BACKGROUND:Defective outgrowth of chondrocyte mitophagy causes degenerative chondrocyte pathological changes such as apoptosis and matrix loss.OBJECTIVE:To investigate the effects and possible mechanism of Juanbi Decoction-containing serum on interleukin-1β-induced inflammatory response and apoptosis of rat knee joint chondrocytes.METHODS:Fifty male Sprague-Dawley rats were randomly given saline,Juanbi Decoction in low,medium and high doses(1.24,2.48 and 4.96 g/kg),and celecoxib(positive drug)group.Drug-containing serum was obtained by continuous gavage for 2 weeks.(1)Chondrocytes were isolated and randomly divided into control group,interleukin-1β group,Juanbi Decoction low,medium and high dose containing serum groups and positive drug serum group.Cell counting kit-8 assay was used to detect cell viability;immunofluorescence double staining was used to detect mitophagy level;immunofluorescence was used to detect the phosphorylated AMP-activated protein kinase level;western blot was used to detect the expression of PTEN induced putative kinase 1/Parkin pathway-related protein and cleaved caspase-3;and ELISA was used to detect inflammatory factor level.(2)PTEN induced putative kinase 1 siRNA and Compound C were used for intervention to explore the role of AMP-activated protein kinase/PTEN induced putative kinase 1/Parkin pathway in the regulation of mitophagy by Juanbi Decoction-containing serum.RESULTS AND CONCLUSION:(1)Compared with the control group,chondrocyte survival,type II collagen,phosphorylated AMP-activated protein kinase,PTEN induced putative kinase 1,Parkin,and microtubule-associated proteins 1 light chain 3 protein levels,and mitophagy levels were significantly lower in the interleukin-1β group(P<0.05),while cleaved caspase-3 protein levels,interleukin-6,interleukin-8 and tumor necrosis factor-α levels were significantly higher(P<0.05).Compared with the interleukin-1β group,opposite changes in all the above indexes were observed in Juanbi Decoction low,medium and high dose containing serum groups and positive drug serum group(P<0.05).(2)PTEN induced putative kinase 1 siRNA significantly inhibited the effect of Juanbi Decoction-containing serum on mitophagy in interleukin-1β-treated chondrocytes and reduced the protective effect of Juanbi Decoction-containing serum on interleukin-1β-induced inflammation and apoptosis in chondrocytes;Compound C reversed the effect of Juanbi Decoction-containing serum on the PTEN induced putative kinase 1/Parkin signaling pathway in interleukin-1β-treated chondrocytes.To conclude,Juanbi Decoction-containing serum inhibits chondrocyte inflammation and apoptosis by affecting the level of mitochondrial autophagy,thereby attenuating interleukin-1β-induced chondrocyte degradation by a mechanism that may be related to the regulation of the AMP-activated protein kinase/PTEN induced putative kinase 1/Parkin pathway.

OBJECTIVE To screen and obtain nanobodies with neutralizing activity against granulo-cyte-macrophage colony stimulating factor(GM-CSF)to contribute to investigations into the mecha-nism of inflammation interventions and the development new drugs.METHODS Recombinant human GM-CSF was subcutaneously injected to immunize camels.Peripheral blood was collected after five immunizations,and mononuclear cells were isolated.Total mRNA was extracted,and the variable domains of the heavy chain of heavy-chain antibody(VHH,also called nanobody)genes were obtained by PCR amplification after reverse transcription.The genes were cloned into the pADSCFV-S phage display vector and electrotransformed into TG1 competent cells to construct a nanobody immune library that was screened with recombinant human GM-CSF immobilized on a solid phase.The VHH genes specifi-cally binding to human GM-CSF were cloned into the pABG eukaryotic expression vector before VHH-Fc samples were prepared by using the human embryonic kidney 293 fibroblast expression system.The binding activity of candidate VHH-Fc molecules to GM-CSF was investigated through ELISA response curves,and binding colorimetric values with different antigens were detected to determine their specificity.The binding affinity between VHH-Fc candidates and GM-CSF was measured using biolayer interferom-etry(BLI).The inhibition rate curve of VHH-Fc candidates on GM-CSF was detected through cell prolif-eration assays to determine its neutralization activity.The Uncle system was used to investigate its thermal stability.100 μg of VHH-Fc was injected into mice via the tail vein,and the serum concentration of VHH-Fc was quantitatively detected by ELISA to examine its pharmacokinetic curve in mice.RESULTS The camel serum titer of anti-human GM-CSF antibodies was higher than 1:800 000 after the fifth immuni-zation,and the capacity of the constructed nanobody library was about 5.55×107.Following the screening process,five candidate VHH-Fc molecules specifically binding to human GM-CSF were obtained.Among these,22N10 effectively neutralized the cell proliferation-promoting activity of GM-CSF(the IC50 value was 17.23 nmol·L-1).Subsequent studies revealed that 22N10 interacted with human GM-CSF with an affinity of 1.97×10-8 mol·L-1,blocked the binding of GM-CSF to its receptor CSF2Rα,exhibited good thermal stability(Tm1=59.2℃),and showed favorable metabolic characteristics in mice.CONCLU-SION A new candidate nanobody molecule 22N10 targeting human GM-CSF is obtained which is expected to facilitate the drug development and mechanism investigations.

