[Objective] To compare next generation sequencing (NGS) library construction technology between probe hybridization capture and amplicon methods, and analyze the influencing factors of HLA genotyping resolution level and its prospects in clinical applications. [Methods] A total of 207 clinical samples with known typing results and samples from the proficiency testing plan were selected. The conformity rate of HLA genotyping results, allele coverage and typing data analysis indicators were confirmed, and the effects of two library construction methods on the level of HLA genotyping discrimination were compared. [Results] The concordance rate of 207 samples with the feedback results of PT or prior well-characterized HLA genotypes was 100%. Among them, 91 samples were captured using hybridization probe capture method. Compared with the original amplicon method, the hybridization probe capture method can distinguish the alleles of DRB1 and DPB1 that cannot be determined in 13 samples. The allelic imbalance of DRB1, DPA1, and DQB1 loci in 6 samples was resolved. Three samples were found to have missed detection of alleles at the DQA1 and DQB1 loci. [Conclusion] The performance indicators of hybridization probe capture and amplicon performance confirmation meet the requirements of clinical detection of HLA genotyping, which provides an experimental method and basis for clinical application.

Objective: To investigate the role of butyric acid-producing bacteria in long bone homeostasis in mice with periodontitis under a high-fat/high-sugar diet and to provide new insights for the prevention and treatment of periodontitis and related bone metabolic diseases. Methods: This study has been approved by the Animal Welfare and Ethics Committee of the Experimental Animal Center. Initially, 14 mice were randomly divided into the CON group (the control group) and the LIG group (the periodontitis group). Mice in the LIG group had experimental periodontitis induced by ligating the second maxillary molars bilaterally and were fed a high-fat and high-sugar diet. After 8 weeks, samples were collected. Micro-computed tomography (Micro-CT) was used to analyze alveolar bone resorption and various parameters of the proximal tibia trabecular bone, including bone mineral density (BMD), bone volume per tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). After decalcification, hematoxylin and eosin (HE) staining was performed on maxillary bone sections to assess periodontal tissue inflammation and connective tissue destruction. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect related genes in the distal femur and proximal tibia bone tissues, including osteocalcin (OCN), osteogenic transcription factor (Osterix), osteoprotegerin (OPG), tartrate resistant acid phosphatase (TRAP), osteoclast-associated receptor (OSCAR), receptor activator of nuclear factor kappa-B (RANK), and receptor activator of nuclear factor kappa-B ligand (RANK-L). Subsequently, the other 28 mice were randomly divided into the CON group (the control group), LIG group (the periodontitis group), CON + butyric acid-producing bacteria (BP) group, and LIG + BP group. The breeding, sampling, and sample detection methods remained the same. Finally, the other 28 mice were randomly divided into the CON group (the control group), LIG group (the periodontitis group), CON + sodium butyrate (SB) group, and LIG + SB group. The breeding, sampling, and sample detection methods remained the same. Results: ①Periodontitis modeling was successful. Compared with the CON group, the LIG group exhibited significant alveolar bone resorption of the maxillary second molar, aggravated periodontal tissue inflammation, and connective tissue destruction. ②Periodontitis exacerbated long bone resorption in mice fed a high-fat high-sugar diet. Compared with the CON group, the LIG group had significantly lower BMD, BV/TV, Tb.N, and Tb.Th (P<0.05), and significantly higher Tb.Sp (P<0.05). HE staining of the proximal tibia showed that the trabeculae in the LIG group were sparse and disordered, with some areas showing fractures or dissolution. The expression of osteoblast markers (OCN, Osterix, OPG) was significantly lower in the LIG group (P<0.05), while the expression of the osteoclast marker TRAP showed an increasing trend (P>0.05). The ratio of RANK-L/OPG was significantly higher in the LIG group compared with the CON group (P<0.05). ③ Supplementation with butyric acid-producing bacteria alleviates periodontitis-induced disruption of long bone homeostasis in mice fed a high-fat/high-sugar diet. Compared with the LIG group, BMD and Tb.Th were significantly higher in the LIG + BP group. HE staining of the proximal tibia showed that bone resorption was mitigated in the LIG + BP group compared with the LIG group. The expression of OCN and Osterix was significantly higher in the LIG + BP group, while the expression of osteoclast-specific genes (OSCAR, RANK, RANK-L) was significantly lower (P<0.05). ④ Supplementation with butyrate alleviates periodontitis-induced disruption of long bone homeostasis in mice fed a high-fat/high-sugar diet. Compared with the LIG group, BV/TV and Tb.N were significantly higher in the LIG + SB group, and Tb.Sp was significantly lower (P<0.05). HE staining of the proximal tibia showed that bone resorption was mitigated in the LIG + SB group compared with the LIG group. The expression of Osterix, OPG, OSCAR, TRAP, and RANK was significantly lower in the LIG + SB group compared with the LIG group (P<0.05). Conclusion Periodontitis disrupts the long bone homeostasis of mice fed a high-fat high-sugar diet, aggravating long bone resorption. Supplementation with butyric acid-producing bacteria or butyrate can effectively alleviate the disruption of long bone homeostasis caused by periodontitis.

