practitioners to master the basic techniques of TC and learn how to perform various actions with the fan.(iii) TCSB: This is a TC fitness tool typically composed of two metal balls connected by a rope. Practitioners rotate the balls in their hands to perform TC-style movements. The use of this tool helps improve hand–eye coordination, balance, and flexibility, with a focus on actions involving the hands and wrists.Baseline characteristics of each subject group were recorded prior to experimentation, and homogeneity of the data was tested to ensure scientific and effective subsequent study. Mean and standard deviation data are presented in Table 2. The error coefficients for BMD measurements were 1–2% for lumbar vertebrae, femoral neck, and greater trochanter, and 2.5–5% for Ward's triangle [This study utilized dual-energy X-ray absorptiometry (DXA) to measure BMD at four anatomical sites  over five consecutive noncardiac rate scans in g/cmtriangle . The coetriangle . BMD meaAll BMD data and intervention results were subject to meticulous two-person cross-entry and verification. The intervention effect was assessed via the formula of (after intervention-before intervention)/before intervention × 100%, with percentages before and after intervention systematically observed. Given that BMD is a continuous variable in this context, we express it as mean ± standard difference (M ± SD) and determine 95% confidence intervals (CI).P < 0.05, the statistical results were deemed significant, while P < 0.01 represented very significant differences. ANOVA was performed for each group, followed by post hoc multiple comparisons. Least significant difference (LSD) was used where P > 0.05 to determine data homogeneity of variance, while Tamhane 2 (T2) test was employed when P ≤ 0.05 indicated variance heterogeneity.Statistical analysis of data processing was performed using SPSS 26.0 software. Abnormal data and normal distribution were detected using the Shapiro–Wilk test in Explore, and any such data were systematically verified and eliminated. To determine the change rate of a given index between groups before and after the intervention, one-way analysis of variance (ANOVA) was utilized. Where GraphPad Prism 8.0.2 software was utilized to generate graphical representations of BMD change rates in L2-L4, Ward's triangle, femoral neck, and greater trochanter regions before and after intervention in each group.From February to April in 2023, 64 subjects were recruited for this study, with 60 subjects identified as "positive" by OMOST after excluding 4 subjects who did not meet the inclusion criteria. These subjects were randomly allocated to 24TCCG, TCKFFG, TCSBG, and CG, each consisting of 15 cases. A total of 49 cases completed the trial after staff communication and consultation, with 11 cases lost. 24TCCG had 13 cases (86.7% compliance), TCKFFG had 12 patients (80% compliance), TCSBG had 11 patients (73.3% compliance), and CG had 13 patients (86.7% compliance). Details of the subject recruitment process are presented in Fig. P > 0.05), ensuring a scientifically rigorous implementation of the follow-up study.Baseline data for each group are given in Table The recorded data on BMD changes are given in Table Figure P > 0.05) while a highly significant difference (P < 0.01) was observed between TCKFFG and CG. Furthermore, there was no significant difference between TCKFFG and TCSBG (P > 0.05) while a highly significant difference (P < 0.01) was observed between TCSBG and CG. Notably, highly significant differences (P < 0.01) were observed between TCKFFG and CG as well as TCSBG and CG. At weeks 8, 12, and 16, there were significant differences (P < 0.01) among 24TCCG and the other 3. Similarly, significant differences (P < 0.01) were observed between TCKFFG and CG while insignificant differences (P > 0.05) were observed between TCSBG and the other groups. Finally, significant differences (P < 0.01) were observed between TCSBG and CG during the same period.During week 4, the comparison between 24TCCG and TCKFFG resulted in insignificant difference (P > 0.05). However, a highly significant difference was found in comparison to CG (P < 0.01). Additionally, no significant difference was detected between TCKFFG and TCSBG (P > 0.05), yet a highly significant difference was evident when compared to CG (P < 0.01). A highly significant difference was also observed between TCKFFG and TCSBG (P < 0.01) and between TCSBG and CG (P < 0.01). However, at week 8, a significant difference was found between 24TCCG and TCSBG (P < 0.05).At weeks 4, 12, and 16, no significant difference was observed between 24TCCG, TCKFFG, and TCSBG (P > 0.05), while a highly significant difference was evident in comparison with CG (P < 0.01). Additionally, substantial differences were detected between TCKFFG and TCSBG as well as CG (both with P < 0.01). Following the 8th, 12th, and 16th week, no significant disparities were found among 24TCCG, TCKFFG, and TCSBG (P > 0.05), yet highly significant distinctions remained evident compared to CG (P < 0.01). Furthermore, no notable differences existed between TCKFFG and TCSBG (P > 0.05); however, highly significant differences persisted when compared to CG (P < 0.01). TCSBG and CG exhibited significant discrepancies (P < 0.01).At the 4th week, no significant difference was observed among 24TCCG, TCKFFG, and TCSBG (P > 0.05) while a highly significant difference was found in comparison to CG (P < 0.01). Additionally, there was no significant difference between TCKFFG and TCSBG (P > 0.05), yet a highly significant difference was noted when compared with CG (P < 0.01). A highly significant difference between TCSBG and CG was also evident (P < 0.01).Following the assessments at the 4th, 8th, 12th, and 16th week intervals, there was no significant difference observed between 24TCCG, TCKFFG, and TCSBG . This temporal variation in TC exercise efficacy on BMD improvement aligns with Cheng et al. . Indeed,In this study, the number of males and females varies among different groups. Based on the existing research, females are more susceptible to OP due to hormonal differences, changes in menopausal hormones, and variations in peak bone mass . DependiThe present study utilized Plot trend analysis of BMD data by site to reveal a decrease in BMD in subjects, possibly due to the risk of OP. It has been suggested that a negative correlation exists between BMI and the risk of OP prevalence, with a higher risk of OP prevalence in subjects with lower BMI . Though In our study, we observed a better effect of 24TCC on BMD improvement, which may be attributed to a combination of biomechanical, physiological, and potential biochemical factors. Several potential mechanisms can be proposed:The superior effect of 24TCC and other TC exercise programs on BMD improvement may be attributed to their movement characteristics. Firstly, the 24TCC program consists of 24 different movements, focusing on the smoothness and continuity of the overall movements. These movements involve various joint motions and provide more diverse and intense mechanical stimuli to the skeletal system compared to other TC programs . The greThe superiority of 24TCC in improving BMD can be attributed to specific movements and biomechanical characteristics inherent in the program. The 24TCC training program consists of 24 different movements involving various joint motions, including weight shifting, rotations, and weighted postures. Some of these movements, such as single-leg standing and transitions between different stances, require a higher level of balance and strength compared to TCKFF and TCSB, which may impose greater mechanical loads on the skeletal system . The weiThe superiority of 24TCC in improving BMD may involve physiological mechanisms. The execution of movements in 24TCC involves the integration of body alignment, breathing techniques, and mindful coordination. The precise execution of movements and the combination of mental focus may enhance the activation of neuromuscular activity and recruitment of skeletal muscle groups, potentially stimulating bone metabolism and enhancing BMD. Furthermore, previous research has suggested that specific movements in 24TCC, such as brush knee twist step and wave hands like clouds, generate intensity and impact that can promote the release of growth factors and hormones associated with bone remodeling. These signaling molecules, including IGF-1  and TGF-This study employed a RCT to investigate the effects of TC exercise on the prevention and treatment of OP in middle-aged and elderly individuals. While the results provide insight into the impact of TC exercise on BMD improvement, it is imperative to elucidate the underlying mechanism, which potentially involves mechanical loading-induced osteogenesis . In thisThe underlying mechanism of how TC exercise enhances BMD in middle-aged and elderly individuals remains elusive. Nonetheless, it is established that the deterioration of bone metabolism in adults can result in an imbalance between bone resorption and bone formation, leading to loss of bone mineral . This dyThe engagement in TC exercise has been demonstrated to generate mechanical stress on the skeletal system, subsequently stabilizing bone metabolism and enhancing BMD. Notably, this exertion may also potentially elicit hormonal, cytokine, noncoding RNA, and signaling pathway responses, thereby fostering osteogenesis.TC exercise may induce hormones and cytokines that promote osteogenesis:The theoretical hypothesis proposes that performing TC exercise can stimulate the production of hormones and cytokines, ultimately leading to the promotion of osteogenesis , 49. PreTC exercise may induce noncoding RNAs to promote osteogenesis:Noncoding RNAs, including siRNA, microRNA, lncRNA, and circRNA, have been identified as important modulators of bone metabolism. Specifically, noncoding RNAs are implicated in regulating osteoblast and osteoclast proliferation and differentiation, and TC exercise has been shown to stimulate the production of these molecules . TC exerTC exercise may induce relevant signaling pathways to promote osteogenesis:The primary signaling pathways that play a crucial role in bone metabolism include the Wnt/β-catenin, BMP, OPG/RANKL/RANK, and Notch pathways . ResearcAdditionally, factors such as bone-specific alkaline phosphatase (BLP), procollagen type I N-terminal propeptide (PINP), β C-terminal telopeptide of type I collagen (BCTX), and N-terminal telopeptide of type I collagen (NTX) have been shown to impact bone metabolism and remodeling. Exercise has been demonstrated to modulate these factors as well , 62.TC exercise improves bone metabolism by enhancing fat contentExisting research suggests that physical activity may influence bone metabolism by improving fat content . StudiesResearch has demonstrated the high