ObjectiveTo investigate the risk factors for pyogenic liver abscess （PLA） comorbid with sepsis by analyzing clinical features， and to construct a predictive model. MethodsA retrospective analysis was performed for 489 patients who were hospitalized and diagnosed with PLA in The Affiliated Hospital of Xuzhou Medical University from January 2019 to December 2023， and according to the presence or absence of sepsis， they were divided into sepsis group with 306 patients and non-sepsis group with 183 patients. Related data were collected， including general information， laboratory markers， and outcome measures. The patients were further divided into a training set of 342 patients and a validation set of 147 patients at a ratio of 7∶3， and the training set was used for screening of variables and construction of a predictive model， while the validation set was used to test the performance of the model. An LASSO regression analysis was used for the screening of variables， and a multivariate Logistic regression analysis was used to construct the predictive model and plot a nomogram. The calibration curve， the receiver operating characteristic （ROC） curve， and the decision curve analysis were used for the validation of the model， and internal validation was performed for assessment. The independent-samples t test was used for comparison of normally distributed continuous data between two groups， and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups； the chi-square test was used for comparison of categorical variables between groups. ResultsThere were significant differences between the sepsis group and the non-sepsis group in pulse rate， mean arterial pressure， duration pf symptoms， comorbidity of liver cirrhosis or malignant tumor， leukocyte count， neutrophil count， lymphocyte count， platelet count （PLT）， activated partial thromboplastin time， fibrinogen， C-reactive protein， aspartate aminotransferase， alanine aminotransferase， albumin， total bilirubin （TBil）， creatinine， potassium， and prognostic nutritional index （PNI） （all P<0.05）. In the training set， the LASSO regression analysis identified four predictive factors of pulse rate， PLT， TBil and PNI， and the multivariate Logistic regression analysis showed that pulse rate （odds ratio ［OR］=1.033， 95% confidence interval ［CI］： 1.006‍ ‍—‍ ‍1.061， P=0.018）， PLT （OR=0.981， 95%CI： 0.975‍ ‍—‍ ‍0.987， P<0.001）， TBil （OR=1.086， 95%CI： 1.053‍ ‍—‍ ‍1.125， P<0.001）， and PNI （OR=0.935， 95%CI： 0.882‍ ‍—‍ ‍0.988， P=0.019） were independent influencing factors for the risk of sepsis in patients with PLA. The model constructed based on these factors showed a good predictive ability， with an area under the ROC curve of 0.948 （95%CI： 0.923‍ ‍—‍ ‍0.973） in the training set and 0.912 （95%CI： 0.848‍ ‍—‍ ‍0.976） in the validation set. The decision curve analysis showed that the model has a good net benefit within the range of 0.3‍ ‍—‍ ‍0.9 for threshold probability. ConclusionThe nomogram prediction model constructed based on pulse rate， PLT， TBil， and PNI has a certain clinical value and can well predict the risk of sepsis in patients with PLA.

