OBJECTIVES: To explore the therapeutic effect of LuoFuShan Rheumatism Plaster (LFS) on neuropathic pain (NP) and its molecular mechanism. METHODS: Mouse models of sciatic nerve chronic constriction injury (CCI) were treated with low, medium, and high doses (2.2, 4.4, and 8.8 cm², respectively) of LFS by topical application for 14 consecutive days. The therapeutic effects were assessed by evaluating the mechanical withdrawal threshold (MWT), paw withdrawal latency (PWL), plasma IL-6 and TNF-α levels, and histopathology of the sciatic nerve. Network pharmacology and molecular docking were used to identify the key targets and signaling pathways. The key targets were verified by RT-qPCR and immunohistochemistry. The biosafety of LFS was evaluated by measuring the organ indices and damage indicators of the heart, liver, and kidneys. RESULTS: Compared with the CCI group, LFS dose-dependently increased MWT and PWL, reduced plasma IL-6 and TNF-α levels, and alleviated sciatic nerve inflammation in the mouse models. Network pharmacology identified 378 bioactive compounds targeting 279 NP-associated genes enriched in TLR and TNF signaling. Molecular docking showed that quercetin and ursolic acid in LFS could stably bind to TLR4 and TNF‑α. In the mouse models of sciatic nerve CCI, LFS significantly downregulated the mRNA expression levels of Tlr4 and Tnf-α in the spinal cord in a dose-dependent manner and lowered the protein expressions of TLR4 and TNF-α in the sciatic nerve. LFS treatment did not cause significant changes in the organ indices or damage indicators of the heart, liver and kidneys as compared with those in the CCI model group and sham-operated group. CONCLUSIONS LFS alleviates NP in mice by suppression of TLR4/TNF-α-mediated neuroinflammation with a good safety profile.
Animals ; Toll-Like Receptor 4/metabolism* ; Neuralgia/metabolism* ; Mice ; Signal Transduction/drug effects* ; Tumor Necrosis Factor-alpha/metabolism* ; Drugs, Chinese Herbal/pharmacology* ; Sciatic Nerve/injuries* ; Male ; Molecular Docking Simulation ; Disease Models, Animal ; Interleukin-6

Objective:To investigate the activation level of neutrophil extracellular trap (NET) in the bile of patients with choledocholithiasis and its clinical significance.Methods:From May 2021 to October 2022, 130 patients underwent endoscopic retrograde cholangiopancreatography (ERCP) at the Department of Gastroenterology, the First Affiliated Hospital of Anhui Medical University were enrolled. The patients were divided into choledocholithiasis group (90 cases) and non-choledocholithiasis group (40 cases), and the choledocholithiasis group was further divided into large stone group (maximum diameter >1 cm, 36 cases) and small stone group (maximum diameter≤1 cm, 54 cases). The bile samples were collected from 130 patients during operation and 16 choledocholithiasis patients with nasobiliary drainage at 24 h after operation.The levels of myeloperoxidase(MPO), neutrophilelastase(NE), and citrullinated histone H3(CitH3) in bile were detected by enzyme-linked immunosorbent assay.The levels of MPO, NE, and CitH3 were compared between choledocholithiasis group and non-choledocholithiasis group, between large stone group and small stone group, as well as between choledocholithiasis patients before ERCP and after ERCP. Mann-Whitney U test and Wilcoxon signed rank test were used for statistical analysis. Results:The levels of MPO, NE and CitH3 in the bile of choledocholithiasis group were 32.6 U/L(28.5 U/L), 30.6 ng/L(35.2 ng/L) and 0.37 μg/L(0.73 μg/L), respectively, which were all higher than those of non-choledocholithiasis group (19.9 U/L(36.4 U/L), 18.2 ng/L(27.4 ng/L), and 0.10 μg/L(0.25 μg/L)), and the differences were statistically significant ( Z=2.91, 3.20 and 3.34; P=0.004, 0.001 and 0.001). The levels of MPO, NE and CitH3 of large stone group were 47.0 U/L(49.4 U/L), 48.4 ng/L(39.5 ng/L) and 0.83 μg/L(1.08 μg/L), respectively, which were all higher than those of small stone group (29.3 U/L(17.5 U/L), 24.0 ng/L(25.8 ng/L), and 0.27 μg/L(0.45 μg/L)), and the differences were statistically significant ( Z=2.01, 3.58 and 3.63; P=0.044, <0.001 and <0.001). The levels of MPO, NE and CitH3 in the bile of choledocholithiasis patients after ERCP significantly decreased compare with those before ERCP (19.4 U/L(19.8 U/L) vs. 33.6 U/L(36.7 U/L), 12.7 ng/L(15.1 ng/L) vs. 22.7 ng/L(25.9 ng/L), 0.05 μg/L(0.12 μg/L) vs. 0.14 μg/L(0.27 μg/L)), and the differences were statistically significant ( Z=3.52, 3.30 and 3.18; all P<0.001). Conclusion:The activation level of NET in the bile of patients with choledocholithiasis increase, while the activation level of NET decrease after ERCP, which indicate that NET may be involved in the formation of choledocholithiasis.