prevalence of OP in our country . StudiesThere exist several advantages to the present study: (i) Subjects in this study underwent OMOST and tested positive, thereby mitigating the error in baseline characteristics and selection bias, resulting in increased credibility of the RCT outcomes. (ii) In the statistical analysis of this study's RCT data, the exclusion of baseline characteristics and measured indexes in the attrition population led to a reduction in result dispersion and an increase in result credibility. (iii) By rigorously establishing inclusion and exclusion criteria for subjects, bias in subject selection was reduced and the credibility of study results was enhanced; the implementation of such criteria contributed to reducing bias further and increasing the reliability of the RCT outcomes.There exist certain limitations to the present study: (i) Sample size and attrition: The sample size in this study was limited, and attrition led to a reduction in the number of participants. Future studies should aim for larger sample sizes to increase statistical power and minimize the impact of attrition on the results. (ii) Baseline characteristics: Although efforts were made to ensure comparable baseline characteristics, there may still be variations among individuals. Future studies could consider stratifying participants based on baseline characteristics to reduce confounding factors and enhance result validity. (iii) Adherence to TC exercise: It is possible that the adherence to TC exercise interventions varied among participants, potentially affecting the outcomes. Future studies should consider monitoring adherence rates and potential influencing factors to better understand the impact of adherence on the results. (iv) Generalizability: The participants in this study were self-selected, which may limit the generalizability of the findings. Future studies could consider recruiting participants from a wider range of populations to improve the external validity of the results. (v) Outcome measurements: Although DXA was used to measure BMD in this study, there is a margin of error associated with this method. Future studies could consider using additional measurement techniques or incorporating multiple outcome measures to provide a more comprehensive evaluation of BMD.(i) Larger sample size: Conduct studies with a larger sample size to increase statistical power and enhance the generalizability of the findings. (ii) Long-term interventions: Implement longer intervention periods to investigate the effects of TC exercise programs on BMD over extended durations. (iii) Stratification based on baseline characteristics: Ensure baseline characteristic homogeneity by stratifying participants to minimize potential confounding factors. (iv) Adherence monitoring: Monitor participants' adherence to TC exercise interventions and investigate factors influencing adherence rates. (v) Diverse population inclusion: Expand the study population to include individuals with different demographic characteristics to improve external validity. (vi) Alternative outcome measures: Consider using additional measurement techniques or incorporating multiple outcome measures to provide a comprehensive evaluation of BMD and its changes.In conclusion, all 3 TC exercise programs yielded comparable positive outcomes on BMD in the femoral neck and greater trochanter. Nevertheless, 24TCC, the long-term (≥ 8 weeks) TC exercise intervention, exhibited superior improvements in BMD at L2-L4 lumbar vertebrae when compared to the other programs. Furthermore, 24TCC was more effective than TCSB in enhancing BMD at Ward's triangle in 8 weeks. Lastly, the short-term (≤ 4 weeks) TC exercise intervention, TCKFF, was found to be more efficacious than TCSB in augmenting BMD at the femoral neck region. This study presents proof supporting the efficiency of TC exercise in enhancing BMD and inhibiting OP in individuals of middle-aged and elderly who are at a higher risk.Additional file 1. Ethical Materials.


The recommended operation for cecum cancer (CC) is right hemicolectomy (RH) in some Western countries while the principle of D3 lymphadenectomy in Japan recommends resecting approximately 10 cm from the tumor edge. Therefore, the optimal surgical approach for cecum cancer (CC) remains controversial. We conducted this retrospective study to explore the pattern of lymph node metastasis and better surgical procedures for CC.A total of 224 cecum cancer patients from January 1, 2014, to December 31, 2021, were retrospectively included in the final study. The pattern of lymph node metastasis (LNM) was investigated.P = 0.028) was the only independent risk factor associated with worse disease-free survival (DFS).A total of 113  patients had pathologically confirmed LNM. The most frequent metastatic site was no. 201 lymph node , while 20  patients had LNM in no. 202 lymph node, and 8  patients had LNM in no. 203 lymph node. Only 1  patient had LNM in no. 221 lymph node, four  patients had LNM in no. 223 lymph node, and no patients had LNM in no. 222 lymph node. LNM in no. 223 lymph node was significantly associated with a poor prognosis. Multivariate analysis indicated that LNM in no. 223 lymph node (HR = 4.59, 95% CI 1.18–17.86, The LNM in no. 223 lymph node for cecum cancer was rare. Therefore, standard right hemicolectomy excision is too extensive for most CC cases. Colorectal cancer (CRC) is the third most common malignancy and the fourth leading cause of cancer-related deaths worldwide . A previIn some western countries , 5 and CInterestingly, in the 8th edition Cancer Staging Manual of American Joint Committee on Cancer (AJCC), the regional LNs for cecum cancer only include LNs distributed in the pericolic, ileocolic artery, and right colic artery , which iTo find a better extent of LN dissection for CC, we conducted this retrospective study to explore the distribution feature of metastatic LN for CC and the oncologic outcomes of CC.This was a retrospective study conducted at the Fujian Medical University Union Hospital (FMUUH). Patients who were diagnosed with cecum cancer underwent primary resection from January 1, 2014 to December 31, 2021 were reviewed. The inclusion criteria of our study were as follows: (1) pathologically proven cecal adenocarcinoma; (2) without distant metastatic disease; and (3) received RH without neoadjuvant therapy. The exclusion criteria were as follows: (1) metachronous colorectal cancer; (2) familial adenomatous polyposis (FAP); (3) multiple primary tumors; (4) palliative surgery; and (5) emergent operation. Patients met the criteria and were included in the final study. The baseline clinicopathological characteristics were collected from the patients’ medical records.This retrospective study was approved by the institutional research ethics committee of FMUUH (2022KY095), and the study was conducted in accordance with the Helsinki Declaration .All patients underwent the RH procedure with the principle of CME with CVL by laparoscopic or open surgery, and the operations were performed by experienced senior surgeon of colorectal cancer. Usually, the surface of the superior mesenteric vein (SMV) was exposed, and the main LN around the root of the ileocolic artery, the right colic artery (if present), and the middle colic artery were dissected. The right branch of the MCA was ligated with the left branch of the MCA preserved Fig. . The lymAfter the specimen was removed from the body, a colorectal surgeon classified lymph nodes according to their positions in the resected specimens, and each sample was assessed histologically. The stations of the LNs were classified according to the JSCCR  as folloThe outcomes measured were operative time, the number of LN dissections, the rate of LN metastasis, and the incidence of LN metastasis according to its positions . Long-term results investigated were disease-free survival (RFS) rate and overall survival (OS) rate. Recurrence was defined as a radiologically or pathologically proven local or systemic metastasis, while OS was defined as death by any cause reported during follow-up.P value < 0.05 was considered statistically significant.Categorical variables were presented as frequencies and percentages (%), and continuous variables were described as the mean with standard deviation (mean ± SD) or median. The Kaplan‒Meier method and the log-rank test were used for survival analysis. Multivariate analysis was conducted using a Cox proportional hazards model. All analyses were performed with SPSS software program, version 23.0 for MAC . A two-tailed There were 302 patients who underwent the RH procedure for cecal carcinoma from January 1, 2014 to December 31, 2021 in our hospital, and 224 patients were included in the final analysis  patients had pathologically confirmed lymph node metastasis, and the mean number of metastatic LNs was 4.5 ± 5.8 (range from 1 to 43). The most frequent metastatic site was no. 201 lymph node , while 20  patients had metastases in No.202 lymph node, and 8  patients had positive LNs in no. 203 lymph node  by preoperative radiologic examination.The median follow-up time was 45.4 months (1.9–101.0 months), with a 5-year overall survival (OS) of 86.2% and a 5-year disease-free survival (DFS) rate of 80.5% for all 224 patients , the number of negative LNs , vascular invasion , adjuvant chemotherapy , lymph node metastasis in no. 201 , lymph node metastasis in no. 202 , and lymph node metastasis in no. 223  were associated with DFS (Table P = 0.028) was the only independent risk factor associated with DFS after multivariate analysis , and it did not explain how that number was calculated . In the Although many studies found that the CME + CVL procedure did not increase the perioperative complications in right-sided colon cancer compared with “conventional” colectomy , 16, 17,There were some limitations to our current study. Firstly, the study was subject to information and selection biases because this was a single-institution, retrospective study. Secondly, the peri-operative and oncological results were not compared between the CME + CVL group and the D3 group because CME + CVL is a routine procedure for right-side colon cancer in our department. We are comparing the two surgical procedures in different medical centers in China and Japan in our future studies. Finally, the sample of patients with LN metastasis was too small to perform a proper oncological comparison, and the follow-up period was short (range from 12.7 to 28.7 months), so the prognosis analysis exhibited bias.In conclusion, the incidence of no. 223 LN metastasis in cecum cancer is very rare. The standard right hemicolectomy which involve resection of no. 223 for CC patients may result in excessive excision of the distal colon and lymphadenectomy. Further study is needed to explore the best surgical strategy for CC.


