1.Qishao Capsules Improve Diabetic Renal Injury in db/db Mice by Inhibiting Podocyte Apoptosis via Regulating Caspase-8 and Caspase-3
Jingwei LIU ; Zhenhua WU ; Bing YANG ; Fengwen YANG ; Miao TAN ; Tingting LI ; Jinchuan TAN
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(2):126-135
ObjectiveTo observe the effect of Qishao capsules on renal injury in db/db mice with diabetic kidney disease (DKD),and explore its mechanism of protecting the kidney by inhibiting podocyte apoptosis. Methodsdb/m mice (7 mice) were used as the normal group,and db/db mice (35 mice) were randomly divided into a model group,a dapagliflozin group (0.001 g·kg-1·d-1),and low-,medium-,and high-dose groups of Qishao capsules (0.341 3,0.682 5,and 1.365 g·kg-1·d-1,respectively). Drug intervention lasted for 8 consecutive weeks. After sampling,the serum renal function indicators [creatinine(SCr),and urea nitrogen(BUN)],fasting blood glucose (FBG),24 h urinary protein quantification (24 h-UTP), and other indicators of the mice were measured. The pathological tissue morphology of the kidney was observed by periodic acid-silver methenamine (PASM) and Masson's trichrome (Masson) staining. Immunohistochemical detection of cysteine-dependent aspartate-specific protease (Caspase)-3 and B-cell lymphoma 2 (Bcl-2) was performed. Western blot was used to detect the protein expression of Caspase-8,Caspase-7,Caspase-3, and other molecules. Terminal deoxynucleotidyl transferase dUTP nick End labeling (TUNEL) staining was used to observe apoptosis in renal tissue. Immunofluorescence staining of Wilms tumor suppressor gene-1
2.Treatment of Attention Deficit Hyperactivity Disorder with Comorbid Tic Disorder in Children from the Perspective of Ministerial Fire Scorching Yin and Internal Stirring of Deficient Wind
Hongsheng YANG ; Junhong WANG ; Meifang LI ; Wei LI ; Zhenhua YUAN ; Rui ZHAI ; Yuan LI ; Kangning ZHOU
Journal of Traditional Chinese Medicine 2026;67(1):79-82
Attention deficit hyperactivity disorder (ADHD) is often accompanied by tic disorder. The core pathogenesis is considered to be ministerial fire scorching yin and internal stirring of deficient wind, which leads to disharmony between the body and spirit, resulting in clinical manifestations. The treatment principles emphasize nourishing yin fluids, calming ministerial fire, and extinguishing endogenous wind (内风). The method of nourishing yin fluids is applied throughout the entire treatment process, commonly using ingredients such as Shudihuang (Rehmanniae Radix Praeparata), Shanzhuyu (Corni Fructus), Gouqizi (Lycii Fructus), Wuweizi (Schisandrae Chinensis Fructus), and Tusizi (Cuscutae Semen). These are combined with approaches to harmonize the zang-fu organs, primarily including extinguishing liver wind, clearing heart fire, nourishing kidney water, and strengthening spleen earth, thereby stabilizing ministerial fire and extinguishing endogenous wind. Additionally, emotional regulation and smoothing emotional constraint are essential to improve clinical symptoms in children with ADHD comorbid with tic disorder.