Objective:To study the value and advantage of color Doppler and cervical multi-slice spiral CT (MSCT) in the diagnosis of cervical lymphadenopathy.Methods:A total of 130 patients with cervical lymphadenopathy diagnosed and treated in the Chenzhou First People′s Hospital from January 2019 to December 2020 were selected and received color Doppler ultrasound examination and MSCT examination. The results of pathological examination were used as the gold standard to compare the efficacy of the two methods in the differential diagnosis of benign and malignant cervical lymphadenopathy.Results:Ultrasound examination of malignant lymph nodes showed irregular boundaries, uneven internal hypoecho, and abundant blood flow signals in lymph nodes; ultrasound examination of benign lymph nodes showed uniform fine dot echo, uniform growth of endothelial medulla, clear and smooth boundary, no blood flow signal or scattered dot blood flow signal. The MSCT images of malignant lymph node showed irregular shape, blurred edge, obvious and uneven enhancement and higher rate of calcification. The aspect ratio of lymph nodes in benign lymph node was significantly higher than that in malignant lymph node (2.14 ± 0.48 vs. 1.92 ± 0.43), and the maximum blood flow velocity (V max), resistance index (RI) and blood flow (BF) in systolic period were significantly lower than those in lymph node [(21.38 ± 3.61) cm/s vs. (23.17 ± 2.55) cm/s, 0.62 ± 0.14 vs. 0.71 ± 0.17, (48.82 ± 13.51) ml/(min·100 g) vs. (65.61 ± 14.64) ml/(min·100 g)], there were statistical differences ( P<0.05). The most common blood flow types was lymphatic hilum type in benign lymph node, the proportion was 51.79% (29/56), while the most common type in malignant lymph node was marginal type and central type, the proportion was 44.59% (33/74) and 25.68% (19/74). The sensitivity, specificity, accuracy and Kappa value of ultrasound combined with MSCT in diagnosis were 92.86%, 95.95%, 94.62% and 0.890. Conclusions:Both color Doppler ultrasonography and MSCT can differentiate the benign and malignant of cervical lymph node lesions with better parameters such as lymph node imaging characteristics and blood flow distribution pattern, but the combined diagnosis has higher sensitivity, specificity and accuracy.

Objective: To investigate the molecular and signal pathway mechanism of Interleukin-27 affecting the anti-tumor effect of NK92 cells. Methods: NK92 cells were cultured with different concentrations of IL-27 (10, 20, 30 and 60 ng/ml) for 24 hours. The cytotoxicity of NK92 cells to target cells was detected by LDH assay. The expressions of NKG2D, NKp30 and NKp46 on the surface of NK92 cells and the secretion of perforin and granzyme B were detected by Flow cytometry. The expression and phosphorylation level of STATs protein was detected by WB. The DU145 cell transplanted tumor model of prostatic carcinoma in NOD-PrkdcscidIl2rgem1/Smoc mice was established and treated with the combination of NK92 cells and IL-27 to evaluate their anti-tumor efficacy. Results: IL-27 at concentrations of 10, 20 and 30 ng/ml could significantly increase the cytotoxicity of NK92 cells to target cells, and 30 ng/ml exerted the best effect (P<0.05 or P<0.01). 30 ng/ml IL-27 could significantly promote the expressions of NKG2D, NKp30 and NKp46 on surface of NK92 cells, as well as elevate the secretion of perforin (all P<0.05), but didn’t affect the secretion of granzyme B (P>0.05); moreover, it also up-regulated the phosphorylation of STAT1, STAT3 and STAT5 protein (all P<0.01). The combined treatment of IL-27 and NK92 cells obviously extended the survival time of tumor-bearing mice (P<0.05). Conclusions: IL-27 can promote the cytotoxicity of NK92 cells against solid tumor cells and blood tumor cells by promoting expressions of NKG2D, NKp30 and NKp46 on the surface of NK92 cells and the secretion of perforin, which might be related with the phosphorylation of STAT1, STAT3 and STAT5 in JAKSTAT pathway.

