1.Study on activation mechanism of SGCs in representation along the distribution of stomach meridian in IBS-D mice.
Na LI ; Junhui REN ; Lu GUAN ; Yashuang XU ; Liyan ZHONG ; Xiaomei SHAO ; Jianqiao FANG ; Junying DU ; Junfan FANG
Chinese Acupuncture & Moxibustion 2025;45(5):593-600
OBJECTIVE:
To explore the peripheral neural mechanism underlying representation along the distribution of stomach meridian induced by intestinal inflammatory reaction using diarrhea predominant-irritable bowel syndrome (IBS-D) mice.
METHODS:
Among 62 healthy male C57BL/6 mice of clean grade, 12 mice were randomly selected and divided into a control group and a model group, 6 mice in each group, additionally, 12 mice were randomly selected and divided into a Tianshu group, a Liangqiu group and a Zusanli group, 4 mice in each group. In the model group, citrobacter was administered orally to establish IBS-D model. In the control group and the model group, the visceral pain threshold was observed using fecal colorectal distension (fCRD) induced electromyography of external oblique muscle, the positive cell number of neutrophil in the colonic muscularis was detected by myeloperoxidase (MPO) staining, the number, location and distribution rule of Evans blue (EB) extravasation points were observed by injection of EB staining solution into the tail vein. In the Tianshu group, the Liangqiu group and the Zusanli group, fluorescent dye Dil was injected at bilateral "Tianshu" (ST25), "Liangqiu" (ST34) and "Zusanli" (ST36) respectively, to observe the dye-positive cell number in different dorsal root ganglion (DRG) segments. In the control group and the model group, the activation of satellite glial cells (SGCs) in different DRG segments was observed by immunofluorescence.
RESULTS:
Compared with the control group, in the model group, the area under curve of electromyography of external oblique muscle was increased at fCRD of 25, 50 and 75 μL distilled water (P<0.001, P<0.01); the MPO-positive cell number of neutrophil in the colonic muscularis was increased (P<0.01). Few EB extravasation points could be found in the control group, while there were much more EB extravasation points observed in the model group, which was specially distribution in the area of stomach meridian, from "Huaroumen" (ST24) to "Zusanli" (ST36), as well as the surface area dominated by L2-L5 segment of the spinal cord. The Dil-positive cells were mainly exhibited in the DRG of T11, L5 and L4 segments in the Tianshu group, the Liangqiu group and the Zusanli group, respectively. Compared with the control group, the ratio of glial fibrillary acidic protein (GFAP)/glutamine synthetase (GS) co-expression was increased in the DRG of T11, L4 and L5 segments in the model group (P<0.05, P<0.01).
CONCLUSION
The activation of SGCs within DRG of T11, L4 and L5 segments may relate closely to the occurrence of the representation along the stomach meridian distribution in IBS-D mice.
Animals
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Male
;
Mice
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Irritable Bowel Syndrome/therapy*
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Mice, Inbred C57BL
;
Meridians
;
Stomach/physiopathology*
;
Humans
;
Acupuncture Points
;
Disease Models, Animal
2.The relationship between the triglyceride-glucose index and its modified index and colorectal cancer:A prospective cohort study
Yi LU ; Shilong DAI ; Mingjun WANG ; Jing ZHOU ; Junying HAO ; Chen ZHENG ; Xinbo XU ; Shan DING ; Qingsong ZHANG
The Journal of Practical Medicine 2025;41(15):2362-2371