Objective To study the correlation between traditional Chinese medicine(TCM)constitution and pathogenic factors in patients with ankylosing spondylitis(AS).Methods One hundred patients of AS and their family members who had medical consultation in the Fifth Hospital of Xi'an(i.e.,Shaanxi Hospital of Integrated Traditional Chinese and Western Medicine)in August 2019 and September 2020 were selected as the study subjects.The guidelines of Classification and Determination of Traditional Chinese Medicine Constitution issued by the China Association of Chinese Medicine were adopted to determine the traditional Chinese medicine(TCM)constitution types of the study subjects.The sociodemographic information,living habits,clinical symptoms,and TCM constitution types of the AS patients and their family members were collected by means of questionnaires and clinical investigations,and then the pathogenic factors of the patients with AS were investigated.The binomial Logistic regression model was used to analyze the correlation between TCM constitution types and pathogenic factors in patients with AS.Results(1)Among the 100 AS patients,the majority of them had the biased constitutions,and the biased constitutions with the occurrence frequency in descending order were yang deficiency constitution,qi deficiency constitution,and damp-heat constitution,which accounted for 33.00%,14.00%,and 18.00%,respectively.(2)The prevalence rates of AS in the first-,second-,and third-degree relatives of AS patients were 56.25%,40.00%and 25.00%,respectively.For the positive rates of human leukocyte antigen B27(HLA-B27)in AS patients and their family members,HLA-B27 in AS patients was all positive,while the positive rates of HLA-B27 in the first-,second-,and third-degree relatives of AS patients were 44.31%,30.67%and 15.63%,respectively.(3)The results of regression analysis showed that the disease duration of AS patients was significantly correlated with qi deficiency constitution,the grading of sacroiliac arthritis was correlated with qi stagnation constitution,and age was correlated with blood stasis constitution(P＜0.05 or P＜0.01).The results indicated that disease duration and age were the important factors affecting the constitution types of AS patients,and disease duration was closely related to qi deficiency while age was closely related to blood stasis.Conclusion AS is a highly hereditary autoimmune disease,and its onset is associated with HLA-B27.Yang deficiency is the basic constitution type of AS,and damp-heat constitution is the main constitution type in the progression of AS(especially in the active stage of the disease).The prolongation of the disease will exacerbate the illness condition of AS and then the manifestations of qi deficiency will be more obvious.

Objective To investigate the effect of artesunate on airway remodeling induced by ovalbumin in asthmatic young rats and its mechanism.Methods SD rats were randomly divided into normal group,model group(ovalbumin activation),positive control group(on the basis of model group,injected with 0.2 mg·kg-1 dexamethasone),low-dose group(on the basis of model group,injected with 10 mg·kg-1 artesunate),high-dose group(on the basis of model group,injected with 20 mg·kg-1 artesunate)and agonist group(on the basis of model group,injected with 20 mg·kg-1 artesunate and 100 mg·kg-1 nigerin sodium salt),with 10 rats in each group.Airway remodeling related indexes were detected after treatment.Western blot and real-time fluorescence quantitative polymerase chain reaction were used to detect Nod-like receptor protein 3(NLRP3)pathway-related protein and mRNA expression;enzyme-linked immunosorbent assay were used to detect inflammatory factors expression level,and the classification and count of inflammatory cells in alveolar lavage fluid were detected by Wright's-Giemsa Staining.Results The mRNA expression levels of NLRP3 in normal group,model group,positive control group,high-dose group and agonist group were 1.00±0.10,2.36±0.26,1.08±0.11,1.33±0.12,2.14±0.26,respectively;cysteine aspartate proteinase 1(caspase-1)mRNA expression levels were 1.00±0.13,1.92±0.22,1.22±0.12,1.44±0.10,1.82±0.14,respectively;interleukin-17(IL-17)inflammatory factor expression quantity were(22.41±1.15),(56.74±6.54),(28.72±2.75),(32.69±3.73),(58.40±4.46)pg·mL-1;neutrophil count were(4.04±0.32)×106,(12.70±1.05)×106,(4.53±0.30)×106,(4.67±0.18)× 106,(10.19±0.58)× 106 cell·mL-1.Compared the model group with the normal group,the positive group,the high dose group compared with the model group,the agonist group compared with the high dose group,the differences of the above indicators were statistically significant(all P＜0.05).Conclusion Artesunate can significantly improve airway remodeling in ovalbumin induced asthmatic pups,which may be achieved by inhibiting the NLRP3/caspase-1/interleukin 1 β(IL-1β)inflammatory pathway.