Objective To analyze the result of proficiency testing(PT)of detection activities for Laboratory animal pathogenic bacteria in 2011 and 2013-2017. To further improve the detection capacity of laboratory animal testing agency,and promote PT to be carried out in future. Methods During the six years(2011 and 2013 -2017), the National Institutes for Food and Drug Control conducted a total of six(seven projects)PT activities of laboratory animal pathogen bacteria. We analyzed the overall trend and the exposed problems by summarizing the result data of the PT in 6 years. Results A total of 45 laboratories in the country including 20 provinces and cities participated in the PT. The PT projects included Mycoplasma pulmonis, Clostridium piliformis, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella spp.,Klebsiella pneumoniae and Bordetella bronchiseptica. The satisfaction rates were 75%,87.5%,80.0%, 78.6%,93.3,96.2% and 88.0%, respectively. The main reasons of unsatisfactory results were for lack of incubation time,select errors of suspicious bacteria, biochemical identification errors, report writing errors and not timely feedback results. Conclusions The level of domestic laboratory animal pathogenic bacteria detection is gradually increased to achieve the desired goal through continuous proficiency testing activities.

Objectives To understand the effects of different doses of vitamin D supplementation on serum calcium,phosphorus,alkaline phosphatase and 25-hydroxyvitamin D levels in very low birth weight infants (VLBWI) and to provide guidance for early prevention of metabolic bone disease in VLBWI.Methods A total of 90 VLBWI hospitalized in the Department of Neonatology,Huzhou Maternal and Child Healthcare Hospital between January 2014 and January 2016 were enrolled and randomly divided into two groups:highdose group and low dose group.High-dose group was given vitamin D 900 U/d orally and low-dose group was given 400 U/d since the eighth day after birth.Serum calcium,phosphorus and alkaline phosphatase levels were detected at 1,7,21 and 42 days of age and serum 25-hydroxyvitamin D levels were detected at 7,21and 42 days of age.Two-sample t-test,Chi-square test,one-way analysis of variance and LSD or Dunnett's T3 test were used for statistical analysis.Results No significant differences in serum calcium,phosphorus and alkaline phosphatase levels were found between the two groups at 1 and 7 days of age,nor were found in serum 25-hydroxyvitamin D level at 7 days of age (all P＞0.05).At 21 days of age,high dose group had higher serum calcium,phosphorus and 25-hydroxyvitamin D levels than low-dose group [(2.38 ± 0.09) vs (2.04 ± 0.15) mmol/L,t=2.421;(1.80±0.50) vs (1.71 ±0.60) mmol/L,t-0.637;(45.58± 18.43) vs (42.53± 16.33) nmol/L,t=0.421],but lower alkaline phosphatase level [(505.12± 185.61) vs (588.32± 168.72) U/L,t=5.314] (all P＜0.05).The same trends were found at 42 days of age.In high-dose group,serum calcium level increased at 7,21 and 42 days of age as compared with that at 1 day of age [(2.43±0.13),(2.38±0.09),(2.39±0.08) vs (2.06±0.57) mmol/L];serum phosphorus level at 7 days of age was lower than that at 1,21 and 42 days of age [(1.31 ±0.26) vs (1.89±0.39),(1.80±0.50),(1.98±0.30) mmol/L];serum alkaline phosphatase level at 7,21 and 42 days of age was higher than that at 1 day of age [(475.18± 133.73),(505.12± 185.61),(538.43 ± 168.16) vs (296.15 ± 99.41) U/L] and a significant increase was observed at 42 days of age as compared with that at 7 days of age;serum 25 hydroxyvitamin D level at 21 days of age was higher than that at 7 days of age,and that at 42 days of age was higher than that at 7 and 21 days of age [(73.55±23.65) vs (30.63± 12.66) and (45.58 ± 18.43) nmol/L];the differences were all statistically significant (LSD or Dunnett's T3 test,all P＜0.05).Conclusions Vitamin D supplementation from the eighth day after birth can improve calcium and phosphorus metabolism in VLBWI and the dose of 900 U/d is more effective than 400 U/d.

Objective To establish a rapid, simple, sensitive, and specific multiplex real-time PCR method for quantitative detection of Campylobacter jejuni, Salmonella and Shigella in tree shrews.Methods Specific primers and probes were designed, according to the HipO gene of Campylobacter jejuni, inV gene of Salmonella and ipaH gene of Shigella.The primers were confirmed by single pathogen quantitative PCR, and the sensitivity and specificity of the multiplex PCR were analyzed.Finall, the samples of experimental tree shrews were detected by this multiplex PCR method.Results The PCR element of TaqMan-MGB real-time PCR assay was able to quantitatively amplify the Campylobacter jejuni, Salmonella or Shigella.Appropriate standard amplification curves of Campylobacter jejuni, Salmonella and Shigella in the multiplex quantitative PCR were obtained.The sensitivity of this method was 1×103 ng/μL.There was no false positive detection from other bacterial strains.Conclusions This multiplex quantitative real-time PCR method has good application and development prospects in the detection of microorganisms in tree shrews.