Pelteobagrus fulvidraco) were fed with three experimental diets using fishmeal (FM), soybean meal (SBM), and rapeseed meal (RSM) as main protein sources, respectively. Females  were distributed into 9 net cages for feeding trial. Results indicated that 30% dietary SBM improved the reproductive performance for higher gonadosomatic index (GSI), relative fecundity, total egg production, egg diameter, and hatching rate. In addition, SBM and RSM diets resulted in higher estradiol (E2), vitellogenin (VTG), luteinizing hormones (LH), and lower follicle-stimulating hormone (FSH) and testosterone (T) in plasma (P < 0.05) of female broodstock. Dietary SBM and RSM also resulted in lower mesenteric fat index (MFI), plasma total cholesterol (TC), plasma total bilirubin (T-Bil) contents, and gonadal cortisol concentrations, while dietary SBM downregulated the transcription levels of steroidogenesis-related proteins by negative feedback (P < 0.05). The results demonstrated that dietary SBM and RSM could promote sex steroid hormone and VTG biosynthesis and showed hypocholesterolemic effects. Besides, 30% dietary SBM inclusion could improve the reproductive performance of female yellow catfish broodstock.To investigate the effects of different dietary protein sources on the reproductive performance of female broodstock, yellow catfish ( Fishmeal (FM) is the most important protein ingredient used in fish diets for balanced nutrition and high digestibility . HoweverBroodstock nutrition is crucial for gonadal maturation and egg production of the females. Food restriction and nutritional deficiency may result in impaired reproductive performance . NutrienCarassius auratus) [Perca flavescens). As the first restricted amino acid in RSM, lysine could improve the fertilization rate of yellow perch [As for their effects on reproductive performance, reproductive impairment caused by dietary inclusion of SBM was found in goldfish (auratus) , and Kemauratus)  found thow perch . Soy isoow perch . So it iPelteobagrus fulvidraco) is one of the popular special aquaculture species in China. As a kind of high-quality freshwater aquatic food, it possesses huge aquaculture production, which reached 587,822 tons in 2021 and still has a very potential market in China. To provide more larvae, it is effective to improve the spawning performance of females by the regulation of broodstock nutrition. Furthermore, the suitable dietary protein level for female yellow catfish was evaluated, and we found that a diet with SBM and RSM inclusion may be better for reproduction in our previous study [Yellow catfish  to avoid suffering.Three semipurified diets using fishmeal, soybean meal, and rapeseed meal as main protein sources were formulated and were named FM, SBM, and RSM, respectively. For containing much higher protein and less residual oil, solvent-extraction SBM and RSM were used. The protein level was set at 46%, which is suitable for growth and reproduction according to our previous study , and casThe feeding trial was conducted in the outdoor pond aquaculture system of the Haioudao experimental base of HAID Research Institute. Yellow catfish broodstock were obtained from the Bairong farm  and domesticated under experimental conditions for two weeks. After being fasted for 24 h, 270 healthy females  were equally distributed to 9 culture net cages (1.0 m × 1.0 m × 1.0 m), which were randomly divided into three treatments. Males were fed with a mixture of the three diets. It took six weeks for the feeding trial to conclude, and broodstock were fed to apparent satiation twice a day at 7 : 30 and 17 : 30. Meanwhile, feed intake was recorded every day and water quality parameters were collected once a week. Conditions were kept stable during the whole feeding trial .At the end of the experiment, females were sampled after being fasted for 24 hours. All females in each cage were counted and weighed. Then, six females for sample collection were selected randomly and euthanized with 80 mg/L MS-222, of which two females were frozen for whole-body proximate analysis. The body length and weight of two females were collected to obtain the condition factor (CF), and then, their liver, viscera, mesenteric fat, and gonad were dissected and weighed for the hepatosomatic index (HSI), viscerosomatic index (VSI), mesenteric fat index (MFI), and gonadosomatic index (GSI). The remaining two fish were dissected after blood sampling from the caudal vein using heparinized syringes. Then, gonadal and hepatic samples were quick-frozen in liquid nitrogen and stored at -80°C for gene expression determination and lipidomics analysis. Plasma samples were obtained by centrifugation of collected blood samples  and then stored at -80°C for analysis of biochemical indexes and hormone contents.The AOAC methods  were useμg luteinizing hormone-releasing hormone A2 (LHRH-A2), and 1500 IU human chorionic gonadotropin (HCG)  per kg body weight. Meanwhile, the males were performed with half of the dosage. Two rounds of injection were performed for all broodstock, with only a quarter injected in the first round and the rest injected in the second ten hours later. As the females matured for spawning, five mature males weighing 100~120 g were chosen, and testes were collected after males were being anesthetized with MS-222. Sperm were obtained by a grinder and preserved in sperm preservative solution for 10 times dilution. Then, eggs were collected, and sperm were added for insemination at a ratio of 1 mL of sperm diluent to 1 kg of eggs. Each group of eggs was then placed in different labeled white porcelain trays and immersed in clean, oxygen-rich water for incubation.After the feeding trial, two females from each net cage and six males were randomly selected and kept in indoor tanks for breeding. Artificial reproduction was performed based on the methods of our previous study in late May . The femAbout 0.5 g gonadal samples of two females from each cage were collected and counted for the determination of egg weight, egg diameter, and total egg production. After artificial propagation, about 300 eggs were randomly selected from each female at the early gastrula stage (12 hours past fertilization) for calculating the fertilization rate. After hatching, the number of larvae was counted for calculating the hatching rate.Fish vitellogenin (VTG), luteinizing hormones (LH), follicle-stimulating hormone (FSH), testosterone (T), and estradiol (E2) ELISA kits  were performed to detect the contents of VTG and hormones in plasma samples according to the instruction.μg of extracted RNA was added to the reaction system for reverse transcription using a synthesis kit . RT–PCR was carried out on a LightCycler 480 II system , and β-actin and ef1a were selected as housekeeping genes. The PCR program was set as follows: 5 min preincubation at 95°C, 40 cycles with 10 s at 95°C, 20 s at melting temperature, and 10 s at 75°C. Based on the method of Vandesompele et al. [TRIzol reagent  was used to extract total RNA from gonad samples. After ensuring purity and integrity, the concentration was measured, and 1 n = 6) were weighed and mixed with 800 μL of extract  in a 1.5-milliliter Eppendorf tube. After evaluating the repeatability and stability of high-performance liquid chromatography-mass spectrometry (LC-MS) analysis, a LC-MS system consisting of ultrahigh-performance liquid phase  and Q Exactive high-resolution mass spectrometer  was used for the separation and detection of lipid metabolites in this experiment. The procedures and detailed parameters were in accordance with the method of Bai et al. [Liver tissues (25 mg) from the FM, SBM, and RSM groups  from each group were quantitatively detected by a liquid-mass spectrometry system consisting of ACQUITY UPLC I-Class ultrahigh-performance liquid chromatography  and QTRAP 6500 triple quadrupole mass spectrometry .Gonadal samples . Significant differences between groups were detected using Duncan's new multiple range test, and the significance level was set at idSearch  was impoidSearch  for dataP > 0.05) (P < 0.05). In addition, the FM group had significantly higher HSI than the SBM group (P < 0.05). Meanwhile, the FM group shows the highest MFI and the lowest BL among all groups (P < 0.05).No significant difference was found in the growth of female yellow catfish broodstock ( > 0.05) . HoweverP < 0.05). However, the RSM group showed significantly lower crude lipid contents than the FM group (P < 0.05). As shown in 2+ contents and lower TC and T-Bil contents in plasma (P < 0.05). In addition, the SBM group showed the highest HDL-C contents and the lowest AST and ALT contents (P < 0.05). No significant difference in plasma TG, LDL-C, AKP, TP, and ALB contents was found among all groups (P > 0.05).Effects of different protein sources on the body composition of female yellow catfish broodstock are shown in P < 0.05). Consequently, the hatching rate of the SBM group was also higher than that of the FM group (P < 0.05), even though no significant difference was found in fertilization rates among all groups (P > 0.05). Moreover, the difference in reproductive performance between the RSM group and the FM group was not significant (P > 0.05).As shown in erβ of female yellow catfish broodstock fed experimental diets are shown in P < 0.05). We also found that the relative mRNA expression of gonadal erβ was significantly upregulated in the SBM group than in the FM group (P < 0.05). SBM and RSM showed similar effects on plasma hormone and VTG contents.Plasma hormone contents, VTG contents, and relative mRNA expression of gonadal