3.Measurement and analysis of radiation doses received by the human body and radiation levels in the CT room under digital miniature CT scanning conditions
Lin YIN ; Zhenhua YANG ; Yaqi XI ; Wenlong FAN ; Rui YANG ; Qisheng XIA ; Qiaoling WU
Chinese Journal of Radiological Health 2025;34(3):373-377
Objective To evaluate the radiation impact of a self-developed digital miniature CT on the human body and the environment under simulated scanning conditions, and verify its safety and regulatory compliance. Methods Under typical head scanning conditions with the digital miniature CT (70 kV/10 mA), the equivalent doses received at the body surface sites corresponding to the thyroid, breast, stomach, liver, kidney, and gonads of the phantom were measured without protection and with 0.5 mmPb equivalent protection using LiF (Mg, Cu, P) thermoluminescent dosimeters. The ambient dose equivalent rates at the bed level inside the CT room at different directions and distances from the scanning center were measured using a model AT1121 X/γ dosimeter. The equivalent doses of organs on both sides of the phantom and the ambient equivalent dose rates on the left and right sides of the longitudinal axis of the bed in the CT room were compared. The Mann-Whitney test was used at a significance level of P < 0.05. Results During a single scan of the head with the digital miniature CT, the equivalent doses at the body surface sites corresponding to the thyroid, breast, stomach, liver, kidney, and gonads without protection were 1.04, 0.95, 0.55, 0.57, 0.40, and 0.12 mSv, respectively, which were only 0.84% to 8.24% of the doses inside the irradiation field. With 0.5 mm Pb equivalent protection, the equivalent dose of the thyroid decreased from 8.24 mSv to 3.27 mSv with a reduction of 60.3%, and the doses of the other organs were reduced to 1.5-11.5 μSv with the maximum reduction of 14 times. In the longitudinal axis direction of the CT bed, the ambient dose equivalent rate at a distance of 2 m from the scanning center was reduced to 0.066 mSv/h, which was only 9.6% of the ambient equivalent dose rate at a distance of 50 cm from the scanning center. Conclusion The digital miniature CT has advantages in ensuring patient safety, optimizing imaging quality, and promoting technological development, demonstrating promising application potential. However, the radiation protection of personal and CT room should not be ignored.
4.Research on medical needles excavated from the tomb of LIU He, Marquis of Haihun in Nanchang.
Zhenhua WU ; Chuning WANG ; Qi ZHOU ; Jun YANG
Chinese Acupuncture & Moxibustion 2025;45(6):861-866
Several metal needles were unearthed from the tomb of LIU He, the Marquis of Haihun in Nanchang, Jiangxi province. Based on the inscription on the signboard unearthed from the tomb and the handed down literature, these needles should be closely related to the "nine needles" mentioned in the handed down medical classics. These needles should be used in acupuncture and moxibustion at that time, and their diameters are close to the specifications and dimensions of the needles in the modern time, which provides the important cultural relics data of the Han Dynasty for the research of early acupuncture and moxibustion of traditional Chinese medicine. This article uses the method of mutual verification of unearthed cultural relics, unearthed documents, and handed down documents to investigate the acupuncture method of medical needles unearthed from the tomb of Marquis Haihun, and to sort out and demonstrate the development of acupuncture and moxibustion in the Western Han Dynasty. The medical needles unearthed from the tomb of Marquis Haihun and their needling techniques can be confirmed by the "nine needles" in the Lingshu (Miraculous Pivot), the "acupuncture treatment" in the bamboo slips of the Han Dynasty in the Northwest, and the "acupuncture atlas" on the Han portrait stone. It proves that the manufacturing of needles for acupuncture and moxibustion and the therapeutic methods and techniques of acupuncture had been greatly developed in the middle and late stages of the Western Han Dynasty.
Acupuncture Therapy/instrumentation*
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Needles/history*
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China
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Humans
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History, Ancient
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Moxibustion/instrumentation*
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Medicine, Chinese Traditional/instrumentation*
5.Upregulation of NR2A in Glutamatergic VTA Neurons Contributes to Chronic Visceral Pain in Male Mice.
Meng-Ge LI ; Shu-Ting QU ; Yang YU ; Zhenhua XU ; Fu-Chao ZHANG ; Yong-Chang LI ; Rong GAO ; Guang-Yin XU
Neuroscience Bulletin 2025;41(12):2113-2126
Chronic visceral pain is a persistent and debilitating condition arising from dysfunction or sensitization of the visceral organs and their associated nervous pathways. Increasing evidence suggests that imbalances in central nervous system function play an essential role in the progression of visceral pain, but the exact mechanisms underlying the neural circuitry and molecular targets remain largely unexplored. In the present study, the ventral tegmental area (VTA) was shown to mediate visceral pain in mice. Visceral pain stimulation increased c-Fos expression and Ca2+ activity of glutamatergic VTA neurons, and optogenetic modulation of glutamatergic VTA neurons altered visceral pain. In particular, the upregulation of NMDA receptor 2A (NR2A) subunits within the VTA resulted in visceral pain in mice. Administration of a selective NR2A inhibitor decreased the number of visceral pain-induced c-Fos positive neurons and attenuated visceral pain. Pharmacology combined with chemogenetics further demonstrated that glutamatergic VTA neurons regulated visceral pain behaviors based on NR2A. In summary, our findings demonstrated that the upregulation of NR2A in glutamatergic VTA neurons plays a critical role in visceral pain. These insights provide a foundation for further comprehension of the neural circuits and molecular targets involved in chronic visceral pain and may pave the way for targeted therapies in chronic visceral pain.
