1.Scutellarin inhibitting BV-2 microglia-mediated neuroinflammation via the cyclic GMP-AMP synthase-stimulator of interferon gene pathway
Zhao-Da DUAN ; Li YANG ; Hao-Lun CHEN ; Teng-Teng LIU ; Li-Yang ZHENG ; Dong-Yao XU ; Chun-Yun WU
Acta Anatomica Sinica 2024;55(2):133-142
Objective To explore the effect of scutellarin on lipopolysaccharide(LPS)induced neuroinflammation in BV-2 microglia cells.Methods BV-2 microglia were cultured and randomly divided into 6 groups:control group(Ctrl),cyclic GMP-AMP synthetase(cGAS)inhibitor RU320521 group(RU.521 group),LPS group,LPS+RU.521 group,LPS+scutellarin pretreatment group(LPS+S)and LPS+S+RU.521 group.The expressions of cGAS,stimulator of interferon gene(STING),nuclear factor kappa B(NF-κB),phosphorylated NF-κB(p-NF-κB),neuroinflammatory factors PYD domains-containing protein 3(NLRP3)and tumor necrosis factor α(TNF-α)in BV-2 microglia were detected by Western blotting and immunofluorescent double staining(n= 3).Results Western blotting and immunofluorescent double staining showed that compared with the control group,the expression of cGAS,STING,p-NF-κB,NLRP3 and TNF-α in BV-2 microglia increased significantly after LPS induction(P<0.05),while the expression of cGAS,STING,p-NF-κB,NLRP3 and TNF-α in LPS+S group were significantly lower than those in LPS group(P<0.05).Treatment with cGAS pathway inhibitor RU.521 showed similar effects as the pre-treatment group with scutellarin.In addition,the change of NF-κB in each group was not statistically significant(P>0.05).Conclusion Scutellarin inhibits the neuroinflammation mediated by BV-2 microglia cells,which may be related to cGAS-STING signaling pathway.
2.Effects of ligustrazine on cognitive function in mice with post-traumatic stress disorder
Ling GUO ; Yong-Quan CHEN ; Can LIU ; Wei-Dong YAO ; Yue YAO ; Ping-Ping CHENG ; Zhao-Fang LIU
The Chinese Journal of Clinical Pharmacology 2024;40(19):2880-2884
Objective To investigate the effect and mechanism of tetramethylpyrazine(TMP)on cognitive function in mice with post-traumatic stress disorder(PTSD).Methods The mice were randomly divided into normal group,model group and experimental group.Except for the normal group,the PTSD mouse model was established by single prolonged stress(SPS).The experimental group was intraperitoneally injected with 10 mg·kg-1 TMP,and the normal group and the model group were intraperitoneally injected with an equal amount of 0.9%NaCl.The Morris water maze,open field and elevated plus maze tests were used to evaluate the cognitive behavior of the mice.The apoptosis of neurons was detected by Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL).The expression of ionized calcium binding adapter molecule-1(Iba-1)protein was detected by immunofluorescence(ICC).The content of oxidative stress inflammatory factors was detected by enzyme-linked immunosorbent assay(ELISA).Results The escape latency of the normal group,model group,and TMP group were(56.50±9.89),(87.16±10.48)and(68.63±10.19)s,respectively;the corner residence time of the open field were(190.37±40.64),(260.39±40.54)and(218.63±38.27)s,respectively;the apoptosis rates were(18.28±2.35)%,(39.36±3.65)%and(30.74±3.58)%,respectively;the fluorescence intensities of Iba-1 were(8.01±2.23)%,(50.87±7.31)%and(7.49±1.41)%;malondialdehyde contents were(5.46±0.95),(12.98±2.06)and(8.31±1.28)nmol·mg-1,respectively;tumor necrosis factor-α contents were(53.59±9.91),(115.46±11.53)and(74.38±10.77)pg·mL-1,respectively.The above indexes in the normal group and the experimental group were statistically significant compared with the model group(P<0.05,P<0.01).Conclusion TMP can improve the cognitive function of PTSD mice,and the mechanism may be related to the regulation of inflammation and oxidative stress.