Objective To investigate the association between the TyG index,its modified variants,and the risk of developing colorectal cancer(CRC).Methods This study included a total of 93,177 participants from the 2006 Kailuan Group health examination cohort.Participants were categorized into four quartiles(Q1-Q4)according to their TyG and modified TyG indices.Follow-up began at the baseline examination,with incident CRC as the primary outcome.Participants were censored at the time of CRC diagnosis,death,or the end of the study,whichever occurred first.The dose-response relationship between TyG and its modified indices and the risk of CRC was evalu-ated using restricted cubic splines(RCS)in conjunction with Cox proportional hazards regression models,yielding hazard ratios(HRs)and 95%confidence intervals(CIs).To compare the strength of associations between TyG and its modified versions(TyG-BMI,TyG-WC,TyG-WHR,TyG-WHtR,TyG-WWI)and CRC risk,HRs for CRC per one standard deviation increase in each index were calculated and compared.Results Both the TyG index and its modified variants demonstrated a significant dose-response relationship with the risk of CRC incidence.Specifically,for the TyG index,each 1-standard deviation(SD)increase was associated with a 1.17-fold(95%CI:1.09~1.27)higher risk of CRC.Compared with the first quartile(Q1),the third quartile(Q3)and fourth quartile(Q4)exhibited a 1.25-fold(95%CI:1.01~1.55)and 1.26-fold(95%CI:1.01~1.57)increased risk,respectively.For TyG-BMI,each 1-SD increase was linked to a 1.20-fold(95%CI:1.07~1.35)elevated CRC risk.Compared with Q1,Q3 and Q4 showed a 1.32-fold(95%CI:1.06~1.64)and 1.51-fold(95%CI:1.21~1.88)increase,respectively.Regarding TyG-WC,each 1-SD increment was associated with a 1.22-fold(95%CI:1.13~1.32)higher CRC risk,with Q3 and Q4 showing a 1.35-fold(95%CI:1.08~1.70)and 1.56-fold(95%CI:1.24~1.96)increased risk compared to Q1.For TyG-WHtR,each 1-SD increase was associated with a 1.24-fold(95%CI:1.08-1.42)higher CRC risk.Compared with Q1,Q2,Q3,and Q4 demonstrated a 1.31-fold(95%CI:1.03~1.66),1.55-fold(95%CI:1.23~1.95),and 1.60-fold(95%CI:1.27~2.02)increase,respectively.In the case of TyG-WHR,each 1-SD increase was associated with a 1.19-fold(95%CI:1.10~1.29)higher CRC risk,with Q4 showing a 1.42-fold(95%CI:1.14~1.77)increased risk compared to Q1.Finally,for TyG-WWI,each 1-SD increase was associated with a 1.22-fold(95%CI:1.13~1.32)elevated CRC risk,with both Q3 and Q4 showing a 1.58-fold increase(Q3:95%CI:1.26~1.98;Q4:95%CI:1.25~1.99).Stratified analyses by sex and age consistently revealed significant associations between the TyG index and its modified variants and CRC risk.Furthermore,these indices were independently associated with the incidence of both colon cancer and rectal cancer.Conclusions(1)Elevated levels of the TyG index and its modified variants are independent risk factors for CRC.(2)Both the TyG index and its modified forms demonstrate a significant dose-response association with the incidence of CRC.(3)Among the modified TyG indices,TyG-WWI,TyG-WHtR,TyG-BMI,TyG-WC,and TyG-WHR showed stronger correlations with CRC risk compared to the original TyG index.
3.The relationship between the triglyceride-glucose index and its modified index and colorectal cancer:A prospective cohort study
Yi LU ; Shilong DAI ; Mingjun WANG ; Jing ZHOU ; Junying HAO ; Chen ZHENG ; Xinbo XU ; Shan DING ; Qingsong ZHANG
The Journal of Practical Medicine 2025;41(15):2362-2371
Objective To investigate the association between the TyG index,its modified variants,and the risk of developing colorectal cancer(CRC).Methods This study included a total of 93,177 participants from the 2006 Kailuan Group health examination cohort.Participants were categorized into four quartiles(Q1-Q4)according to their TyG and modified TyG indices.Follow-up began at the baseline examination,with incident CRC as the primary outcome.Participants were censored at the time of CRC diagnosis,death,or the end of the study,whichever occurred first.The dose-response relationship between TyG and its modified indices and the risk of CRC was evalu-ated using restricted cubic splines(RCS)in conjunction with Cox proportional hazards regression models,yielding hazard ratios(HRs)and 95%confidence intervals(CIs).To compare the strength of associations between TyG and its modified versions(TyG-BMI,TyG-WC,TyG-WHR,TyG-WHtR,TyG-WWI)and CRC