StAR, sf-1, 3β-hsd, 17β-hsd, 20β-hsd, cyp11a1, and cyp17a1 were significantly downregulated in the SBM group than in the FM group (P < 0.05) .The gonadal mRNA expression levels of  < 0.05) . RSM dieP < 0.05). And TG (triglyceride) contents were significantly higher in the RSM group than in the SBM group (P < 0.05). PLS-DA discriminant analysis model score map was performed to investigate comparative interpretations and variation among groups. The FM group was clearly distinguished from the SBM and RSM groups, but the SBM group and the RSM group overlapped each other  and extremely significantly lower in the RSM group than in the FM group (P < 0.01).As presented in Sparus aurata), higher SBM inclusion level to 42.6% in the diet also did not result in depressed growth performance [Trachinotus carolinus, L.) [Pseudobagrus ussuriensis [The present study revealed that 30% inclusion of SBM and RSM in the diets did not suppress the growth of female yellow catfish broodstock. For gilthead sea bream  , respectnus, L.) , and incuriensis . Even thThe quality of dietary protein can not only have an impact on feed utilization but also reproductive performance, since adequate intake of essential nutrients supports growth, metabolism, and gonadal development , 30. BroInterestingly, dietary SBM also could regulate the endocrine system of reproduction. Plasma concentrations of E2 and VTG increased significantly in the SBM group, and it was in accordance with the results on female gibel carp, of which soy isoflavone increased the serum E2 and VTG levels . HypothaAs the precursor of egg yolk, VTG could transport protein and some lipids from the liver through the blood to the growing oocytes in the ovary . Then, nCynoglossus semilaevis) [Dietary SBM and RSM promoted the secretion of E2. In return, the steroid hormone may regulate the release of gonadotropins, including LH and FSH, through feedback effect. However, plasma LH contents were increased by dietary SBM and RSM, while plasma FSH contents were decreased by dietary SBM and RSM. This may be due to their different functions and biological activities. FSH is usually secreted in large quantities at the early stage of gonadal development and plays a major role in stimulating the secretion of E2 and T in the gonad. But LH is secreted in large quantities in the late gonadal development and the maturity stage and stimulates the production of steroid hormones that induce oocytes to mature at last. Steroidogenic enzymes are vital for the biosynthesis of E2, and sf-1 is an important transcription factor in steroidogenesis . Besidesilaevis) .Lipids are essential for gametogenesis and gonadal maturation. The lowest crude lipid content of female yellow catfish was observed in the RSM group, possibly indicating that RSM affects the absorption and utilization of dietary lipids. With more and larger eggs in the gonads, the whole-body crude lipid content was higher in the SBM group than in other groups. Thus, VSI and LDE were higher in the SBM group than in the RSM group. Furthermore, the dietary SBM and RSM resulted in lower MFI. The FM diet may provide excess nutrients, which are deposited in the fish as mesenteric fat. Excessive lipid accumulation usually impairs female reproductive performance . ConsequCortisol accumulates easily in the growing oocyte, and it has a negative effect on the surrounding follicles . Higher In conclusion, the present study revealed that dietary SBM inclusion could improve the reproductive performance of female yellow catfish broodstock. And dietary SBM and RSM could promote the secretion of E2 and VTG and showed hypocholesterolemic effects. Hence, FM can be partially replaced with 30% SBM in the broodstock diet. But further research should be conducted to determine the effects of dietary soy isoflavones on reproduction and estrogenic activity in female yellow catfish.


P = 0.007) and hypogonadal state  were independently associated with osteoporosis. According to the 2014 AWGS criteria, the prevalence of overall sarcopenia and severe sarcopenia was 44.6% and 13.4%, respectively. Severe sarcopenia was strongly associated with fragility fractures . In conclusion, osteoporosis, fragility fractures, and sarcopenia were prevalent in adults with TDT. Severe sarcopenia was associated with fragility fractures. Early osteoporosis and sarcopenia screening and prevention may reduce fracture risk and its complications in these patients.Patients with transfusion-dependent thalassemia (TDT) have an increased risk of osteoporosis and fractures. They also have several potential factors associated with sarcopenia. There has been currently no study on sarcopenia and its association with falls and fractures in TDT. This study aims to determine the prevalence of and factors associated with osteoporosis, fragility fractures, and sarcopenia in adults with TDT. A cross-sectional study was conducted at the hematologic clinic at King Chulalongkorn Memorial Hospital, Bangkok, Thailand. Clinical data and laboratory testing were collected. Bone mineral density and morphometric vertebral fracture were assessed. Sarcopenia was defined using the 2014 and 2019 Asian Working Group for Sarcopenia (AWGS) criteria. We included 112 TDT patients aged 35.1 ± 12.5 years. The prevalence of osteoporosis was 38.4%. Fragility fractures were found in 20.5% of patients. Lower BMI (OR 0.29; 95% CI 0.12–0.72,  In the most severe form of thalassemia, transfusion-dependent thalassemia (TDT), patients require regular lifelong blood transfusions to survive4. Despite advances in transfusion and chelation therapy, high rates of morbidity and mortality from the disease itself as well as multiple end-organ dysfunctions caused by iron excess persist4.Thalassemia is one of the most common genetic disorders in the world characterized by ineffective erythropoiesis, hemolysis, and anemia due to defective globin production5. Patients with TDT may experience pain as a result of fractures, which can develop over time6. In comparison to the general population, pain-related quality of life declines significantly with age7. Patients with more pain locations reported higher levels of anxiety and depression8.Thalassemia bone disease (TBD) is characterized by bone pain, deformity, decreased bone mineral density (BMD), and fragility fractures. It has emerged as the major challenge in managing the growing morbidity burden associated with severe thalassemia phenotype9. Depending on the study population, osteoporosis definition, and co-morbidities, the prevalence of osteoporosis in adults with TDT has been reported to range from 17.6 to 74.6%13. Compared to controls, adolescents and young adults with TDT had lower trabecular volumetric BMD, cortical area, cortical thickness, and periosteal circumference14. A recent meta-analysis found a higher prevalence of fractures in TDT  16–19%) than in non-TDT 15. Although osteoporosis was strongly associated with increased risk of fracture in postmenopausal women and aging men, it was unknown whether decreasing BMD predicted fracture in TDT19.The pathogenesis of TBD is incompletely understood but believed to be multifactorial, including childhood growth retardation, bone marrow expansion, iron and chelator toxicity, physical inactivity, low body weight, nutritional deficiencies, and hormonal deficiencies21. Muscle and bone are anatomically and functionally connected. The evidence associating sarcopenia, low BMD, and fracture with an elevated risk of mortality and morbidity has been widely characterized in the general population22. TDT patients presented various potential risk factors for sarcopenia, including low body weight, poor nutrition, limited physical activity, and chronic inflammatory stage9. As a result, it is critical to investigate sarcopenia in these patients.Sarcopenia is a progressive clinical syndrome characterized by general loss of skeletal muscle mass and impaired muscle functionCurrently, there have been no studies examining the prevalence of sarcopenia and its associated negative health outcomes in TDT patients. It is also unclear whether sarcopenia is associated with TDT-related falls and fractures. Therefore, the current study aimed to determine the prevalence of osteoporosis and sarcopenia in adults with TDT and examine their association with fracture risk. Our findings will contribute to a greater comprehension of the pathogenesis of bone abnormalities in TDT, which may aid in the development of fracture prevention strategies in order to decrease fracture risks and their complications.2 respectively. One-third of them (9 men and 27 women) had hypogonadism. All of the men and 14 of the 27 women had secondary hypogonadism, and 13 women reached natural menopause. All of the patients received regular blood transfusions and iron chelation therapy, and 41.1% underwent splenectomy. Seven patients (6.3%) had a history of falls in the past year. Vitamin D deficiency was prevalent among women (50.9%). Endocrinopathies included low IGF-1 levels (33.9%), subclinical/secondary hypothyroidism (32.2%), and secondary hypogonadism (21.4%). Men and women with hypogonadism had significantly lower IGF-1 levels than eugonadal patients. The demographic, clinical, and laboratory characteristics of the study population are shown in Table A total of 112 patients were included in the final analysis. Study recruitment flow is summarized in Supplementary Fig. Fifty-eight percent of the patients had BMD below the expected range for their age. Based on the BMD T-score, 55.4% of the patients had osteopenia and 38.4% had osteoporosis. The majority of patients had either