3.Evaluation of the efficacy of cryopreservation of human adipose tissue with novel cryoprotective agents
Runlei ZHAO ; Guanhuier WANG ; Yang AN ; Danli YANG ; Mingjie YAO ; Qilong CAO ; Xiangwu CHU ; Dong LI
Chinese Journal of Plastic Surgery 2024;40(2):202-211
Objective:To investigate the effectiveness of new cryoprotective agents in preserving and transplanting human adipose tissue.Methods:The adipose tissue samples were obtained from healthy adult females who underwent liposuction at the Department of Plastic Surgery of Peking University Third Hospital from January to March 2022. The adipose tissue samples were centrifuged and then randomly divided into 9 groups. These groups were cryopreserved in liquid nitrogen using different cryoprotective agents [group A, group B, and dimethyl sulfoxide (DMSO) group] and cryopreservation times (1-month, 2-month, and 3-month groups), respectively. The cryoprotective agent formulation in group A was dextrose glycoside 40 (DEX), amino acids, vitamins, and inorganic salts. In group B, the formulation included DMSO and DEX. The ratio of cryoprotective agent in the DMSO group was 10% DMSO, 20% fetal bovine serum (FBS), and 70% DMEM-12. For cryopreservation, 5 ml cryogenic tubes were used with a fat to cryoprotective agent ratio of 3∶2, and each group contains 6 tubes for cryopreservation. After thawing the adipose tissue, HE staining was used to observe the histological morphology. Immunohistochemical staining was employed for the quantitative analysis of lipid droplet-encapsulated protein (Perilipin), and the Perilipin positivity rate was calculated by the ratio of the number of positive cells to the total number of cells. Adipocyte viability was assessed using the CCK-8 method. Thirty-eight healthy, clean nude mice were selected and divided into 3 groups of 12 mice each according to the use of different cryoprotective agents (groups A, B, and DMSO), while the other 2 mice were used as the day 0 control group. The mean fat freezing duration for all groups was 3 months. After nude mice were anesthetized intraperitoneally, 0.9 ml of thawed cryopreserved fat was injected into the dorsum bilaterally. The rate of adipose tissue retention was calculated by MRI scanning and three-dimensional software at 1, 2, and 3 months after transplantation, and compared between the groups. The fat grafts were explanted from the mice after they were sacrificed, and then subjected to histological morphology and quantitative analysis of Perilipin by using HE staining and immunohistochemical staining. GraphPad Prism 8.0 software was used for statistical analysis of the data. The data that conformed to a normal distribution were expressed as Mean ± SD. The overall comparison between multiple groups used analysis of variance for repeated measures. The comparison of data between groups at the same time point used Tukey’s multiple comparison test.Results:The morphology of adipose tissue in different cryoprotective agent groups closely resembled that of normal fresh adipose tissue after being cryopreserved in liquid nitrogen for 1-3 months. The difference in the proportion of Perilipin-stained positive cells in each group was not statistically significant ( P>0.05). The CCK-8 method indicated that the effect of the DMSO group was superior to groups A and B at 1 and 3 months of cryopreservation ( P<0.01), and that the DMSO group and group B were superior to group A at 2 months of cryopreservation ( P<0.01). In the animal experiments, there was no statistically significant difference between the groups in the volume retention rate 1-3 months after cryopreserved fat transplantation ( P>0.05). Additionally, the adipose tissues in each group exhibited varying degrees of localized necrosis accompanied by an inflammatory reaction 1-3 months after transplantation. There was no statistically significant difference in the Perilipin staining positivity between the groups ( P>0.05). Conclusion:The use of new cryoprotective agents for cryopreserving adipose tissue does not show a significant difference compared to the traditional cryoprotective agent. However, it is theoretically safer as it avoids the potential toxic effects of using DMSO or FBS on the human body.