BACKGROUND:Defective outgrowth of chondrocyte mitophagy causes degenerative chondrocyte pathological changes such as apoptosis and matrix loss.OBJECTIVE:To investigate the effects and possible mechanism of Juanbi Decoction-containing serum on interleukin-1β-induced inflammatory response and apoptosis of rat knee joint chondrocytes.METHODS:Fifty male Sprague-Dawley rats were randomly given saline,Juanbi Decoction in low,medium and high doses(1.24,2.48 and 4.96 g/kg),and celecoxib(positive drug)group.Drug-containing serum was obtained by continuous gavage for 2 weeks.(1)Chondrocytes were isolated and randomly divided into control group,interleukin-1β group,Juanbi Decoction low,medium and high dose containing serum groups and positive drug serum group.Cell counting kit-8 assay was used to detect cell viability;immunofluorescence double staining was used to detect mitophagy level;immunofluorescence was used to detect the phosphorylated AMP-activated protein kinase level;western blot was used to detect the expression of PTEN induced putative kinase 1/Parkin pathway-related protein and cleaved caspase-3;and ELISA was used to detect inflammatory factor level.(2)PTEN induced putative kinase 1 siRNA and Compound C were used for intervention to explore the role of AMP-activated protein kinase/PTEN induced putative kinase 1/Parkin pathway in the regulation of mitophagy by Juanbi Decoction-containing serum.RESULTS AND CONCLUSION:(1)Compared with the control group,chondrocyte survival,type II collagen,phosphorylated AMP-activated protein kinase,PTEN induced putative kinase 1,Parkin,and microtubule-associated proteins 1 light chain 3 protein levels,and mitophagy levels were significantly lower in the interleukin-1β group(P<0.05),while cleaved caspase-3 protein levels,interleukin-6,interleukin-8 and tumor necrosis factor-α levels were significantly higher(P<0.05).Compared with the interleukin-1β group,opposite changes in all the above indexes were observed in Juanbi Decoction low,medium and high dose containing serum groups and positive drug serum group(P<0.05).(2)PTEN induced putative kinase 1 siRNA significantly inhibited the effect of Juanbi Decoction-containing serum on mitophagy in interleukin-1β-treated chondrocytes and reduced the protective effect of Juanbi Decoction-containing serum on interleukin-1β-induced inflammation and apoptosis in chondrocytes;Compound C reversed the effect of Juanbi Decoction-containing serum on the PTEN induced putative kinase 1/Parkin signaling pathway in interleukin-1β-treated chondrocytes.To conclude,Juanbi Decoction-containing serum inhibits chondrocyte inflammation and apoptosis by affecting the level of mitochondrial autophagy,thereby attenuating interleukin-1β-induced chondrocyte degradation by a mechanism that may be related to the regulation of the AMP-activated protein kinase/PTEN induced putative kinase 1/Parkin pathway.

OBJECTIVE To screen and obtain nanobodies with neutralizing activity against granulo-cyte-macrophage colony stimulating factor(GM-CSF)to contribute to investigations into the mecha-nism of inflammation interventions and the development new drugs.METHODS Recombinant human GM-CSF was subcutaneously injected to immunize camels.Peripheral blood was collected after five immunizations,and mononuclear cells were isolated.Total mRNA was extracted,and the variable domains of the heavy chain of heavy-chain antibody(VHH,also called nanobody)genes were obtained by PCR amplification after reverse transcription.The genes were cloned into the pADSCFV-S phage display vector and electrotransformed into TG1 competent cells to construct a nanobody immune library that was screened with recombinant human GM-CSF immobilized on a solid phase.The VHH genes specifi-cally binding to human GM-CSF were cloned into the pABG eukaryotic expression vector before VHH-Fc samples were prepared by using the human embryonic kidney 293 fibroblast expression system.The binding activity of candidate VHH-Fc molecules to GM-CSF was investigated through ELISA response curves,and binding colorimetric values with different antigens were detected to determine their specificity.The binding affinity between VHH-Fc candidates and GM-CSF was measured using biolayer interferom-etry(BLI).The inhibition rate curve of VHH-Fc candidates on GM-CSF was detected through cell prolif-eration assays to determine its neutralization activity.The Uncle system was used to investigate its thermal stability.100 μg of VHH-Fc was injected into mice via the tail vein,and the serum concentration of VHH-Fc was quantitatively detected by ELISA to examine its pharmacokinetic curve in mice.RESULTS The camel serum titer of anti-human GM-CSF antibodies was higher than 1:800 000 after the fifth immuni-zation,and the capacity of the constructed nanobody library was about 5.55×107.Following the screening process,five candidate VHH-Fc molecules specifically binding to human GM-CSF were obtained.Among these,22N10 effectively neutralized the cell proliferation-promoting activity of GM-CSF(the IC50 value was 17.23 nmol·L-1).Subsequent studies revealed that 22N10 interacted with human GM-CSF with an affinity of 1.97×10-8 mol·L-1,blocked the binding of GM-CSF to its receptor CSF2Rα,exhibited good thermal stability(Tm1=59.2℃),and showed favorable metabolic characteristics in mice.CONCLU-SION A new candidate nanobody molecule 22N10 targeting human GM-CSF is obtained which is expected to facilitate the drug development and mechanism investigations.