osteoporosis at both the lumbar spine and the hip (48.8%) or only at the lumbar spine 37.2%), while a minority had isolated hip osteoporosis (14%). At all sites, osteoporosis was prevalent in hypogonadal patients of both sexes. The prevalence of fragility fracture and morphometric VF were 20.5% (23/112) and 8.0% (9/112), respectively. The majority of patients had a single fracture with the lumbar spine having the highest prevalence, followed by the forearms, humerus, femurs, and clavicle. Women tended to have fractures more than men, and although not statistically significant, hypogonadal women had more fractures than eugonadal women . Using the 2019 revised AWGS criteria, the prevalence of overall sarcopenia and severe sarcopenia increased to 61.6% and 24.1%, respectively (Table P = 0.007) and hypogonadal state  were independently associated with osteoporosis  and IGF-1 levels  were independently associated with fragility fracture (Table P = 0.032) and deferasirox usage (P = 0.031) were significantly associated with overall sarcopenia, while a history of falls in the past year (P = 0.032) was associated with severe sarcopenia. However, these associations were not found significant in multivariate analyses D levels greater than 12 ng/dL, which was unlikely to result in osteomalacia. In addition, there was no correlation between low 25(OH)D and high intact parathyroid hormone, suggesting that they may have relative hypoparathyroidism. These factors may partially explain why vitamin D deficiency was not associated with bone loss and/or fracture.Vitamin D plays an important role in calcium and skeletal homeostasis, and vitamin D deficiency results in secondary hyperparathyroidism, bone loss, and mineralization defects44. In addition to elderly populations, sarcopenia was substantially more common in various patient categories with the prevalence ranging from 18% in diabetic individuals to 66% in patients with unresectable esophageal cancer. A high prevalence of sarcopenia has also been found in patients with renal and liver disease who require surgery, as well as in individuals with various site-specific malignancies45. According to the 2014 AWGS definition, we found that sarcopenia was prevalent in young to middle-aged adults with TDT (44.6%), and that 30% of these individuals had severe sarcopenia. Even though there have been no comparative studies of sarcopenia in patients with thalassemia, the above prevalence was much higher than reported among community-dwelling older Thai people (ranging from 10 to 30%)48, indicating that TDT accelerated the development of sarcopenia. We suggested that physicians should be aware of the risk of sarcopenia in TDT patients, not just the elderly, but also the middle-aged.Sarcopenia is an age-related disease, characterized by a general loss of skeletal muscle mass and impaired muscle function, and is associated with a wide range of adverse health outcomes, including falls and fractures44. Underlying mechanisms leading to sarcopenia in TDT have not been adequately studied. We believe that they are multifactorial, including both risk factors found in the general elderly population and risk factors specific to thalassemia.Several mechanisms, including aging, oxidative stress, inflammation, mitochondrial dysfunction, and apoptosis may be involved in the pathogenesis of sarcopenia49. Recent clinical data have consistently suggested a correlation between iron accumulation and sarcopenia. Kim et al.50 demonstrated that serum ferritin levels were substantially associated with sarcopenia in middle-aged and elderly Korean women. Patients with elevated serum ferritin levels had a twofold increased risk of sarcopenia compared to those with normal serum ferritin levels. Another study by Perna et al.51 also found that elderly patients with sarcopenia had significantly elevated serum ferritin levels and serum inflammatory markers. These findings suggested that excess iron may play a role in the development of sarcopenia in TDT. The presence of chronic, severe anemia may contribute to sarcopenia in TDT patients. In a previous study involving middle-aged to elderly individuals54, a negative association between hemoglobin level and sarcopenia was observed. In one report, a 1 g/dL increase in hemoglobin was associated with a 5% reduction in the risk of sarcopenia 53. In the general population, an association between hormonal deficiencies, such as vitamin D deficiency, hypogonadism, growth hormone deficiency, and sarcopenia has been described55. However, there have been few studies investigating the effect of these factors on muscle mass and/or function in TDT patients56.Age-related iron accumulation in skeletal muscles causes muscle atrophy and is likely associated with oxidative damage and mitochondrial dysfunction, according to animal studiesIn our study, we found no significant association between sarcopenia (based on either the 2014 or 2019 AWGS criteria) and age, BMI, vitamin D level, IGF-1 level, hypogonadism, pre-transfusion hemoglobin level, or serum ferritin level. A relatively small sample size and distinctions in the study population may have resulted in insufficient power to detect any associations in the multivariate analyses.59. In one retrospective cohort of adults with TDT, there was a positive correlation between skeletal muscle mass and BMD60. No association between sarcopenia and BMD was demonstrated in our study, however, we found that a significant proportion of TDT patients with osteoporosis had sarcopenia and vice versa. Therefore, sarcopenia should be routinely assessed in TDT patients, especially for those with osteoporosis.In the general population, the effect of decreased muscle mass and strength on fall risk, BMD, and fractures has been well described P < 0.001) and fractures  than non‐sarcopenic individuals61. Although we found no association between sarcopenia and falls in TDT, patients with severe sarcopenia, according to the 2014 AWGS criteria had a 4.6-fold increased risk of fragility fracture .A recent meta-analysis of prospective studies in older adults found that sarcopenic individuals had a significantly higher risk of falls . All study participants provided written informed consent before any procedures were performed. All procedures were conducted following applicable rules and guidelines. The trial registration number was TCTR 20201008006.At the time of study enrollment, the demographic and clinical information of all participants were collected from an electronic medical record system and interviewed by trained study personnel. A structured questionnaire was used to collect data, including a history of thalassemia, reproduction, and traditional osteoporosis and fracture risk history. We measured anthropometric variables, including weight and current height. The data collection was performed at the same visit as the blood sample collection and BMD measurement or within a month.62. Bone turnover markers including serum procollagen type 1 N-terminal propeptide and C-terminal cross-linking telopeptide of type I collagen were measured using a chemiluminescence immunoassay  in an Elecsys 2010 machine.After at least 8 h of fasting, whole blood samples were collected, and serum and plasma were stored at − 80 °C until analysis. Complete blood count, ferritin, calcium, phosphorus, albumin, creatinine, liver function tests, fasting plasma glucose, insulin-like growth factor-1 (IGF-1), intact parathyroid hormone , serum 25-hydroxyvitamin D (25(OH)D) levels , free thyroxin (T4) and thyroid stimulating hormone (TSH)  were measured in all participants. A fasting plasma glucose level of ≥ 126 mg/dL was used to define diabetes. Subclinical hypothyroidism was defined as a TSH level above the upper limit of the reference range and a normal level of free T4, and overt hypothyroidism was defined as a free T4 level below the lower limit of the reference range with any TSH concentration. In this study, low IGF-1 was defined as IGF-1 less than the lower limit of normal IGF-1 level of that age. Age-specific reference ranges of IGF-1 levels were provided by SIEMENS (IMMULITE® 2000 systems). For the 25(OH)D assay, the intra- and interassay coefficients of variation were less than 6%. Vitamin D deficiency was defined as serum 25(OH)D levels of < 20 ng/mL63. In women with irregular menstruation, FH, LH, and estradiol levels were measured. Secondary hypogonadism was diagnosed in women with irregular or absent menstruation for at least 12 months with serum estradiol concentrations < 50 pg/mL and inappropriately low or normal serum FSH concentrations. Woman over the age of 45 years who had not had a menstrual period for a year and had FSH levels > 30 mIU/mL was diagnosed as menopause64.Serum FSH, LH, total testosterone, and sex hormone binding globulin levels were measured in all men. Men with calculated free testosterone concentration of < 0.245 nmol/L based on Vermeulen’s formula were defined as hypogonadism65, they recommended using BMD T-score according to the WHO operational definition of osteoporosis in young adults with a chronic disorder or taking medications known to affect bone metabolism, unless it appears that he/she is still growing. In this study, osteoporosis was defined as a T-score of ≤  − 2.5 standard deviation (SD), whereas osteopenia was defined as a T-score between − 1 and − 2.5 SD below the young adult mean value. BMD Z-scores were also calculated. Individuals with a Z-score ≤  − 2.0 SD were defined as having low bone mass compared to a healthy person of the same age and gender66.BMD at the lumbar spine, femoral neck, and total