risk,HRs for CRC per one standard deviation increase in each index were calculated and compared.Results Both the TyG index and its modified variants demonstrated a significant dose-response relationship with the risk of CRC incidence.Specifically,for the TyG index,each 1-standard deviation(SD)increase was associated with a 1.17-fold(95%CI:1.09~1.27)higher risk of CRC.Compared with the first quartile(Q1),the third quartile(Q3)and fourth quartile(Q4)exhibited a 1.25-fold(95%CI:1.01~1.55)and 1.26-fold(95%CI:1.01~1.57)increased risk,respectively.For TyG-BMI,each 1-SD increase was linked to a 1.20-fold(95%CI:1.07~1.35)elevated CRC risk.Compared with Q1,Q3 and Q4 showed a 1.32-fold(95%CI:1.06~1.64)and 1.51-fold(95%CI:1.21~1.88)increase,respectively.Regarding TyG-WC,each 1-SD increment was associated with a 1.22-fold(95%CI:1.13~1.32)higher CRC risk,with Q3 and Q4 showing a 1.35-fold(95%CI:1.08~1.70)and 1.56-fold(95%CI:1.24~1.96)increased risk compared to Q1.For TyG-WHtR,each 1-SD increase was associated with a 1.24-fold(95%CI:1.08-1.42)higher CRC risk.Compared with Q1,Q2,Q3,and Q4 demonstrated a 1.31-fold(95%CI:1.03~1.66),1.55-fold(95%CI:1.23~1.95),and 1.60-fold(95%CI:1.27~2.02)increase,respectively.In the case of TyG-WHR,each 1-SD increase was associated with a 1.19-fold(95%CI:1.10~1.29)higher CRC risk,with Q4 showing a 1.42-fold(95%CI:1.14~1.77)increased risk compared to Q1.Finally,for TyG-WWI,each 1-SD increase was associated with a 1.22-fold(95%CI:1.13~1.32)elevated CRC risk,with both Q3 and Q4 showing a 1.58-fold increase(Q3:95%CI:1.26~1.98;Q4:95%CI:1.25~1.99).Stratified analyses by sex and age consistently revealed significant associations between the TyG index and its modified variants and CRC risk.Furthermore,these indices were independently associated with the incidence of both colon cancer and rectal cancer.Conclusions(1)Elevated levels of the TyG index and its modified variants are independent risk factors for CRC.(2)Both the TyG index and its modified forms demonstrate a significant dose-response association with the incidence of CRC.(3)Among the modified TyG indices,TyG-WWI,TyG-WHtR,TyG-BMI,TyG-WC,and TyG-WHR showed stronger correlations with CRC risk compared to the original TyG index.
4.Neuroprotective effects of Ginkgo biloba dropping pills in Parkinson's disease
Dingyi YU ; Pengli ZHANG ; Junying LI ; Ting LIU ; Yaodan ZHANG ; Qingqing WANG ; Jianbing ZHANG ; Xiaoyan LU ; Xiaohui FAN
Journal of Pharmaceutical Analysis 2021;11(2):220-231
Parkinson's disease(PD)is the second most common neurodegenerative disease in the world;however,it lacks effective and safe treatments.Ginkgo biloba dropping pill(GBDP),a unique Chinese G.biloba leaf extract preparation,exhibits antioxidant and neuroprotective effects and has a potential as an alternative therapy for PD.Thus,the aims of this study were to evaluate the effects of GBDP in in vitro and in vivo PD models and to compare the chemical constituents and pharmacological activities of GBDP and the G.biloba extract EGb 761.Using liquid chromatography tandem-mass spectrometry,46 GBDP constitu-ents were identified.Principal component analysis identified differences in the chemical profiles of GBDP and EGb 761.A quantitative analysis of 12 constituents showed that GBDP had higher levels of several flavonoids and terpene trilactones than EGb 761,whereas EGb 761 had higher levels of organic acids.Moreover,we found that GBDP prevented 6-hydroxydopamine-induced dopaminergic neuron loss in zebrafish and improved cognitive impairment and neuronal damage in methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced PD mice.Although similar effects were observed after EGb 761 treatment,the neuroprotective effects were greater after GBDP treatment on several endpoints.In addition,in vitro results suggested that the Akt/GSK3β pathway may be involved in the neuroprotective effects of GBDP.These findings demonstrated that GBDP have potential neuroprotective effects in the treatment of PD.