Objective To investigate the feasibility of inserting and detaining nasointestinal feeding tube in small bowl guided by bedside ultrasound(US)in critically ill elderly patients.Methods This was a retrospective study.Sixty four aged patients(≥ 60 years)in general ICU,the Second Affiliated Hospital of Jiaxing College,received the US-guided nasointestinal feeding tubes inserting and detaining.Feeding tubes passed through nasal and went into the stomach by manual blind method.Under US-guiding condition,the tube passed through the pyloric sphincter and further into the duodenum or jejunum.Finally the correct position of the tube head was assessed by bedside X-ray examination.Results The US-guided nasointestinal feeding tube-detaining technique was successfully operated in 57 patients(89.1％).The feeding tube heads were in the duodenum in thirty four cases(53.1 ％),and in proximal jejunum in twenty-three cases(35.9％).The untoward reaction included the bleeding of nasal cavity in 1 case,and hypotension in another case.Conclusions Bedside US-guided nasointestinal feeding tube placement is safe and feasible in aged critical patients.

Objective To acquire the prevalence and molecular identification data on Syphacia muris and provide reference for the revision of national standard. Methods 923 batches of 5199 SPF animals ( including one batch of 5 monkeys, 3 batches of 25 mini?pigs, 28 batches of 55 rabbits, 13 batches of 248 hamsters, 37 batches of 198 guinea pigs, 93 batches of 459 rats, 742 batches of 4179 mice, 5 batches of 25 chickens and one batch of 5 ducks) and 145 batches of 1389 clean animals ( including one batch of 3 rabbits, 4 batches of 31 hamsters, 16 batches of 157 guinea pigs, 32 batches of 268 rats and 92 batches of 930 mice ) came from 50 different manufactures in China. Direct microscopy real?time dynamic video recording techniques in combination with morphological identification method were applied to screen the Syphacia muris infestation. A multiple polymerase chain reaction ( multiple?PCR ) testing of the isolate based on amplification of the conserved portions of the Syphacia muris internal transcribed spacer (ITS), 28S ribosomal RNA (28S rRNA), NADH dehydrogenase subunits 1 (nad1) and cytochrome c oxidase subunit 1 (cox1) genes, and the molecular sequencing of the multiple?PCR amplicons was used to confirm the Syphacia muris infection. Results Syphacia muris eggs, larvae and adults were detected by using direct microscopy real?time dynamic video recording technique. Syphacia muris were detected based on the morphology and size of ovum, larvae, and female and male adult worms. Multiple?PCR and sequencing were performed to identify ITS, 28S rRNA, nad1 and cox1 genes of DNA extracted from the single egg, larva and adult parasite Syphacia muris. This approach allowed the specific identification with no amplicon being amplified from heterogeneous DNA samples, and sequencing confirmed the identity of the amplified sequences. Molecular characterization by multiple?PCR amplification and sequencing of the ITS, 28S rRNA, nad1 and cox1 genes demonstrated the presence of Syphacia muris. Multiple?PCR followed by sequencing confirmed 285 of 5199 SPF and 135 of 1389 clean animal samples classified as positive by using direct microscopy real?time dynamic video recording technique in the study as containing Syphacia muris?specific DNA. Comparison of the partial sequences of the ITS, 28S rRNA, nad1 and cox1 genes revealed 100% similarity amongst Syphacia muris from different animals. The prevalence of Syphacia muris infection in SPF and clean animals were 5?5% (285/5199) and 9?7% (135/1389), respectively. Conclusions Direct microscopy real?time dynamic video recording technique, multiple?PCR and sequencing can be used to rapidly detect and accurately identify Syphacia muris. The zoonotic nature of Syphacia muris can be regard as a public health alter, hence the good quality control of animal has an important role in protecting human health and safeguarding people safety. This is the first molecular identification and infection investigation of Syphacia muris in SPF and clean animals in China.

Objective Through the proficiency validation of testing of mammalian orthoreovirus 3 (Reo3)antibody in laboratory mice, to investigate the capacity of experimental animal quality control laboratories, so as to improve the de-tection capacity.Methods According to the program approved by CNAS, serum samples after calibration were tested for stability and homogeneity, and numbered randomly.The random samples were issued to the participant laboratories with the Standard Operation Procedure( SOP) .The participants must submit the test reports and original records in time.The feed-back results were judged by the concordance rate with the anticipated results.Results 27 laboratories from 17 provinces were enrolled in this evaluation project, all of them submitted detection results on time.Results All the 27 laboratories were marked as pass or excellent, with a pass rate of 100%.ELISA was used in 26 laboratories, and immunofluorescence assay was used in one laboratory.Conclusions The ability for detection of Reo3 antibody in laboratory mice in animal test laboratories is high.The implementation of proficiency testing can reflect the inspection level of quality control laborato-ries.