hip were measured using dual energy X-ray absorptiometry (DXA) on a QDR 4500 bone densitometer . BMD Z- and T-scores were calculated using the manufacturer-supplied reference databases for Asian populations. According to the IOF Committee of Scientific Advisors Working Group on Osteoporosis Pathophysiology 201267, morphometric vertebral fracture (VF) was diagnosed by three independent radiologists. Disputes regarding the value of a joint were settled by consensus. The VF was determined by evaluating vertebral bodies from the T4 to L4 levels.A fragility fracture is defined as a fracture sustained from force comparable to a fall from a standing position or less, excluding fractures of the skull, face, fingers, and toes. For morphometric VF assessment, in accordance with standard protocol, a lateral thoraco-lumbar X-ray radiograph was obtained with a tube-to-film distance of 101.6 cm, which included information regarding the positioning of the participants and the radiographic technique used. The radiographs were obtained from the left lateral position, centered at the L1 level. Using Genant’s semi-quantitative method68 observed that the significantly associated factors with sarcopenia were not the same when sarcopenia was classified using different criteria. Most factors associated with the 2014 AWGS criteria showed no consistent pattern with the 2019 AWGS, and the validity of the AWGS 2019 consensus requires more research. Since there is no current study on sarcopenia in TDT patients, we decided to investigate its prevalence and associated variables using both the 201421 and 201969 revised AWGS criteria. A DXA whole-body scan was used to determine skeletal muscle mass . A skeletal muscle mass index of < 7.0 kg/m2 for men and < 5.4 kg/m2 for women was used to define muscle mass loss. Handgrip strength was measured using a grip dynamometer . The participant was instructed to squeeze the dynamometer with their dominant hand, adducted arm beside the body, and elbow flexed to a 90° angle for 3 s at their maximum force. Handgrip strength was measured twice, with the highest values being recorded. For the 2014 AWGS criteria, low handgrip strength was defined as < 26 kg for men and < 18 kg for women, whereas the 2019 AWGS criteria used < 28 kg for men and < 18 kg for women. Physical performance was determined by the usual 6-m gait speed. Low physical performance was defined as a gait speed cut-off of < 0.8 (the 2014 AWGS criteria) and < 1.0 (the 2019 AWGS criteria) meter per second. Sarcopenia was diagnosed in patients who met the criteria for low muscle mass and either low handgrip strength or low gait speed. Participants with low muscle mass, low handgrip strength, and low gait speed were diagnosed with severe sarcopenia. In this study, overall sarcopenia was defined as the combination of sarcopenia and severe sarcopenia.The Asian Working Group for Sarcopenia (AWGS) criteria were used to define sarcopenia. The updated 2019 consensus, when compared to the 2014 AWGS criteria, may increase the estimated prevalence of sarcopenia due to an increase in cutoff values for handgrip strength and gait speed. Liu et al.11, with a sampling variability of 7%, it was estimated that a sample size of at least 114 was needed for statistical adequacy to estimate the true prevalence of osteoporosis.Based on a prevalence estimate of the prevalence of osteoporosis in Thai adult with TDT (17.6%)P values were two-sided and considered significant if they were < 0.05.Data analysis was performed using SPSS, version 26 for Windows Evaluation Software . Descriptive statistics were used to categorize and summarize demographic data. Numbers and percentages were used to present gender-specific category data. The Kolmogorov–Smirnov test was used to determine the normality of a continuous data distribution. Data having a normal distribution were presented as means with standard deviations (SD), whereas data with a non-normal distribution were presented as a median with interquartile range (IQR). Comparisons of continuous data between study groups were performed using the unpaired t-test or non-parametric equivalents (Mann–Whitney U test); categorical comparisons were performed using Chi-square or Fisher’s exact test as appropriate. All P values < 0.20 in the univariate models and known potential confounders were entered. A P value < 0.05 was considered statistically significant.Logistic regression analysis was performed to calculate the odds ratio (OR) and 95% CI between predictive variables and osteoporosis, sarcopenia, and fragility fracture. The variance inflation factor (VIF) was used to quantify multicollinearity among independent variables. If VIF is > 10, multicollinearity was evaluated. We considered that there was no multicollinearity concern because none of the VIF values exceeded 10. In the multivariable logistic regression , all variables with Supplementary Information.


Neutrophil to Lymphocyte Ratio (NLR) is a simple biomarker of systemic inflammatory response. We investigated predictability of NLR for early adverse outcome after surgery for hip fracture in elderly population.We reviewed a total of 971 elderly patients with hip fracture who underwent emergency surgery between January 2017 and July 2020 in the Department of Orthopaedics Surgery at the Wollongong Hospital. After considering exclusion criteria, data from a total of 834 patients included in our study. Socio-demographic data, NLR in admission, updated Charlson comorbidity index (uCCI), biochemical markers, mortality rate and 15 short term post-operative complications were collected to determine predictability of NLR for post-operative complications and mortality.P-value < 0.001, OR 1.05) and uCCI≥4  are associated with post-operative complications. Moreover, decreased haemoglobin was associated with adverse effects . No association was found for any of these variables with in-patient mortality except for albumin . In addition, despite significant association, ROC analyses showed a low predictability for each of the above variables including NLR (AUC 0.59) for post-operative complications.After hip surgery, Hospital in-patient case fatality rate was 3.7% (31). In addition, 63.1% (511) of the patients had at least one complication or more. Logistic regression demonstrated that raised NLR (Despite significant association, NLR was unable to prognosticate early adverse outcomes. However, it can be considered as a risk factor in admission for postoperative complications in combination with other risk factors and clinical context. There are about 28,007 hip fractures a year in people over 65 years old in Australia . This coThis study is following the STROBE guideline .Retrospective observational single centre study.We included all emergency patients with hip fractures who underwent surgery between January 2017 and July 2020 at Wollongong Hospital orthopaedic department. Patients’ information was extracted manually by the Author from electronic Medical Records (eMR). Anonymous patient data was used retrospectively and no blood test was performed on any patient for this study. Follow up finished at the date of patient discharge to peripheral hospital from acute ward or death.A total of 971 consecutive patients who required emergency surgery after hip fracture with age of 65 years old or older were included in the study. We excluded patients with pathological fractures, active cancer or steroid treatment because of the potential bias of diluting results due to the established NLR prognostication in oncologic mortality in previous research –14. Of nThe surgical protocol consisted of early discharge from the Emergency department and surgery within 48 hours of arrival to the hospital unless there was an acute medical reason. After the operation, early mobilization and optimal pain control were performed with help of a multidisciplinary team of Geriatricians, Orthopaedic surgeons, acute pain team, physiotherapists and occupational therapists familiar with orthogeriatric medicine.The first Peripheral venous blood EDTA based sample was collected from each patient upon admission to the emergency department before surgery. These samples were analysed using a blood analyser in our laboratory for electrolytes, renal  markers and albumin . Full blood count was analysed using standard automated laboratory methods  for haemoglobin, neutrophil and leucocytes counts. Age, gender, type of surgery and postoperative complications were collected manually from patients’ records in eMR. The NLR ratio was calculated by dividing absolute neutrophil counts by lymphocyte counts. In order to capture a wide variety of adverse outcomes, fifteen different types of complications were identified including (1) Severe arterial hypertension requiring medical intervention, (2) Decompensated heart failure requiring medical intervention, (3) Pulmonary oedema requiring medical intervention, (4) ST Elevation Myocardial Infarction(STEMI) and Non-STEMI, (5) Arrhythmia, (6) Thrombo-embolic complications, (7) Infections documented by radiological, biochemical and/or microbiological evidence, (8) Acute respiratory distress , (9) Neurological dysfunction , (10) Electrolytes disturbance requiring treatment, (11) Anaemia- New onset requiring red blood cell transfusion, (12) Acute renal failure (evidenced by a new decline in eGFR), (13) Acute liver failure, (14) Direct Surgical complication and (15) Non- specified other complications which are not included in above categories. The rate of in-hospital death was calculated for the duration of the total length of orthopaedic admission to qualify