Animals
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Male
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Visceral Pain/metabolism*
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Up-Regulation/physiology*
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Ventral Tegmental Area/metabolism*
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Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors*
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Neurons/drug effects*
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Mice, Inbred C57BL
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Mice
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Proto-Oncogene Proteins c-fos/metabolism*
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Chronic Pain/metabolism*
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Glutamic Acid/metabolism*
6.Effect of Liangxue Tuizi Formula (凉血退紫方) on RAF/MEK/ERK Pathway in Skin Tissue and Serum NETs Biomarkers in Henoch-Schönlein Purpura Model Rats with Blood Heat Syndrome
Yingying JIANG ; Manxiang YANG ; Zhenhua YUAN ; Leying XI ; Mingyang CAI ; Diya MA ; Yifan LI ; Yuhang NIU ; Runze LIU ; Jiawen CAO ; Xilin CHEN ; Xianqing REN
Journal of Traditional Chinese Medicine 2025;66(23):2475-2483
ObjectiveTo investigate the potential mechanism of Liangxue Tuizi Formula (凉血退紫方, LXTZF) in treating Henoch-Schönlein Purpura (HSP) by examining its regulatory effect on neutrophil extracellular trap (NETs) dysregulation via the rapidly accelerated fibrosarcoma kinase (RAF)/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway. MethodsSeventy Wistar rats were randomly allocated into a blank control group (n=14) and a modeling group (n=56). Rats in the modelling group underwent an eight-week modelling period to establish HSP rat models with blood-heat syndrome via modified ovalbumin (OVA) induction method combined with oral administration of heat-property Chinese herbal medicine. Fifty successfully modeled rats were subsequently randomly divided into five groups (n=10 per group), model group, compound glycyrrhizin group, LXTZF group, RAF inhibitor group, and LXTZF + RAF agonist group. Additionally, 10 rats were selected from the original blank control group for the final experiment. From the 11th week of modelling, rats in the blank control group and the model group received 1 ml/(100 g·d) ultrapure water via oral administration, in addition to 0.5 ml/(kg·d) 0.9% sodium chloride solution via intraperitoneal injection. The LXTZF group and the compound glycyrrhizin group received 7.5 g/(kg·d) LXTZF granule suspension via gavage, 13.5 mg/(kg·d) compound glycyrrhizin suspension via gavage, respectively. The RAF inhibitor group received 1 mg/(kg·d) GW5074 suspension via intraperitoneal injection and ultrapure water via oral administration; the LXTZF + RAF agonist group received 7.5 g/(kg·d) LXTZF granule suspension via gavage and 1 mg/(kg·d) paclitaxel suspension via intraperitoneal injection. All administrations were performed once daily for 4 weeks. After intervention, skin tissue histopathology was examined by hematoxylin and eosin (H&E) staining, immunoglobulin A (IgA) deposition was assessed via immunofluorescence, serum levels of neutrophil elastase (NE), tumor necrosis factor-α (TNF-α), and vascular cell adhesion molecule-1 (VCAM-1) were measured using enzyme-linked immunosorbent assay (ELISA), serum myeloperoxidase (MPO) level was determined by a colorimetric assay; the mRNA expression levels of RAF, MEK, and ERK in skin tissue were detected by real-time quantitative polymerase chain reaction (RT-qPCR); and the protein expression of RAF, MEK, ERK, as well as phosphorylated MEK (p-MEK) and phosphorylated ERK (p-ERK), were analyzed by Western Blot. ResultsSkin tissue in the blank control group rats remained normal, whereas the model group exhibited neutrophil infiltration and haemorrhage with red blood cell rupture. In all drug intervention groups, neutrophil infiltration and haemorrhagic exudation reduced markedly, with LXTZF group demonstrating the most pronounced improvement. Compared with the blank control group, rats in the model group exhibited enhanced IgA fluorescence intensity in skin tissue, elevated serum levels of NE, MPO, TNF-α and VCAM-1, increased mRNA expression of RAF, MEK, ERK1 and ERK2, as well as heightened RAF protein levels and p-MEK/MEK and p-ERK/ERK ratios (P<0.05). Compared with the model group, the drug intervention groups exhibited reduced IgA fluorescence intensity in skin tissue, along with decreased serum levels of NE, MPO, TNF-α, and VCAM-1 (P<0.05). In LXTZF group and RAF inhibition groups, reduced mRNA expression of RAF, MEK, ERK1, and ERK2 was observed in rat skin tissue, alongside decreased RAF protein levels and reduced p-MEK/MEK and p-ERK/ERK ratios (P<0.05). Compared with LXTZF + RAF agonist group, the compound glycyrrhizin group, LXTZF group, and RAF inhibitior group exhibited reduced IgA fluorescence intensity in skin tissue, decreased serum NE, MPO, TNF-α, and VCAM-1 levels, and decreased MEK mRNA expression and p-MEK/MEK ratio (P<0.05). ConclusionThe potential mechanism by which LXTZF treats Henoch-Schönlein purpura with blood heat syndrome may involve blocking the RAF/MEK/ERK signaling pathway in skin tissue, and suppressing excessive formation of NETs, thereby reducing IgA deposition in dermal microvessels and attenuating systemic inflammatory responses.