5.Effect of kangaroo care on neurological behavior in neonates with neonatal intensive care unit asphyxia
Danqiong LU ; Jinyan WANG ; Xia YU ; Jinya WANG ; Junying SHEN ; Suping WANG
Chinese Journal of Practical Nursing 2020;36(12):909-912
Objective:To explore the effect of kangaroo care on the neurological behavior of neonatal intensive care unit (NICU) neonates with asphyxia.Methods:Totally 76 cases of neonatal asphyxia admitted to NICU from January 2018 to December 2018 were randomly divided into observation group (40 cases) and observation group (36 cases). The control group was given routine nursing care, while the observation group was given kangaroo nursing intervention on the basis of the control group. The neurobehavioral changes, the occurrence of nervous system damage and the physical development after 42 days of birth were compared between the two groups.Results:The scores of behavior ability (11.64 ± 0.26), original reflex (5.89 ± 0.32), passive muscle tension (7.64 ± 0.46), active muscle tension (7.83 ± 0.55), general reaction (6.17 ± 0.46) in the intervention observation group were significantly higher than those in the control group (11.02 ± 0.39), original reflex (5.53 ± 0.31), passive muscle tension (7.21 ± 0.47), active muscle tension (6.17 ± 0.46) Tension (7.41 ± 0.41), general reaction (5.88 ± 0.41) ( t values were 8.227, 4.970, 4.027, 3.740, 2.888, P<0.05). The incidence of neurological damage in the observation group was 2.50% (1/40) which was significantly lower than 16.67% (6/36) in the control group ( χ 2=4.117, P < 0.05). The growth of body weight (2.17 ± 0.42) kg, length (7.15 ± 1.74) cm and head circumference (4.38 ± 0.93) cm in the observation group were significantly higher than those in the control group (1.68 ± 0.39) kg, (5.89 ± 1.81) cm, (3.81 ± 0.79) cm ( t values were 5.252, 3.093, 2.863, P<0.05). Conclusion:Kangaroo nursing can effectively promote the neurological development and physical development of neonatal asphyxia, and reduce the nervous system damage of asphyxiated neonates, and the operation is simple and easy, which is worthy of clinical application.
6.Correlation between peritoneal thickness and baseline peritoneal solute transport function
Meilan QIU ; Yongping CHEN ; Weizeng LIAO ; Yufeng LIANG ; Meirong QIU ; Xinglan LIANG ; Li QIU ; Meijin LAN ; Binsan HUANG ; Juan LU ; Xiaoyan LIN ; Junying WU ; Xuehua ZHANG
Chinese Journal of Nephrology 2020;36(3):197-202
Objective:To investigate the relationship between peritoneal thickness and baseline solute transport function in peritoneal dialysis (PD) patients, and analyze the factors affecting the function of peritoneal transport.Methods:Non-diabetic end-stage renal disease (ESRD) patients admitted to the Second Hospital of Longyan City from January 2017 to June 2019 were enrolled in this study. The thickness of the peritoneal membrane was measured by color ultrasound instrument before the peritoneal catheterization. Standard peritoneal equilibration test (PET) was performed after one month of peritoneal dialysis. The ratio of corrected creatine in 4 h dialysate to 2 h serum creatine (D/Pcr) was used as a solute baseline transport index, and according to the D/Pcr evaluation results, the patients were divided into high/high average transfer (H) group (D/Pcr≥0.65) and low/low average transfer (L) group (D/Pcr<0.65). The clinical data, peritoneal thickness and peritoneal dialysis related indicators between the two groups of patients were compared. Binary logistic regression was used to analyze the factors affecting the function of peritoneal transport.Results:The amount of peritoneal ultrafiltration in H group was significantly lower than that in L group, intraperitoneal creatinine clearance (Ccr) and peritoneal thickness were significantly higher than those in L group (both P<0.05). Pearson and Spearman correlation results showed that the thickness of peritoneal membrane positively correlated with D/Pcr ( r=0.673, P<0.05), peritoneal Ccr ( r=0.261, P<0.05), and negatively correlated with ultrafiltration of peritoneal dialysis ( r=-0.365, P<0.05). Partial correlation analysis showed that the peritoneal thickness was positively correlated with the solute transport index D/Pcr ( r=0.539, P<0.05) and the peritoneal Ccr ( r=0.338, P<0.05). Binary logistic regression results showed that peritoneal thickening was a risk factor affecting peritoneal transport function ( OR=1.175, 95% CI 1.009-1.369, P<0.05). Conclusions:There is a positive correlation between the peritoneal membrane thickness and the baseline solute transport index in patients with non-diabetic peritoneal dialysis. Peritoneal thickening is a risk factor affecting peritoneal transport function.