4.Clinical Study on Huangjing Jiangya Decoction in the Treatment of Patients with Hypertension of Qi-Deficiency Type Accom-panied by Insomnia
Wen SHI ; Haijuan MA ; Jintao HE ; Lei DONG ; Yao LIU ; Huiling ZHAO ; Yuan XING
Journal of Nanjing University of Traditional Chinese Medicine 2024;40(11):1256-1262
OBJECTIVE To observe the effect of Huangjing Jiangya Decoction on blood pressure and sleep in patients with hy-pertension of qi-deficiency type accompanied by insomnia.METHODS 73 patients with hypertension of qi-deficiency type accompa-nied by insomnia who met the inclusion criteria were selected and randomly divided into an observation group of 36 cases and a control group of 37 cases.The control group was treated with amlodipine besylate tablets,and the observation group was given Huangjing Jian-gya Decoction oral treatment on the basis of the control group.Both groups were treated continuously for 8 weeks.The changes in TCM syndrome scores,office blood pressure monitoring(OBPM),home blood pressure monitoring(HBPM),24-hour ambulatory blood pressure monitoring(ABPM),Pittsburgh Sleep Quality Index(PSQI)scores and clinical efficacy of the two groups of patients before and after treatment were observed.RESULTS After treatment,the TCM syndrome scores in the observation group were significantly decreased(P<0.05,P<0.01),which were better than the control group(P<0.01);OBPM and HBMP in both groups were signifi-cantly reduced(P<0.05,P<0.01),the observation group was better than the control group(P<0.05,P<0.01);the ABPM of the observation group was significantly reduced(P<0.01),which was better than the control group(P<0.05,P<0.01);the sleep quali-ty,sleep latency,sleep duration,daytime dysfunction score and PSQI total score of the observation group were significantly decreased(P<0.01),which were better than those in the control group(P<0.05,P<0.01);the clinical efficacy of hypertension and insomnia in the observation group was both better than the control group(P<0.01).CONCLUSION Huangjing Jiangya Decoction combined with amlodipine can improve the symptoms of patients with hypertension of qi-deficiency type accompanied by insomnia,lower blood pressure,improve sleep quality,shorten sleep latency,alleviate daytime dysfunction,and has good clinical efficacy.
5.TSHR Variant Screening and Phenotype Analysis in 367 Chinese Patients With Congenital Hypothyroidism
Hai-Yang ZHANG ; Feng-Yao WU ; Xue-Song LI ; Ping-Hui TU ; Cao-Xu ZHANG ; Rui-Meng YANG ; Ren-Jie CUI ; Chen-Yang WU ; Ya FANG ; Liu YANG ; Huai-Dong SONG ; Shuang-Xia ZHAO
Annals of Laboratory Medicine 2024;44(4):343-353
Background:
Genetic defects in the human thyroid-stimulating hormone (TSH) receptor (TSHR) gene can cause congenital hypothyroidism (CH). However, the biological functions and comprehensive genotype–phenotype relationships for most TSHR variants associated with CH remain unexplored. We aimed to identify TSHR variants in Chinese patients with CH, analyze the functions of the variants, and explore the relationships between TSHR genotypes and clinical phenotypes.
Methods:
In total, 367 patients with CH were recruited for TSHR variant screening using whole-exome sequencing. The effects of the variants were evaluated by in-silico programs such as SIFT and polyphen2. Furthermore, these variants were transfected into 293T cells to detect their Gs/cyclic AMP and Gq/11 signaling activity.
Results:
Among the 367 patients with CH, 17 TSHR variants, including three novel variants, were identified in 45 patients, and 18 patients carried biallelic TSHR variants. In vitro experiments showed that 10 variants were associated with Gs/cyclic AMP and Gq/11 signaling pathway impairment to varying degrees. Patients with TSHR biallelic variants had lower serum TSH levels and higher free triiodothyronine and thyroxine levels at diagnosis than those with DUOX2 biallelic variants.
Conclusions
We found a high frequency of TSHR variants in Chinese patients with CH (12.3%), and 4.9% of cases were caused by TSHR biallelic variants. Ten variants were identified as loss-of-function variants. The data suggest that the clinical phenotype of CH patients caused by TSHR biallelic variants is relatively mild. Our study expands the TSHR variant spectrum and provides further evidence for the elucidation of the genetic etiology of CH.