7.Contrast of the therapeutic effect of CT/MRI image fusion technology and CT guided radiofrequency thermocoagulation of trigeminal semilunar ganglion
Jing SHEN ; Mengge WANG ; Yang SONG ; Zhenhua REN
Acta Universitatis Medicinalis Anhui 2024;59(3):538-541,546
Objective To evaluate the therapeutic effect of CT/MRI image fusion and usual CT guided percutane-ous radiofrequency thermocoagulation of trigeminal semilunar ganglion.Methods The medical information of 88 patients diagnosed with primary trigeminal neuralgia were assembled.In accordance with different imaging guidance means,they were equally divided into the control group(trigeminal semilunar ganglion radiofrequency thermo-coagulation with CT guidance)and the fusion group(trigeminal semilunar ganglion radiofrequency thermocoagula-tion with assistance of CT/MRI image fusion technology)at random.The puncture time,intraoperative discomfort rate,preoperative,intraoperative and postoperative visual analogue scale(VAS)score,Barrow neurological insti-tute(BNI)pain score and postoperative complication rate were contrasted.Results The puncture operation time of the fusion group was shorter than that of the control group(P<0.05);the intraoperative and postoperative VAS and BNI scores,occurrence rate of intraoperative discomfort and postoperative complications in the fusion group were lower than those in the control group(P<0.05).Conclusion In respect of improving therapeutic effect and diminishing intraoperative discomfort and postoperative complications,CT/MRI image fusion technique is superior to CT guidance.
8.Cell softness reveals tumorigenic potential via ITGB8/AKT/glycolysis signaling in a mice model of orthotopic bladder cancer
Shi QIU ; Yaqi QIU ; Linghui DENG ; Ling NIE ; Liming GE ; Xiaonan ZHENG ; Di JIN ; Kun JIN ; Xianghong ZHOU ; Xingyang SU ; Boyu CAI ; Jiakun LI ; Xiang TU ; Lina GONG ; Liangren LIU ; Zhenhua LIU ; Yige BAO ; Jianzhong AI ; Tianhai LIN ; Lu YANG ; Qiang WEI
Chinese Medical Journal 2024;137(2):209-221
Background::Bladder cancer, characterized by a high potential of tumor recurrence, has high lifelong monitoring and treatment costs. To date, tumor cells with intrinsic softness have been identified to function as cancer stem cells in several cancer types. Nonetheless, the existence of soft tumor cells in bladder tumors remains elusive. Thus, our study aimed to develop a microbarrier microfluidic chip to efficiently isolate deformable tumor cells from distinct types of bladder cancer cells.Methods::The stiffness of bladder cancer cells was determined by atomic force microscopy (AFM). The modified microfluidic chip was utilized to separate soft cells, and the 3D Matrigel culture system was to maintain the softness of tumor cells. Expression patterns of integrin β8 (ITGB8), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) were determined by Western blotting. Double immunostaining was conducted to examine the interaction between F-actin and tripartite motif containing 59 (TRIM59). The stem-cell-like characteristics of soft cells were explored by colony formation assay and in vivo studies upon xenografted tumor models. Results::Using our newly designed microfluidic approach, we identified a small fraction of soft tumor cells in bladder cancer cells. More importantly, the existence of soft tumor cells was confirmed in clinical human bladder cancer specimens, in which the number of soft tumor cells was associated with tumor relapse. Furthermore, we demonstrated that the biomechanical stimuli arising from 3D Matrigel activated the F-actin/ITGB8/TRIM59/AKT/mTOR/glycolysis pathways to enhance the softness and tumorigenic capacity of tumor cells. Simultaneously, we detected a remarkable up-regulation in ITGB8, TRIM59, and phospho-AKT in clinical bladder recurrent tumors compared with their non-recurrent counterparts.Conclusions::The ITGB8/TRIM59/AKT/mTOR/glycolysis axis plays a crucial role in modulating tumor softness and stemness. Meanwhile, the soft tumor cells become more sensitive to chemotherapy after stiffening, that offers new insights for hampering tumor progression and recurrence.