Objective To investigate the effects of total flavones from Oxytropis falcata Bunge on hepatic fibrosis(HF)induced by carbon tetrachloride and liver transforming growth factor(TGF-β)/Smad signaling pathway.Methods Forty-eight male rats were randomly divided into normal group(intraperitoneal injection of peanut oil,intragastric administration of 0.9％NaCl),model group(intraperitoneal injection of 40％CC14 peanut oil solution induced HF model,intragastric administration of 0.9％NaCl),positive control group(modeling,intragastric administration of 0.2 mg·kg-1 of colchicine),experimental-L,-M,-H groups(modeling,intragastric administration of 100,200 and 400 mg·kg-1 of total flavonoid extract of Oxytropis falcata Bunge),8 individuals in each group,for 4 consecutive weeks.The histopathological changes were observed by hematoxylin-eosin and Masson staining.Serum liver function and liver fibrosis were measured;erum inflammatory factors were detected;fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to determine gene expression in liver.Results The pathological injury of liver tissue in the model group was serious,and a large number of inflammatory factors and collagen fibers were accumulated,while the rest of the treatment groups had different degrees of remission.In normal group,model group,positive control group,experimental-L,-M,-H groups,glutamic-pyruvic transaminase levels were(49.28±12.44),(5 885.42±948.37),(4 454.60±489.27),(4 650.47±843.53),(3 761.75±887.30)and(3 544.90±1 066.75)μg·L-1;glutamic-oxaloacetic transaminase levels were(186.90±46.89),(5 936.23±793.81),(3 971.37±780.28),(4 360.30±863.35),(3 943.10±439.47)and(3 971.38±631.08)μg·L-1;hyaluronic acid levels were(45.08±17.16),(104.32±36.06),(66.83±20.09),(70.30±21.07),(60.00±9.68)and(59.02±10.73)μg·L-1;laminin levels were(23.13±3.89),(60.85±13.66),(35.67±9.92),(39.98±9.39),(36.55±12.21)and(34.68±24.83)μg·L-1;type Ⅲ procollagen level were(24.98±5.34),(82.58±30.14),(40.70±16.14),(51.08±23.21),(43.60±12.48)and(44.20±11.66)p±g·L-1;interleukin(IL)-1β levels were(37.63±1.24),(46.10±3.23),(39.22±2.36),(41.33±0.93),(40.25±2.04)and(39.18±2.23)pg·mL-1;tumor necrosis factor-α levels were(314.58±20.56),(383.71±16.97),(349.00±7.93),(348.88±25.11),(325.75±27.84)and(335.07±21.33)pg·mL-1;TGF-β1 mRNA expression of relative quantity respectively were 1.00±0.00,60.99±15.70,9.61±1.59,7.37±1.09,6.41±0.64,6.87±1.09;Smad7 mRNA relative expression were 1.00±0.00,0.34±0.05,0.21±0.03,0.35±0.02,0.38±0.02,0.42±0.03.The above indexes in the model group were compared with the normal group,and the above indexes in the experimental-M,-H groups were compared with the model group,and the differences were statistically significant(P＜0.05,P＜0.01,P＜0.001).Conclusion Total flavonoids of Oxytropis falcata Bunge have protective effects on CC14-induced liver fibrosis in rats,and the mechanism may be related to the regulation of TGF-β/Smad pathway.