Objective:To conduct the genetic analysis of a family with one patient suffering from juvenile Parkinson's disease(JP)and discuss the clinical manifestations,genetic mutation characteristics,and treatment plans prompted by PRKN gene compound heterozygous mutations,and to enhance the clinicians'awareness of this disease.Methods:The clinical data of one patient with JP caused by PRKN gene mutations was analyzed,the clinical manifestations and genetic mutation features of the patient were summarized,and the related literatures were reviewed.Results:The patient,a 16-year-old male,was admitted to the hospital due to unstable gait,trembling limbs with rigidity in both lower limbs for three years.The examination results revealed a panic gait,clear consciousness,fluent speech,normal muscle strength in limbs,increased"gear-like"muscle tone in both upper limbs,and"lead-pipe"rigidity in both lower limbs;the sensory functions and tendon reflexes were normal.The head,neck,and thoracic magnetic resonance imaging(MRI)results showed no abnormalities.18F-fluorodeoxyglucose(18F-FDG)positron emission tomography/computed tomography(PET/CT)results showed that the head size and shape were normal,the glucose metabolism in the left cerebellum and middle temporal gyrus was slightly decreased,and the glucose metabolism in bilateral thalami,right frontal lobe,parietotemporal lobe,and left medial frontal lobe was increased.The dopamine transporter(DAT)PET/CT results showed that there was no radioactive distribution in the brain cortex and the DAT distribution in the posterior part of both striata was decreased.The whole-exome sequencing results showed the patient had two PRKN gene mutations,such as codons c.8T>A and c.850G>C compound heterozygous mutations,and each mutation was from one parent;the patient's father carried the c.8T>A mutation,the patient's mother carried the c.850G>C mutation,and the patient's sister had the same genetic mutation site as the patient's father.Conclusion:PRKN gene compound heterozygous mutations may be the basis of the disease in this family.Identification of the mutation c.8T>A expands the mutation spectrum of the PRKN gene,and provides the valuable information for the research on the pathogenic genetic mutations of the JP patients.

Objective:The authors introduced the change point analysis of normal distribution based on the likelihood ratio principle, analyzed the number of outpatients in a hospital of Luohu District, Shenzhen, to provide scientific basis for rational allocation of health resources.Methods:The authors collected totally 636 number of outpatients' data from 8: 00 to 12: 00 a.m. at 48 time windows at 5 minutes intervals in a hospital of Luohu District, Shenzhen, and analyzed it with single change point analysis of simultaneous change of mean and variance, and discussed when the change point occurred.Results:The average number of outpatient was 13.250 0 for every time window, the change point occurred at 8: 50, the probability was 0.000 025, the average number of outpatient per 5 minutes from 8: 00 to 8: 50 was 7.000 0, the average number of outpatient per 5 minutes from 8: 50 to 12: 00 was 14.897 4, and the ratio of number of outpatients before and after the change point occurred was 1∶2.Conclusion:In the case of no need to determine the base period or compare the data, the change point analysis of normal distribution based on the principle of likelihood ratio provides a new way of statistical analysis and statistical monitoring for the rational allocation of health resources based on the number of patients.

Objective To investigate effect of Dahuang Fuzi decoction on alveolaur epithelial barrier in rats with lung injury with severe acute pancreatitis. Method Ninty-six health SD rats were randomly divided into three groups: sham operation group, SAP-ALI group, Dahuang Fuzi decoction group, and then according to the time point of sacrifice after operation, each group was subdivided into 3,6,12,24 hour subsets ( each, n = 8). After the belly of a rat in the sham operation group was cut open, the pancreas was flipped several times,and then a stoma was made in the jejunum to form its fistula. In the SAP-ALl group,1 mL/kg sodium taurocholate was reversely injected into the pancreatobile duct to establish the model of SAP, and then the jejimum fistula was performed. The SAP-ALI model in Dahuang Fuzi decoction group was treated by injection of 10ml of Dahuang Fuzi decoctionon into the fistula respectively. Blood was collected from heart to detect serum amvlase and endotoxin (ET) levels before the rat being executed. The lung histopathologic changes, pulmonary injury scores and wet/dry weight(W/D) ratios were observed after the rats were executed. The alveolar liquid clearance rate(ALCR), total lung water content (TLW), extravascular lung water content(EVLW) and alveolar epithelial permeability (AEP) were examined in 3,6, 12,24 h after injury.Results There was continuous increase of AEP,TLW and EVLW,as well as progressive reduction of ALCR compared with sham operation group at 3,6,12,24 h after operation. Compared with SAP-ALI group, there was continuous decrease of AEP,TLW and EVLW, and elevated of ALCR at 3,6,12,24 h after operation.Conclusions Dahuang Fuzi decoction can significantly reduce alveolaur epithelial barrier and degree of lung tissue of SAP-ALI rats by inhibiting the elevation of LPS and inflammation reaction.