9.Influence of childhood abuse on the presence of depressive symptom in junior high school students: the effecting path of peer attachment and emotional resilience
Xinlong TANG ; Yang JIANG ; Zhenhua LU ; Wanqing YU ; Fan SONG ; Jun ZHANG
Sichuan Mental Health 2024;37(6):526-531
BackgroundDepression as a major mental health condition is commonly found in junior high school students. Peer attachment, emotional resilience and childhood abuse have been found to be associated with depressive symptoms, and it has been hypothesized that peer attachment and emotional resilience may play a chained effecting path in the relationship between childhood abuse and depressive symptoms in junior high school students. ObjectiveTo explore the relationship between childhood abuse and depressive symptom in junior high school students, analyze the effecting path of peer attachment and emotional resilience, thus to provide references for improving the mental health of junior high school students. MethodsFrom May to July 2022, a cluster sampling technique was utilized to recruit 1 781 junior high school students from a junior high school in Anhui province. Childhood Trauma Questionnaire Short Form (CTQ-SF), Revised version of Inventory of Parent and Peer Attachment (IPPA-R), Adolescent' Emotional Resilience Questionnaire (AERQ) and Center for Epidemiological Studies Depression Scale (CES-D) were used as the measurement tools. Pearson correlation coefficient was calculated to assess the correlation among above scales. Process4.2 and Bootstrapping method were employed to verify the effecting path of peer attachment and emotional resilience in the relationship between childhood abuse and depressive symptoms. ResultsCTQ-SF score was negatively correlated with IPPA-R peer attachment subscale score and AERQ score (r=-0.527, -0.495, P<0.01) and positively correlated with CES-D score (r=0.669, P<0.01) in junior high school students. IPPA-R peer attachment subscale score was positively correlated with AERQ score (r=0.556, P<0.01) and negatively correlated with CES-D score (r=-0.599, P<0.01) in junior high school students. AERQ score was negatively correlated with CES-D score (r=-0.698, P<0.01) in junior high school students. Childhood abuse in junior high school students was shown to be a positive predictor of depressive symptoms (β=0.675, P<0.01) and a negative predictor of peer attachment (β=-0.824, P<0.01) and emotional resilience (β=-0.305, P<0.01). Peer attachment and emotional resilience were independent effecting path between childhood abuse and depressive symptoms, with indirect effect size of 0.093 (95% CI: 0.066~0.122) and 0.108 (95% CI: 0.084~0.133), respectively. Peer attachment and emotional resilience affected as a chain effecting path between childhood abuse and depressive symptoms, with indirect effect size of 0.087 (95% CI: 0.071~0.105), accounting for 12.89% of the total effect. ConclusionChildhood abuse in junior high school students can affect the presence of depressive symptom both directly and indirectly through either separate or chained effecting path of peer attachment and emotional resilience. [Funded by 2020 Provincial General Scientific Research Project of West Anhui Health Vocational College (number, KJ2020B006); 2024 Provincial University Natural and Humanities Sciences Research Project of West Anhui Health Vocational College (number, 2024AH053467)]
10.Comprehensive evaluation of single-anastomosis duodenal-ileal bypass with sleeve gastrectomy in obese patients based on efficacy and nutrition
Lifu HU ; Lun WANG ; Shixing LI ; Yang LIU ; Zheng ZHANG ; Minghao XIAO ; Zhenhua ZHANG ; Zhiqiang WEI ; Liang CUI ; Tao JIANG
Chinese Journal of Gastrointestinal Surgery 2024;27(9):945-952