6.Influence of curative-intent resection with textbook outcomes on long-term prognosis of gall-bladder carcinoma: a national multicenter study
Zhipeng LIU ; Zimu LI ; Yule LUO ; Xiaolin ZHAO ; Jie BAI ; Yan JIANG ; Yunfeng LI ; Chao YU ; Fan HUANG ; Zhaoping WU ; Jinxue ZHOU ; Dalong YIN ; Rui DING ; Wei GUO ; Yi ZHU ; Wei CHEN ; Kecan LIN ; Ping YUE ; Yao CHENG ; Haisu DAI ; Dong ZHANG ; Zhiyu CHEN
Chinese Journal of Digestive Surgery 2024;23(7):926-933
Objective:To investigate the influence of curative-intent resection with textbook outcomes of liver surgery (TOLS) on long-term prognosis of gallbladder carcinoma (GBC).Methods:The retrospective cohort study was conducted. The clinicopathological data of 824 patients with GBC in the national multicenter database of Biliary Surgery Group of Elite Group of Chinese Journal of Digestive Surgery, who were admitted to 15 medical centers from January 2014 to January 2021, were collected. There were 285 males and 539 females, aged (62±11)years. According to the evalua-tion criteria of TOLS, patients were divided into those who achieved TOLS and those who did not achieve TOLS. Measurement data with normal distribution were represented as Mean± SD, and com-parison between groups was conducted using the independent sample t test. Measurement data with skewed distribution were represented as M( Q1, Q3), and comparison between groups was conducted using the Mann-Whitney U test. Count data were described as absolute numbers, and comparison between groups was conducted using the chi-square test. Comparison of ordinal data were conduc-ted using the Mann-Whitney U test. The Kaplan-Meier method was used to calculate survival rate and draw survival curve, and the Log-rank test was used for survival analysis. The COX stepwise regression model with backward Wald method was used for univariate and multivariate analyses. Results:(1) Achievement of TOLS. Of the 824 patients undergoing curative-intent resection for GBC, there were 510 cases achieving TOLS and 314 cases not achieving TOLS. (2) Follow-up. Of the 824 patients undergoing curative-intent resection for GBC, after excluding 112 deaths within 90 days after discharge, 712 cases were included for the survival analysis. The median follow-up time, median overall survival time and 5-year overall survival rate of the 510 patients achieving TOLS were 22.1(11.4,30.1)months, 47.6(30.6,64.6)months and 47.5%. The median follow-up time, median overall survival time and 5-year overall survival rate of the 202 patients not achieving TOLS were 14.0(6.8,25.5)months, 24.3(20.0,28.6)months and 21.0%. There was a significant difference in overall survival between patients achieving TOLS and patients not achieving TOLS ( χ2=58.491, P<0.05). (3) Analysis of factors influencing prognosis of patients. Results of multivariate analysis showed that TOLS, carcinoembryonic antigen (CEA), CA19-9, poorly differentiation of tumor, T2 stage of eighth edition of American Joint Committee on Cancer (AJCC) staging, T3 and T4 stage of eighth edition of AJCC staging, N1 stage of the eighth edition of AJCC staging, N2 stage of the eighth edition of AJCC staging, adjuvant therapy were independent factors influencing overall survival time of patients undergoing curative-intent resection for GBC ( hazard ratio=0.452, 1.479, 1.373, 1.612, 1.455, 1.481, 1.835, 1.978, 0.538, 95% c onfidence interval as 0.352-0.581, 1.141-1.964, 1.052-1.791, 1.259-2.063, 1.102-1.920, 1.022-2.147, 1.380-2.441, 1.342-2.915, 0.382-0.758, P<0.05). Conclusion:Patients under-going curative-intent resection for GBC with TOLS can achieve better long-term prognosis.
7.The sedative effect of remimazolam on ICU elderly patients undergoing mechanical ventilation and its influence on the circulatory system
Peng ZHAO ; Fangchao YAO ; Yi ZHENG ; Hailing DONG ; Jiuqing CUI ; Hao SUN ; Renjie LI ; Jingpu TIAN
Chinese Journal of Postgraduates of Medicine 2024;47(7):640-646