for short term mortality.Updated Charlson comorbidity index (uCCI) –22 was cThe names of the patient was collected from local hip fracture registry. The data was collected from electronic Medical Records (eMR) which is a state-wide digital patient medical record used in public hospitals in New South Wales, Australia. Demographic data, date and type of surgery, post-operative complications, albumin level, haemoglobin level, NLR Day 0 (NLR0), mortality and complications during hospital stay were collected manually by the Author from eMR.After collecting information, the population was divided into two groups of survived and non-survived according to in-patient mortality. Also, for sake of complications, the survived group was separated into two groups with and without complication.P-value found to be under 0.05. Data analysis were performed using Jamovi 1.6  package [Descriptive analysis was used for quantitative variables such as age, gender and NLR0. Standard deviation and means have been used to describe continuous variables and for categorical variables, frequencies and percentages reported. We compared continuous variables with Students independent t-test and categorical variables with the Chi-square test. Test of equal variances was carried out before t-test for continuous variables. When there was a violation of the assumption of equal variances, we used non-parametric tests (Mann-Whitney). Following testing for the presence of differences, we used multiple logistic regression analysis to investigate the association of NLR with post-operative complications and mortality after adjustment for other variables. We used the updated Charlson index in our model instead of the ASA (American Society of Anaesthesiology) score because it would have increased the chance of collinearity. Updated CCI numbers equal to or more than 4 were considered high in the logistic regression model, based on previous literature . The Per package  and SPSSFrom a total of 971 patients with hip fractures who were admitted during our study timeline, 913 were 65 years old or older. In addition, 79 patients were excluded due to the presence of active malignancy, or steroid treatment. A total of 834 patients were eligible and included in our study  female and 237(28.4%) male patients. The patients’ average age was 84.1 ± 7.55 (range 65–107). A total of 511 (61.3%) patients had at least one complication and more. There was 31 (3.7%) death in hospital postoperatively. The average mean age in surviving patients was 84 ± 7.57 (Range 65–107) versus 86 ± 6.71 in non-survivors. The most common type of hip fractures were intertrochanteric (54.9%) followed by undisplaced intracapsular (23%), displaced intracapsular 16.1% and subtrochanteric (6%). Long Intramedullary nail was the most frequent surgery used (21% long nail vs 6.5% short nail). The next was hemiarthroplasty (19.2% cemented vs 12.8% uncemented) followed by sliding hip screw (12.9%) and total hip replacement (2.1% cemented vs 4.3% uncemented).P-value < 0.01). Also, albumin and haemoglobin were significantly lower in patients with at least one complication. .Table P-value < 0.004).In contrast, as shown in Table P-value: 0.08 and 0.613 respectively).Moreover, Mortality or postoperative complications were not significantly different between male and female patients  and uCCI≥4  are independently associated with postoperative complications. Moreover, lower haemoglobin was associated with adverse effects .Finally, we combined NLR0, age, sex, albumin, uCCI and haemoglobin in a logistic regression model to determine association with early postoperative complications. The results of the model as demonstrated in Table P-value: 0.03)  Table .Table 4MFurthermore, to show prognostication, ROC analyses were performed which showed a low performance for each of the above variables in predicting postoperative complications Fig. .Fig. 2ROAs mentioned in the literature review, a few studies have shown an association of NLR with postoperative complications after hip fracture surgery , 24, 26.P-Value 0.001). However, in their multivariate analysis, they considered NLR at day five as a significant risk factor that has a lower clinical value than NLR in admission. In our study, we demonstrated that NLR in admission can also be considered a risk factor even in the preoperative period in correlation with clinical assessment.Considering NLR as an independent risk factor, our finding broadly supports the work of other research in this area , 24. ForAlthough extensive research has been carried out on the prognostication of NLR in the field of cardiac or vascular surgery, a small number of studies exists in the field of hip fracture surgery. Previously, Fisher and colleagues  reportedP-value 0.12) [Mortality after hip fracture surgery varies from 3.4%  in Austrue 0.12)  which shHowever, This discrepancy between studies shows that these results need to be interpreted with caution and further studies need to be conducted to carefully examine mortality risk.In our study, we showed that higher uCCI is a risk factor for postoperative complications. It is interesting to note that NLR0 and uCCI had the same predictability according to the ROC graph (AUC: 0.59) Fig. . SurprisThe possible mechanism of NLR association with adverse outcomes is not entirely clear. It is demonstrated that Neutrophil extracellular traps (NETs) which contain mitochondrial DNA (mt DNA) are present for 5 days after major orthopaedic surgery . They reP-value 0.029). These results corroborate a great deal of the previous work with d’Engremont et al. [In addition, lymphopenia has been an indicator of malnutrition in hip fracture patients . Blanco t et al.  and Imabt et al.  who refeHowever, it is important to bear in mind that elevated NLR0 in the emergency hip fracture is considered an acute inflammatory response and can be affected by a variety of confounding factors.The most important limitation for this study lies in the fact that firstly, this is a retrospective observational study with limited power for causality. Secondly, we didn’t include postoperative delirium in the list of complications due to retrospective measurement limitations. Some studies consider NLR as a predictor for delirium . ThirdlyIn terms of strengths, we used a large sample of patients to decrease the chance of selection bias. We also excluded patients with cancer and those on steroid treatment which could be a field for confounders due to the known effect of raised NLR in the oncologic population.We demonstrated that NLR can be considered as a risk factor in admission for postoperative complications in combination with other risk factors. However, it failed to predict short term in-patient adverse outcomes accurately as a sole predictor in admission. More research using controlled trials is needed to include serial NLR in perioperative patients as well as looking into other predictors for hip fracture surgery complications.


The third and fourth groups received BLM plus Stachys pilifera hydroalcoholic extract (SPHE) . Vitamin E  was given to group V in addition to BLM. After 14 days, the animals were euthanized to assess biochemical parameters and lung histopathology. Malondialdehyde (MDA), nitric oxide (NO), total thiol (TSH), and glutathione (GSH) levels were measured. In addition, hydroxyproline (HYP) levels along with histological changes in lung tissue were also assessed.  In this experimental study, 35 male Wistar rats (120–180 g) were divided into five groups , and Vit E. SPHE also significantly increased GSH and TSH levels in comparison to the BLM group.HPLC analyses showed the presence of thymol (55.47 ng/mL) and carvacrol (109.91 ng/mL) in SPHE as potential bioactive phenolic compounds.  The results suggest that SPHE alleviates the development of BLM-induced pulmonary fibrosis by inhibiting the proliferation of fibroblasts mediated by antioxidant pathways. Other mechanisms underlying this Effect of SPHE need to be clarified through further research. E are activated [Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease characterized by proliferation and differentiation of fibroblasts and accumulation of extracellular matrix, primarily collagen, leading to irreversible damage to lung architecture , 2. In sctivated . Other activated . Numerouctivated , 9, 10. ctivated .Bleomycin (BLM) is a chemotherapeutic antibiotic used to treat a variety of malignancies. Since one of the most crucial side effects of BLM during the chemotherapy process is pulmonary fibrosis, it is a classic combination for modeling pulmonary fibrosis in laboratory animals , 11. IntStachys pilifera is a perennial and fragrant plant belonging to the Lamiaceae family [Stachys species have been utilized as antinephritis, antidiarrhea, and wound disinfectants [Stachys species have been confirmed [S. pilifera [S. pilifera hydroalcoholic extract (SPHE) on bleomycin-induced pulmonary fibrosis in rats.e family . In tradfectants , 15. Moronfirmed . Differeonfirmed . Farjam pilifera . In our pilifera , 20. HenBleomycin (BLM), 2,4,6-tri(2-pyridyl)-1,3,5-triazine (TPTZ), sulfanilamide, n-(1-naphthyl) ethylenediamine dihydrochloride, thiobarbituric acid (TBA), trichloroacetic acid (TCA), ethylenediaminetetraacetic acid (EDTA) and 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) were purchased from Merk- Germany. Standards of thymol and carvacrol were obtained from Sigma Aldrich Co. . Acetonitrile  was purchased from Merck, Germany.Stachys pilifera were collected from the Kakan region in Kohgiluyeh and Boyer-Ahmad Province in spring 2018. Dr. Azizollah Jafari