Objective To explore the effect of barretinib on mice with acute lung injury(ALI)by regulating the Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3)pathway.Methods The male C57BL/6 mice were randomly divided into control group,model group and experimental-L,-M-H groups.Except for the control group,the other four groups were constructed ALI models by intraperitoneal injection of lipopolysaccharide.The control and model groups were given equal volume 0.9％NaCl by gavage.The experimental-H,-M,-L groups were administered with 1.00,0.50 and 0.25 mg·mL-1 barretinib solution 200 μL by gavage.Five groups were treated for 48 hours with once every 12 hours.The levels of inflammatory cytokine in bronchoalveolar lavage fluid were measured by enzyme linked immunosorbent assay.The expression levels of Occludin,JAK2 and STAT3 in mouse lung tissue were detected by Western blot.Results The levels of tumor necrosis factor-α in bronchoalveolar lavage fluid for experimental-M,-H groups,model group and control group were(228.48±25.41),(198.53±23.11),(317.32±32.85)and(48.93±2.59)ng·L-1;the levels of interleukin-6 were(118.81±14.85),(98.58±13.82),(172.23±25.94)and(49.47±3.06)ng·L-1;the relative expression levels of Occludin were 0.48±0.13,0.49±0.11,0.28±0.09 and 0.69±0.21;the phospho-JAK2/JAK2 ratios were 0.51±0.13,0.32±0.09,0.75±0.21 and 0.16±0.05;the phospho-STAT3/STAT3 ratios were 0.43±0.11,0.27±0.08,0.78±0.21 and 0.17±0.05,respectively.The differences of above indicators were statistically significant between the experimental-M,-H groups,control group and model group(all P＜0.05).Conclusion Barretinib can reduce the lung inflammatory response and improves the level of tight junction protein in lung tissue to protect ALI mice by inhibiting the JAK2/STAT3 signaling pathway.

Objective To investigate the effects of oxymatrine on proliferation and apoptosis of diffuse large B-cell lymphoma and its molecular mechanism.Methods Human diffuse large B lymphoma cells OCI-LY19 were randomly divided into control group(normal culture),experimental-L group(25.00 μmol·L-1 oxymatrine),experimental-M group(50.00 μmol·L-1 oxymatrine),experimental-H group(100.00 μmol·L-1 oxymatrine),Oxymatrine+si-NC group(transfected with si-NC+100.00 μmol·L-1oxymatrine),Oxymatrine+si-PD-L1 group[transfected with si-programmed death receptor ligand 1(PD-L1)+100.00 μmol·L-1 oxymatrine],Oxymatrine+Vector group(transfected with si-NC+100.00 μmol·L-1 oxymatrine)and oxymatrine+PD-L1 group(transfected with PD-L1+100.00 μmol·L-1 oxymatrine).5-acetylidene-2'-deoxyuridine(EdU)assay was used to detect cell proliferation;Western blot assay was used to detect protein expression;flow cytometry assay was used to detect cell apoptosis.Results The EdU cell proliferation rates in control group,experimental-H group,oxymatrine+si-NC group,oxymatrine+si-PD-L1 group,oxymatrine+Vector group and oxymatrine+PD-L1 group were(33.88±2.79)％,(15.23±1.32)％,(15.84±1.58)％,(10.13±0.90)％,(16.14±1.20)％and(20.84±1.88)％,respectively;the expressions of Cyclin-dependent kinase 4(CDK4)protein were 0.92±0.11,0.37±0.04,0.35±0.04,0.24±0.03,0.38±0.06 and 0.71±0.06,respectively;the apoptosis rates were(3.20±0.07)％,(25.35±2.01)％,(24.81±1.91)％,(30.27±1.65)％,(24.39±2.73)％and(17.97±1.27)％,respectively.The above indicators:Experimental-H group were compared with the control group respectively,and the differences were statistically significant(all P＜0.05);there were significant differences between oxymatrine+si-PD-L1 group and oxymatrine+si-NC group(all P＜0.05);there were significant differences between oxymatrine+PD-L1 group and oxymatrine+Vector group(all P＜0.05).Conclusion Oxymatrine can inhibit the proliferation of OCI-LY19 cells and induce apoptosis by down-regulating the expression of PD-L1.PD-L1 may be a potential target of oxymatrine in the treatment of diffuse large B lymphoma.