7.Role of cerebrospinal fluid chimerism in predicating central nervous relapse surveillance for patients of acute leukemia after allogeneic hematopoietic stem cell transplantation
Junying LI ; Zhaodong ZHONG ; Yong YOU ; Liang TANG ; Xuan LU ; Han YAN ; Huafang WANG ; Linghui XIA ; Yu HU ; Wei SHI
Chinese Journal of Organ Transplantation 2019;40(3):138-143
Objective To explore the role of cerebrospinal fluid chimerism in central nervous relapse surveillance for patients of acute leukemia after allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods The follow-up data were retrospectively collected and analyzed in 104 patients with acute leukemia after allo-HSCT.Comparisons were made between patients with complete chimerism and mixed chimerism in cerebrospinal fluid.The role of recipient DNA percentage and its changing trend in predicting central nervous relapse were also explored.Analysis was conducted for determining the risk factors of central nervous relapse.And the effectiveness of prophylaxis with intrathecal injection was also examined.Results The incidence of relapse was higher in patients with mixed chimerism (P<0.001),high percentage of recipient DNA (P<0.05) and higher mixed chimerism (P<0.001).Hyperleukocytosis at an initial diagnosis was a risk factor of central nervous relapse.Whether or not intrathecal injection prophylaxis was applied showed no significant difference in relapsing rate.Conclusions Monitoring cerebrospinal fluid chimerism can effectively help predict central nervous relapse among patients of acute leukemia after allo-HSCT.Yet intrathecal injection prophylaxis failed to benefit recipients.
8. Lentivirus media miR-1246 knockdown inhibits tumor growth and promotes apoptosis of SiHa cells
Ping DU ; Yuehua LAI ; Desheng YAO ; Yan LU ; Junying CHEN ; Nan DING
Chinese Journal of Obstetrics and Gynecology 2018;53(7):481-486
Objective:
To study the effect of lentivirus-mediated microRNA (miR) -1246 RNA interference (RNAi) on biological characteristics and behaviors in cervical cancer cells as well as to identify the downstream signaling pathways affected.
Methods:
MiR-1246 specific cDNA was synthesized and cloned into the recombinant lentiviral vector (LV-miR-1246-inhibitor) . The SiHa cells were devided into three groups: no viral infection (negative control, NC) , infection with control virus (LV-NC) , and infection with miR-1246-inhibitor virus (LV-miR-1246-inhibitor) . The expression of the miR-1246 was detected by reverse transcription (RT) -PCR. Cell growth was analyzed by cell counting kit 8 (CCK-8) assay. The invasion was dectected by transwell matrige gel. Cell apoptosis was detected by flow cytometer. The growth of xenograft tumors was also investigated. Expression of thrombospondin-2 (THBS2) , matrix metalloproteinase (MMP) 2, 9 were also evaluated in the cells.
Results:
(1) The expression level of miR-1246 in SiHa cells (0.11±0.13) was significantly lower in group LV-miR-1246-inhibitor than those in the group LV-NC and the group NC (1.14±0.86 and 1.30±0.73, respectively;
9.Investigation of clinical application for severe segmental calcification by subtraction technique of coronary artery CT angiography
Weifeng GUO ; Mengsu ZENG ; Juying QIAN ; Zheyong HUANG ; Junying GU ; Lijun ZHANG ; Xiuliang LU ; Shuai GUO ; Shan YANG
Fudan University Journal of Medical Sciences 2017;44(3):274-279
Objective To investigate the feasibility of subtraction coronary computed tomography angiography (Sub-CCTA) for the diagnosis of coronary heart disease in the segment with severe calcification.Methods A retrospective analysis was performed on 27 patients who underwent clinically indicated digital subtraction angiography (DSA) and CCTA using a 320-detector row CT.Compared with the results of DSA,sensitivity,specificity,positive predictive value,negative predictive value and accuracy of Con-CCTA and Sub-CCTA were calculated.The clinical diagnostic accuracy of the two imaging methods was evaluated using the receiver operating characteristic (ROC) curve.The stenosis of coronary segments was divided into four grades (Ⅰ,Ⅱ,Ⅲ,Ⅳ).Kappa coefficient was used to measure agreement between two imaging methods.Image quality of 4-scale grade scoring method was used