Objective:To investigate the sedative effect of remimazolam on ICU elderly patients undergoing mechanical ventilation and its influence on circulatory system.Methods:Using a prospective research approach, 189 ICU elderly patients undergoing mechanical ventilation in Hebei Petro China Central Hospital from October 2021 to June 2023 were selected. The patients were divided into remimazolam group, dexmedetomidine group and propofol group by random number table method with 63 cases in each group. The patients in remimazolam group, dexmedetomidine group and propofol group were sedated with remimazolam, dexmedetomidine and propofol, respectively. The sedation standard time, sedation standard rate, sedation maintenance time and recovery time after drug withdrawal were compared among the three groups. The heart rate, mean arterial pressure (MAP), respiratory rate and pulse oxygen saturation (SpO 2) before medication (T 0) and medication for 15 min (T 1), 30 min (T 2), 1 h (T 3), 6 h (T 4), 12 h (T 5) were recorded. The incidences of bradycardia, hypotension, respiratory depression, body movement and delirium during sedation were recorded. Results:The sedation standard time and recovery time after drug withdrawal in remimazolam group were significantly shorter than those in dexmedetomidine group and propofol group: (22.27 ± 5.31) min vs. (29.45 ± 6.24) and (30.12 ± 5.87) min, (28.66 ± 7.06) min vs. (32.22 ± 6.85) and (34.34 ± 7.24) min, and there were statistical differences ( P<0.05); there were no statistical difference between dexmedetomidine group and propofol group ( P>0.05). The sedation standard rate in remimazolam group and dexmedetomidine group was significantly higher than that in propofol group: 87.43% (661/756) and 83.60% (632/756) vs. 72.49% (548/756), and there was statistical difference ( P<0.016 7); there was no statistical difference between remimazolam group and dexmedetomidine group ( P>0.016 7). There was no statistical difference in sedation maintenance time among the three groups ( P>0.05). There were no statistical difference in T 0 heart rate, MAP, respiratory rate and SpO 2 among the three groups ( P>0.05). The T 1 to T 5 heart rate and MAP in remimazolam group were significantly higher than those in dexmedetomidine group and propofol group, the T 2 to T 5 heart rate and MAP in dexmedetomidine group were significantly lower than those in propofol group, and there were statistical differences ( P<0.05). The T 2 to T 5 respiratory rate in remimazolam group was significantly lower than that in dexmedetomidine group, the T 1 to T 5 respiratory rate in remimazolam group and dexmedetomidine group was significantly higher than that in propofol group, and there were statistical differences ( P<0.05). The T 2 to T 5 SpO 2 in remimazolam group and dexmedetomidine group was significantly higher than that in propofol group, and there was statistical difference ( P<0.05). The incidence of bradycardia in remimazolam group was significantly lower than that in dexmedetomidine group: 7.94% (5/63) vs. 25.40% (16/63), the incidence of hypotension was significantly lower than that in propofol group: 6.35% (4/63) vs. 23.81% (15/63), and there were statistical differences ( P<0.016 7). The incidence of respiratory depression in remimazolam group and dexmedetomidine group was significantly lower than that in propofol group: 4.76% (3/63) and 1.59% (1/63) vs. 22.22% (14/63), and there was statistical difference ( P<0.016 7). There was statistical difference in incidence of delirium among the three groups ( P<0.05), but there was no statistically significant difference in pairwise comparison ( P>0.016 7). There was no statistical difference in the incidence of body movement among the three groups ( P>0.05). Conclusions:The effect of remimazolam sedation in ICU elderly patients undergoing mechanical ventilation is satisfactory, with little influence on circulation and respiratory system and few adverse reactions.
8.Effects and mechanism of metformin on the wound healing of full-thickness skin defects in diabetic rats
Baohong WANG ; Yanbing ZHANG ; Xianping ZHANG ; Yuting LI ; Zhihui WU ; Rongying HU ; Shiyue ZHAO ; Hongna JIANG ; Yuwei YAO ; Jianda DONG
Chinese Journal of Burns 2024;40(6):579-588