Objective To explore the efficacy of T-cell spot test of tuberculosis infection(T-SPOT.TB)in the differential diagnosis of spinal tuberculosis(STB),and optimize diagnostic efficacy through the optimal cut-off value of receiver operating characteristic(ROC)curve.Methods Clinical data of patients with spinal infection in a hospi-tal from January 2010 to May 2019 were collected,including preoperative T-SPOT.TB test results,white blood cell count,C-reactive protein,erythrocyte sedimentation rate,procalcitonin,and tuberculosis antibodies,etal.Clinical diagnosis was conducted based on diagnostic criteria.The sensitivity and specificity of T-SPOT.TB in preoperative diagnosis of STB and other spinal infection was analyzed,and the diagnostic efficacy of the optimized T-SPOT.TB indicators was evaluated.Results A total of 132 patients were included in this study,out of whom 78 patients(59.09％)were diagnosed with STB,and 54(40.91％)were diagnosed with non-tuberculosis(non-TB)spinal in-fection.The sensitivity and specificity of T-SPOT.TB in differential diagnosis of STB were 67.68％and 66.67％,respectively.Univariate logistic regression analysis showed that compared with non-TB spinal infection,the OR va-lue of T-SPOT.TB test in diagnosing STB was 4.188(95％CI:1.847-9.974,P＜0.001).The optimized T-SPOT.TB evaluation index through ROC curve to determine the optimal cut-off values of ESAT-6,CFP-10,and CFP-10+ESAT-6 for differential diagnosis of STB and non-TB spinal infection were 12.5,19.5,and 36,respec-tively,and area under curve(AUC)values were 0.765 6,0.741 5,and 0.778 6,respectively,all with good diag-nostic efficacy.CFP-10+ESAT-6 had the highest AUC.CFP-10+ESAT-6 specific spot count had higher efficacy in the diagnosis of STB,with a diagnostic accuracy of 75.56％,higher than 67.42％of pre-optimized T-SPOT.TB.Conclusion T-SPOT.TB test has high diagnostic efficacy in differentiating STB from non-TB spinal infection.Posi-tivity in T-SPOT.TB test,especially with spot count of CFP-10+ESAT-6 over 36,indicates a higher likelihood of STB.

Analyzing polysorbate 20(PS20)composition and the impact of each component on stability and safety is crucial due to formulation variations and individual tolerance.The similar structures and polarities of PS20 components make accurate separation,identification,and quantification challenging.In this work,a high-resolution quantitative method was developed using single-dimensional high-performance liquid chromatography(HPLC)with charged aerosol detection(CAD)to separate 18 key components with multiple esters.The separated components were characterized by ultra-high-performance liquid chro-matography-quadrupole time-of-flight mass spectrometry(UHPLC-Q-TOF-MS)with an identical gradient as the HPLC-CAD analysis.The polysorbate compound database and library were expanded over 7-time compared to the commercial database.The method investigated differences in PS20 samples from various origins and grades for different dosage forms to evaluate the composition-process relationship.UHPLC-Q-TOF-MS identified 1329 to 1511 compounds in 4 batches of PS20 from different sources.The method observed the impact of 4 degradation conditions on peak components,identifying stable components and their tendencies to change.HPLC-CAD and UHPLC-Q-TOF-MS results provided insights into fingerprint differences,distinguishing quasi products.

Objective To evaluate the intraoperative identification of ectopic parathyroid tissue in the central neck region using autofluorescence point-spectral analysis(AFPSA)combined with immune colloidal gold technique(ICGT),for guiding total parathyroidectomy(TPTX)or clean parathyroidectomy(CPTX)in the management of secondary hyperparathyroidism(SHPT).Methods Retrospectively collected and compared the clinical data of 64 patients with SHPT from October 2019 to June 2023.In the observation group,TPTX was performed as the initial procedure in 36 cases,followed by sampling of suspicious targets using AFPSA in the central neck area and subsequent detection through ICGT.CPTX was then conducted if a positive result was obtained.On the other hand,the control group consisted of 28 cases where only TPTX was performed without any additional tests during surgery.The surgical data,parathyroid hormone(PTH)levels,blood calcium levels,blood phosphorus levels,alkaline phosphatase(ALP)levels,regression of clinical symptoms,changes in parathyroid function and occurrence of hypocalcemia were compared between these two groups.Results In the observation group,there were 9 cases of AFPSA-ICGT positivity,including 2 left-sided cases,4 right-sided cases,and 3 thymic cases;among these posi-tive cases,there were a total of 10 locations with mildly hyperplastic or nonhyperplastic microscopic parathyroid tissue.The difference in the number of total parathyroid glands removed(including ectopic)between the two groups was statistically significant(P<0.05).At both 3 and 6 months postoperatively,ALP levels in the observation group were significantly lower than those in the control group(P<0.01 and P<0.001 respectively);at 6 months postoperatively,differences in PTH and blood phosphorus levels between the two groups were also statistically significant(P<0.05 and P<0.001 respectively).Joint bone pain and skin itching recurred in some patients within the control group at six months after surgery(P<0.05),whereas recurrence of SHPT was less frequent within the observation group compared to controls(P<0.05);however,no statistically significant differences were observed regarding postoperative hypoparathyroidism or hyperparathyroidism as well as hypocalcemia between either groups.Conclusion The AFPSA-ICGT intraoperative test can be utilized to guide surgery for SHPT,enabling accurate and efficient identification as well as safe targeting of parathyroid tissues that may not exhibit obvious hyperplasia in the central cervical region.