confirmed the collected samples from the Department of Botany, Center for Research in Natural Resource and Animal Husbandry, Yasuj University, Yasuj, Iran, where a voucher specimen (herbarium No. 1897) was deposited. The plant materials were transferred to the laboratory and dried in a dark place.The aerial parts of n-hexane through the liquid-liquid extraction (LLE) method. The remained fraction defatted was stored in the refrigerator for technical HPLC analysis.The air-dried powdered plant (100 g) was macerated in 70% methanol (500 mL) and kept at room temperature for 48 hours (three times 500 mL each). Having filtrated the extract by Whatman No. 1, the solvent was evaporated by rotary apparatus to yield a dark green gummy residue. The extract was stored in the refrigerator at 4°C for further experiments. Briefly, a portion of the extract was dissolved in methanol/water using sonication and stored in a cold place , 22. The18; 250 mm × 3.9 mm i.d., 5 μm). Moreover, the flow rate and the UV-Vis light detector were set at 1.1 mL/min and 220 nm, respectively. In order to have a calibration curve, about 2 mg of standard thymol and carvacrol (each 1 mg) was dissolved in methanol (HPLC grade) to reach the final concentration of 2000 μg/mL. The calibration curves for standards were prepared using concentrations of 0.5 to 2000 μg/mL [In order to calculate the amount of thymol and carvacrol as simple phenolics in SPHE, a high-performance liquid chromatography  apparatus was applied equipped with a quaternary K-1001 controller pumps and a K-2550 UV absorbance detector. A binary mobile phase consisting of 45% water and 55% MeCN was gone through a stainless steel analytical column  with a 12-hour light/dark-light cycle and access to adequate food and water. All ethical issues approved by the Committee for Supervision of Laboratory Animals were followed in this work. The code of IR.YUMS.REC.1396.124 was enclosed by the ethics committee of Yasuj University of Medical Sciences.Animals were sedated with intraperitoneal injections of ketamine (50 mg/kg) and xylazine (5 mg/kg). A single intratracheal instillation of BLM  was administered to anesthetized rats by placing them on a biased panel and suspending them by their upper canine teeth, followed by a single intratracheal instillation of Next . The placebo group received the same volume of intratracheal normal saline as the BLM group. The animals were euthanized two weeks following PLF induction .n = 7):  Group I received a single intratracheal instillation of normal saline (control group)  Group II I received a single intratracheal instillation of BLM solution (7.5 IU/kg in 0.9% NaCl) (BLM-treated group)  Group III was given a single intratracheal instillation of BLM solution (7.5 IU/kg in 0.9% NaCl) and a daily SPHE  for two weeks (BLM/PPX 0.5)  Group IV received a single intratracheal instillation of BLM solution (7.5 IU/kg in 0.9% NaCl) and a daily SPHE  for two weeks (BLM/PPX 1)E  for two weeks (BLM/Vit E)  Group V was given a single intratracheal instillation of BLM solution (7.5 IU/kg in 0.9% NaCl) and a daily Vit Out of fifty male Wister rats, 35 were randomly divided into the following 5 groups  was mixed with 500 t 535 nm , 26.μL of serum or tissue homogenate with 100 μL grease reagent ) was transferred to a 96-well plate and placed in the dark for 15 minutes at room temperature. The absorbance of the samples was then read at 541 nm [Since nitric oxide (NO) is an unstable compound rapidly converted to nitrate and nitrite, its direct measurement is a bit difficult, so the amount of nitrite as a stable NO metabolite was measured by the grease method. In summary, 10 t 541 nm .The Tietze method was used to determine glutathione. In this method, an enzyme cycle in which glutathione reductase (GR) plays a significant role is used to quantify the glutathione present .μL of DTNB, 150 μL of tris-EDTA, and 790 μL of methanol were added to microtubes containing 25 μL of the sample. After incubation at room temperature, the samples were centrifuged, then the adsorption of the supernatant was read at 412 nm, and the amount of TSH was calculated using a molar absorption coefficient of 13,600 M−1·cm−l [The amount of total thiol (TSH) was determined based on the reaction of DTNB with thiol groups and the formation of TNB (2-nitro-5-thiobenzoic acid) and disulfide compounds. Briefly, 10 M−1·cm−l .3+) ions and their conversion to ferrous (Fe2+) at an acidic pH in the presence of tripyridyl-S-triazine, the Fe-TPTZ complex is formed in blue. The resulting color intensity was measured by spectrophotometry at 593 nm [Benzie and strain method was used to measure ferric reducing antioxidant power (FRAP) activity. In this method, the ability of plasma to reduce ferric ions is measured. Following the reduction of ferric  levels in lung tissues .μm sections were prepared. After staining with hematoxylin-eosin, lung tissue was examined by light microscopy.Lung tissue samples were kept in 10% formalin buffer. These samples were dehydrated with alcohol and immersed in paraffin. Tissues were placed in paraffin, and 5 P < 0.05 was considered significant.Data were analyzed by SPSS software for normality and expressed as Mean ± SEM. Statistical calculations were performed for significant differences between the experimental groups using analysis of variance (ANOVA) and Tukey post hoc test. Stachys pilifera aerial parts showed the presence of low-weight phenolics, thymol (55.47 ng/mL), and carvacrol (109.91 ng/mL) . However, using SPHE at doses of 300 and 600 mg/kg caused a substantial decrease in these indices. BLM administration significantly reduced TSH and GSH levels compared to the control group. However, in the groups receiving SPHE + BLM, these markers increased significantly compared to the BLM-treated group. Vitamin E treatment also had a similar effect to SPHE . However, SPHE significantly reduced hydroxyproline when compared to the BLM group alone  is one of the most important of its side effects. This compound damages lung tissue by increasing extracellular matrix remodeling and redox imbalance . These dMedicinal plants and their bioactive phenolic and flavonoid compounds have gained more attention due to their beneficial properties, such as antioxidant and anti-inflammatory potencies .The present study investigated the possible protective effects of SPHE against the harmful effects of BLM on pulmonary tissue. In a manner similar to IPF, BLM destroys alveolar epithelial cells and provokes a pronounced inflammatory response. Reference . It is aBased on our results, it was determined that BLM caused significant hyperplasia and alveolar damage in the animals. BLM administration dramatically elevated the concentrations of hydroxyproline (HYP), MDA, and NO, and reduced TSH and GSH levels. Oral treatment with SPHE alleviated BLM-induced pulmonary damage by restoring lung tissue architecture and normalizing the indicated oxidative stress markers.The inflammatory mediators' cascade was activated following inflammatory cell infiltration in the early stages of pulmonary fibrosis. In the later stages, fibroblasts activate and stimulate collagen secretion . As a ref HYP in lung tissue compared to the saline-treated group. Treatment with SPHE significantly reduced the elevation of HYP levels in the lung tissue due to BLM intoxication.In accordance with Bahri et al.  and HuanThis activity could be attributed to phenolic and flavonoid ingredients in SPHE that produce anti-inflammatory and antioxidant effects . In thisThe critical role of oxidative stress during the PF process has been demonstrated. Increased ROS and oxidant/antioxidant imbalances have been shown to play a critical role in lung tissue damage in IPF patients , 40. TheThere is increasing evidence that NO plays an essential role in the pathogenesis of numerous lung diseases. An isoform of NO called iNOS is responsible for producing cytokines in lung diseases. This isoform is involved in the pathogenesis of PF by producing ROS such as peroxynitrite and nitrogen dioxide. A significant increase in NO levels was observed in the BLM-treated group compared with the control group, which was consistent with the results reported by Kseibati et al. . In the SPHE-treated groups had lower FRAP levels than BLM-treated groups, which is in line with our previous findings. As antioxidants, the thiol groups of proteins and glutathione play a significant role in inhibiting ROS and RNS. Oxidative stress reduces -SH reserves by oxidizing the -SH groups into disulfide or sulfonic acid. References , 44. FurE as an antioxidant behaved similarly to SPEH in the evaluated parameters, but it did not significantly affect GSH levels. Polyphenols such as resveratrol, mangiferin, quercetin, and dihydroquercetin mitigate lung damage after BLM induction in animal models [However, Vit l models . It is al models . Carvacrl models –47. Salil models . Hence, Future research could address two significant limitations of the study. First, SPHE is also examined for its effects on apoptosis and inflammation in BLM-induced lung fibrosis. The second step is to prepare and evaluate the extract's different fractions or principal components.The evidence from this study suggests SPHE on BLM-induced fibrosis. Histological examination of lung tissue confirmed the biochemical results. Thus, it can be postulated that a defatted SPHE involving thymol and carvacrol as the significant constituents may effectively reduce BLM damage due to its antioxidant effects. More research is required to determine the precise mechanisms of SPHE in managing pulmonary fibrosis.