Objective To observe the effects of Dahuangfuzi decoction on the intestine barrier functional of acute necrotizing pancreatitis in rats. Methods The 60 rats were randomly divided into sham operation group ( n = 19 ), ANP group ( n = 21 ), and Dahuangfuzi treatment group ( n = 20). The rats of ANP group were induced by injecting 1 ml/kg of 4% sodium taurocholate into the pancreatiobiliary duct, and jejunal fistula was esablished. The rats of treatment group received Dahuangfuzi decoction (2 ml, repeated at 4 and 8 h)through jejunum distal stoma tube 0. 5 h after ANP induction. The other 2 groups received same amount of normal saline. Blood sample was collected through abdominal aorta, 24 h after ANP induction, and the serum amylase, endotoxin, D-lactate, plasma diamine oxidase (DAO) were detected. Pancreas, small intestine tissue was harvested for pathologic examination, index of intestinal epithelial damage was measured and ultrastructural changes in small intestinal mucosa was observed. Results The expression of serum amylase, endotoxin,D-lactate, DAO in sham operation group was ( 152 ± 32 ) U/L, (6.95 ± 2.10) pg/L, ( 3.96 ± 1.08 ) μg/mland ( 14.26 ± 2.67 ) μg/ml, while the corresponding values were ( 1549 ± 93 ) U/L, (40.48 ± 3.41 ) pg/L,( 12.34 ± 1.23 ) μg/ml and ( 80.28 ± 3.54) μg/ml in ANP group, and they were (655 ± 49 ) U/L, ( 19.55 ±2.50) pg/L, (6.75 ± 1.36 ) μg/mland ( 20.69 ± 7.53 ) μg/ml in treatment group. The values in ANP group were significantly higher than those in sham operation group. The values in treatment group were significantly lower than those in ANP group, but significantly higher than those in sham operation group ( P ＜ 0.05 or P ＜0. 01 ). The thickness and height of intestinal mucosa in ANP group were ( 389.44 ± 29.87 )μm and ( 16.52 ±3.73) μm, which were significantly lower than those in treatment group [(501.95 ± 45.38 )μm, (27.82 ±5.17)] μm, and in sham operation group [( 658.72 ± 57.49 ) μm, ( 35.49 ± 6.43 )μm, Index of intestional epitholial donage in ANP group was 3.72 ± 0.65 which is significently higher than those in theatment (2.12 ±0.37 ) and in sham operation group (0.85 ± 0.24). The intestinal mucosa histological and ultrastructural changes in Dahuangfuzi treatment group were better than those in ANP group. Conclusions Dahuangfuzidecoction can significantly decrease the damage of intestine barrier function in ANP rats.

Objective To report the anatomical study and clinical application of the vascularized composite fibula perforating osteoseptocutaneous flap. Methods Latex injection studies were performed on 24 adult cadaveric lower limbs. The presence, prevalence, and location of the peronel artery and its perforators in the distal leg were documented. 16 vascularized fibul osteoseptocutaneous flaps were performed for reconstruction of composite soft-tissue defect of limbs. The flap areas ranged from 6 cm×4 cm to 16 cm× 8 cm. Results A mean of 5.3 perforating vessels of the peroneal artery was noted in the specimen. 40.6% of them were muscularcutanous perforators, and the other were septacutanous perforators. The perforating vessels were all located within 3.1 cm from the postlateral fibula. The average diameter was 1.1 mm at the deep fascial level, and the interval of them was 4-8 cm. There were two constant perforators which located about 15-25 cm and 4-7 cm from the lateral maleolar respectively. All of the 16 clinical cases survived uneventfully and the time to union of the graft-host junction site were 3 to 5 months. The appearance of flaps and the function of the limbs were satisfied during 10 to 36 months following up. Conclusion It is the anatomic basis that there are pedorating vessels of the peroneal constantly. The vnscularized compound fibula perforating osteoseptocutaneous flap has the advantage of flexible design. It is the safe and suitable choice in the cases when compound bone and soft tissue defects of the limbs are required for reconstruction.