and t test was conducted.Results A total of 52 segments with severe calcification were evaluated.The scores of image quality in Con-CCTA and Sub-CCTA were 2.8 ± 0.5 and 3.4 ± 0.7,respectively.There was significant difference between them (t =5.9,P < 0.05).Compared with the result of DSA as the golden standard,the Kappa coefficients were 0.55 and 0.81 respectively in Con-CCTA and Sub-CCTA for the quantitative evaluation of the severe calcified segments.The sensitivity,specificity,positive predictive value and negative predictive value and accuracy of Con-CCTA were 81.0%,63.1%,63.1%,81.1% and 70.8 %;and for Sub-CCTA they were 90.5 %,85.2%,82.1 %,92.0% and 87.5 % respectively.Compared with Con-CCTA,the area under the ROC curve of Con-CCTA and Sub-CCTA were 0.84 (95%CI:0.70-0.93) and 0.96 (95% CI:0.86-1.00),respectively,and the difference was statistically significant (P =0.03).Conclusions Sub-CCTA can improve the diagnostic accuracy of coronary artery stenosis in severe calcified segment.Application of subtraction technique in CCTA can reduce or even eliminate the artifacts caused by severe calcified plaque,and has a good clinical application prospect.
10.The value of umbilical cord blood and amniotic fluid biological markers in predicting brain injury in prema-ture infants after premature rupture of the membranes
Hongyan LU ; Qiang ZHANG ; Ming CHANG ; Qiuxia WANG ; Junying LU
Chinese Journal of Applied Clinical Pediatrics 2016;31(6):452-456
Objective To investigate the relationship between the preterm infants after premature rupture of the membranes(PROM)brain injury and some cellular factors in the umbilical cord blood and amniotic fluid,and ana-lyze the biological markers with great predictive value,and provide a theoretical basis for early monitoring of brain injury in premature infants. Methods One hundred and thirty - nine singleton infants with PROM,their gestation less than 34 weeks,were evaluated. The umbilical cord blood and amniotic fluid of cytokines,including interleukin - 1β(IL - 1β),IL - 4,IL - 6,IL - 8,IL - 10,IL - 17A,tumor necrosis factor alpha(TNF - α),granulocyte colony - stimu-lating factor(G - CSF),monocyte chemotactic protein - 1(MCP - 1),S100B protein and soluble intercellular adhe-sion molecule - 1(sICAM - 1)levels were measured with Luminex liquid chip. All the premature infants underwent brain imaging for the diagnosis of brain damage. All cases were divided into brain injury group and non - brain injury group based on brain imaging examination. Results The concentration of IL - 10 in cord blood was significantly lower in the brain injury group than that in the non - brain injury group,and the difference was statistically significant(P ﹤0. 05). The levels of IL - 1β,IL - 6,IL - 8,TNF - α,G - CSF,MCP - 1,S100B and sICAM - 1 in the brain injury group were significantly higher than those of non - brain injury group,and the differences were statistically significant (all P ﹤ 0. 05). The levels of IL - 1β,IL - 6,IL - 8,TNF - α,G - CSF,MCP - 1 and sICAM - 1 in the amniotic fluid were significantly higher than those of non - brain injury group,and the differences were statistically significant(all P ﹤ 0. 05),but amniotic fluid S100B protein level was similar between 2 groups,which had no statistical significance (P ﹥ 0. 05). To predict the value of brain damage in premature infants,the highest sensitivity in cord blood was S100B protein,the highest specificity was IL - 6. The highest sensitivity in amniotic fluid was IL - 1β,and the highest specificity was IL - 8. The levels of IL - 4 and IL - 17A in the umbilical cord blood and amniotic fluid,IL - 10 in amniotic fluid were very low,and had no predictive value for brain damage. Conclusions Many biological markers in umbilical cord blood and amniotic fluid provide information about the risk of brain injury in premature infants. The highest sensitivity in cord blood was S100B protein,the highest specificity was IL - 6. The highest sensitivity in amniotic fluid was IL - 1β,the highest specificity was IL - 8. Changes in inflammation - related biomarkers suggest that brain damage in the preterm infants might be associated with intrauterine inflammation.

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