Objective:To investigate the effects and mechanism of metformin on the wound healing of full-thickness skin defects in diabetic rats.Methods:This study was an experimental study. Eighteen 8-week-old male Sprague Dawley rats were divided into control group, diabetes group, and diabetes+metformin group according to complete random grouping method, with 6 rats in each group. The latter two groups of rats were used to create diabetic models, and then four circular full-thickness skin defect wounds with a diameter of 5 mm were made on the back of 18 rats. Metformin F-127 hydrogel was applied only to the wounds of rats in diabetes+metformin group. The wound healing status on post injury day (POD) 7 and 13 was observed and the wound healing rate was calculated. The wound tissue on POD 7 and 13 was collected for hematoxylin-eosin staining to measure the length of re-epithelialized epidermis and calculate the change rates in diameters of epidermal and dermal wounds, for immunohistochemical staining to detect the relative expressions of keratin 10 and proliferating cell nuclear antigen (PCNA), and for Western blotting to detect the protein expressions of keratin 10 and PCNA. The sample size in all the above experiments was 8 except that in the last experiment was 3. The correlations between the relative expressions of keratin 10 and PCNA in wound tissue in three groups of rats and their wound healing rates, and the correlation between the relative expressions of keratin 10 and PCNA in wound tissue were analyzed.Results:On POD 7, the wound healing rates of rats in diabetes group and diabetes+metformin group were 81.48% (77.89%, 85.53%) and 93.04% (92.51%, 94.24%), which were significantly lower than 100% (97.17%, 100%) in control group (with Z values of 2.37 and -3.36, respectively, P<0.05); the wound healing rate of rats in diabetes+metformin group was significantly higher than that in diabetes group ( Z=3.45, P<0.05). On POD 13, the wound healing rates of rats in control group and diabetes+metformin group were both 100% (100%, 100%), which were significantly higher than 94.47% (90.68%, 99.82%) in diabetes group (with Z values of 2.90 and -2.90, respectively, P<0.05). On POD 7, the change rates in epidermal wound diameter of rats in control group and diabetes+metformin group were significantly higher than that in diabetes group (with Z values of 3.36 and -2.74, respectively, P<0.05). The change rates in dermal wound diameter of rats in the three groups were similar on POD 7 and 13 ( P>0.05). The lengths of re-epithelialized epidermis of rats in control group and diabetes+metformin group on POD 13 were significantly longer than that in diabetes group (with Z values of 3.34 and -2.64, respectively, P<0.05). The relative expressions of keratin 10 in wound tissue of rats in diabetes group on POD 7 and 13 were significantly higher than those in control group (with Z values of -3.36 and -3.26, respectively, P<0.05) and diabetes+metformin group (with Z values of 3.36 and 3.15, respectively, P<0.05), and the relative expression of keratin 10 in wound tissue of rats in diabetes+metformin group on POD 7 was significantly lower than that in control group ( Z=3.05, P<0.05); the relative expressions of PCNA in wound tissue of rats in diabetes group on POD 7 and 13 were significantly lower than those in control group (with both Z values of 3.36, P<0.05) and diabetes+metformin group (with both Z values of -3.36, P<0.05). The protein expressions of keratin 10 in wound tissue of rats in control group and diabetes+metformin group on POD 7 as well as that in diabetes+metformin group on POD 13 were significantly lower than those in diabetes group ( P<0.05), and the protein expressions of PCNA in wound tissue of rats in control group and diabetes+metformin group on POD 7 were significantly higher than that in diabetes group ( P<0.05). There was a significant positive correlation between the relative expression of keratin 10 in wound tissue and the wound healing rate in control group and diabetes+metformin group of rats (with r values of 0.78 and 0.71, respectively, P<0.05), there was a significant negative correlation between the relative expression of PCNA in wound tissue and the wound healing rate in diabetes+metformin group of rats ( r=-0.60, P<0.05), and there was a significant negative correlation between the relative expressions of PCNA and keratin 10 in wound tissue of rats in diabetes group and diabetes+metformin group (with r values of -0.41 and -0.49, respectively, P<0.05). Conclusions:The diabetic rats with full-thickness skin defect wound exhibit delayed healing, accompanied by up-regulation of keratin 10 and down-regulation of PCNA in keratinocytes in the wound tissue. Metformin can promote wound healing in diabetic rats with full-thickness skin defects by down-regulating keratin 10 expression and up-regulating PCNA expression in keratinocytes in the wound tissue, and the wound healing rate was positively correlated with the expression of keratin 10 and negatively correlated with the expression of PCNA.