Objective:To evaluate the 1-year postoperative efficacy and nutritional indicators of single-anastomosis duodeno-ileal bypass with sleeve gastrectomy (SADI-S) in obese patients.Methods:This retrospective observational study included patients with a body mass index (BMI) of ≥40.0 kg/m 2 regardless of other related metabolic diseases and patients with severe type 2 diabetes and a BMI between 27.5 and 40.0 kg/m 2. The clinical data of 66 obese patients who underwent SADI-S at the Bariatric and Metabolic Surgery Department of China-Japan Union Hospital of Jilin University from November 2018 to May 2022 were collected, including 53 cases of da Vinci robotic surgery and 13 cases of laparoscopic surgery. The patients comprised 38 men and 28 women with a median age of 35 (18–61) years and a mean preoperative BMI of 42.93 ± 6.82 kg/m 2. A total of 38 patients had type 2 diabetes, and 46 had hyperuricemia, 45 had hypertension, 35 had hyperlipidemia, 12 had hypercholesterolemia, and 12 had a high low-density lipoprotein (LDL) level. The main observation indicators were (1) intraoperative and postoperative conditions; (2) weight loss outcomes, including body weight, BMI, excess body weight loss (%EWL), and total body weight loss (%TWL) at 3, 6, and 12 months after surgery; (3) effects of treatment on metabolic disease; and (4) changes in nutrient indicators. Results:(1) Intraoperative and postoperative conditions: All patients successfully underwent SADI-S with neither conversion to laparotomy nor death. Four (6.1%) patients developed postoperative complications, and all of them recovered and were discharged after conservative or surgical treatment. (2) Weight loss outcomes: %EWL at 3, 6, and 12 months after surgery was 62.07 ± 26.56, 85.93 ± 27.92, and 106.65 ± 29.65, respectively, and %TWL was 22.67 ± 4.94, 32.10 ± 5.18, and 40.56 ± 7.89, respectively. Body weight and BMI 3 to 12 months after surgery were significantly lower than those before surgery (all P < 0.001). (3) Effect of treatment on metabolic disease: 3 to 12 months after surgery, fasting blood sugar, HbA1c, uric acid, systolic blood pressure, diastolic blood pressure, triglycerides, total cholesterol, LDL, and other indicators were significantly lower than those before surgery (all P < 0.05). Twelve months after surgery, the remission rates of diabetes, hyperuricemia, hypertension, hypertriglyceridemia, hypercholesterolemia, and high LDL were 100% (38/38), 65.2% (30/46), 62.2% (28/45), 94.3% (33/35), 100% (12/12), and 100% (12/12), respectively. (4) Changes in nutrient indicators: Compared with the preoperative nutrient levels, the hemoglobin and hematocrit levels were lower at 3 to 12 months after surgery, the total protein level was lower at 6 to 12 months after surgery, the albumin level was lower at 6 months after surgery, and the ferritin level was lower at 3 months after surgery. The differences were statistically significant (all P < 0.05). The incidence of anemia was 6.1% (4/66), hypoalbuminemia was 4.5% (3/66), and ferritin deficiency was 4.5% (3/66), all of which were improved or normalized through conservative treatment. Twelve months after surgery, 30 (45.5%) patients had vitamin A deficiency, 17 (25.8%) had vitamin E deficiency, 11 (16.7%) had folic acid deficiency, 2 had potassium deficiency (3.0%), 3 (4.5%) had calcium deficiency, 2 (3.0%) had magnesium deficiency, 9 (13.6%) had iron deficiency, and 16 (24.2%) had zinc deficiency. However, no relevant clinical symptoms occurred. Conclusions:SADI-S has a very significant effect on weight loss and alleviation of metabolic diseases. Nutrient deficiencies after SADI-S mainly involve vitamin A, vitamin E, zinc, and folic acid. The long-term efficacy and safety of SADI-S still need further follow-up observation.


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