9.Evaluation of the efficacy of cryopreservation of human adipose tissue with novel cryoprotective agents
Runlei ZHAO ; Guanhuier WANG ; Yang AN ; Danli YANG ; Mingjie YAO ; Qilong CAO ; Xiangwu CHU ; Dong LI
Chinese Journal of Plastic Surgery 2024;40(2):202-211
Objective:To investigate the effectiveness of new cryoprotective agents in preserving and transplanting human adipose tissue.Methods:The adipose tissue samples were obtained from healthy adult females who underwent liposuction at the Department of Plastic Surgery of Peking University Third Hospital from January to March 2022. The adipose tissue samples were centrifuged and then randomly divided into 9 groups. These groups were cryopreserved in liquid nitrogen using different cryoprotective agents [group A, group B, and dimethyl sulfoxide (DMSO) group] and cryopreservation times (1-month, 2-month, and 3-month groups), respectively. The cryoprotective agent formulation in group A was dextrose glycoside 40 (DEX), amino acids, vitamins, and inorganic salts. In group B, the formulation included DMSO and DEX. The ratio of cryoprotective agent in the DMSO group was 10% DMSO, 20% fetal bovine serum (FBS), and 70% DMEM-12. For cryopreservation, 5 ml cryogenic tubes were used with a fat to cryoprotective agent ratio of 3∶2, and each group contains 6 tubes for cryopreservation. After thawing the adipose tissue, HE staining was used to observe the histological morphology. Immunohistochemical staining was employed for the quantitative analysis of lipid droplet-encapsulated protein (Perilipin), and the Perilipin positivity rate was calculated by the ratio of the number of positive cells to the total number of cells. Adipocyte viability was assessed using the CCK-8 method. Thirty-eight healthy, clean nude mice were selected and divided into 3 groups of 12 mice each according to the use of different cryoprotective agents (groups A, B, and DMSO), while the other 2 mice were used as the day 0 control group. The mean fat freezing duration for all groups was 3 months. After nude mice were anesthetized intraperitoneally, 0.9 ml of thawed cryopreserved fat was injected into the dorsum bilaterally. The rate of adipose tissue retention was calculated by MRI scanning and three-dimensional software at 1, 2, and 3 months after transplantation, and compared between the groups. The fat grafts were explanted from the mice after they were sacrificed, and then subjected to histological morphology and quantitative analysis of Perilipin by using HE staining and immunohistochemical staining. GraphPad Prism 8.0 software was used for statistical analysis of the data. The data that conformed to a normal distribution were expressed as Mean ± SD. The overall comparison between multiple groups used analysis of variance for repeated measures. The comparison of data between groups at the same time point used Tukey’s multiple comparison test.Results:The morphology of adipose tissue in different cryoprotective agent groups closely resembled that of normal fresh adipose tissue after being cryopreserved in liquid nitrogen for 1-3 months. The difference in the proportion of Perilipin-stained positive cells in each group was not statistically significant ( P>0.05). The CCK-8 method indicated that the effect of the DMSO group was superior to groups A and B at 1 and 3 months of cryopreservation ( P<0.01), and that the DMSO group and group B were superior to group A at 2 months of cryopreservation ( P<0.01). In the animal experiments, there was no statistically significant difference between the groups in the volume retention rate 1-3 months after cryopreserved fat transplantation ( P>0.05). Additionally, the adipose tissues in each group exhibited varying degrees of localized necrosis accompanied by an inflammatory reaction 1-3 months after transplantation. There was no statistically significant difference in the Perilipin staining positivity between the groups ( P>0.05). Conclusion:The use of new cryoprotective agents for cryopreserving adipose tissue does not show a significant difference compared to the traditional cryoprotective agent. However, it is theoretically safer as it avoids the potential toxic effects of using DMSO or FBS on the human body.
10.Ropivacaine dosage study for sub-high altitude cesarean spinal anesthesia
Le ZHANG ; Meng-Zhao MA ; Wang-Jun DONG ; Yao-Jun LU ; Ye-Yue GAO ; Shao-Qiang HUANG ; Chen YANG
Fudan University Journal of Medical Sciences 2024;51(5):778-783
Objective To determine the median effective dose(ED50)of ropivacaine for spinal anesthesia in sub-high altitude cesarean sections.Methods A total of 30 parturients from sub-high altitudes received initial 14 mg(1.4 mL)of 1%ropivacaine intrathecally.Effectiveness was defined by sensory block to T6 within 15 minutes without additional epidural anesthesia.Doses were adjusted by±1 mg based on response.ED50 and 95%CI were estimated using Dixon's method and isotonic regression.Adverse reactions were noted.Results Thirty parturients with an average age of(30.88±5.56)years,gestational weeks of(40±1.41)weeks,height of(156.69±5.80)cm,and weight of(67.44±10.48)kg were studied.The ED50 was 10.68 mg(95%CI:9.65-12.58 mg)by Dixon's method and 10.33 mg(95%CI:9.41-12.07 mg)by isotonic regression.Intraoperatively,8 cases of hypotension,1 case of bradycardia,and 7 cases of nausea and vomiting were observed,no hypertension or shivering occurred among the parturients.Conclusion The ED50 for ropivacaine in sub-high altitude cesarean sections is 10.68 mg,which is higher than the currently known ED50 required for patients in plain areas.

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