Objective : To construct a humanized mouse model of the interleukin-9 receptor(IL-9R) coding DNA sequence(CDS) gene and to verify the genotype and IL-9R expression in mice. Methods : The CRISPR/Cas9 genome editing technology was used to replace the exon 2-7 fragment of the il-9r gene in mouse embryonic stem cells with the corresponding human IL-9R sequence. After verifying the completion of the gene fragment replacement, tetraploid embryos were constructed and microinjected back into the oviducts of surrogate mice. Through surrogacy by female mice, homozygous humanized mice were obtained. DNA was extracted from the homozygous humanized mice IL-9R CDS gene, and their genotypes were identified by agarose gel electrophoresis after PCR amplification. Western blot was used to detect the expression of IL-9R in the spleen and thymus of homozygous humanized mice with either wild-type(WT) or IL-9R gene humanization. Results : Gel electrophoresis after PCR amplification showed that mice with only a 1 805 bp band amplified using WT primers were wild-type, while mice with 2 553 bp and 2 340 bp bands amplified using 5KI and 3KI primers, respectively, were homozygous humanized mice with IL-9R CDS gene. Western blot results indicated that the tissues of homozygous humanized mice model with IL-9R CDS gene expressed IL-9R significantly. Conclusion The humanized mouse model with IL-9R CDS gene has been successfully constructed and characterized.

Objective: To construct microRNA(miR)-22 gene knockout(miR-22-/-) mice using CRISPR/Cas 9 technology, to breed miR-22-/- mice and to identify their genotypes. Methods : In this experiment, CRISPR/Cas 9 technology was used to construct miR-22-/- genetically engineered mice. After gene identification, the F0 generation miR-22-/- mice were mated with wild-type mice in the same litter to obtain F1 generation miR-22-/- mice. The miR-22 knockout efficiency was analyzed at the RNA level by real-time fluorescence quantitative polymerase chain reaction(qPCR). Western blot was used to detect the interaction between miR-22 and target genes. Results : miR-22-/- mice were successfully constructed using CRISPR/Cas 9 technology, gene identification was performed on the bred mice, and three stable genotypes of miR-22+/+,miR-22+/-, and miR-22-/- were identified. The real-time fluorescence quantitative PCR detection results confirmed that miR-22-/- mice showed almost no expression of miR-22 in the heart, liver, lung, kidney, spleen, and thymus tissues compared to wild-type mice in the same litter. Western blot analysis showed that the relative expression level of NLRP3 protein in miR-22-/- mouse tissues was lower than that in wild-type mice. Conclusion A miR-22-/- mouse model is successfully constructed, and stable genetic homozygous miR-22-/- mice is obtained. This indicates that miR-22 has an inhibitory effect on the downstream target gene NLRP3.

Objective: To explore the breeding and genotyping of CCR2 knockout mice, and to verify the applicability of the polymerase chain reaction(PCR) method for genotype detection of CCR2 knockout mice. Methods: The introduced CCR2 pure male mice and wild-type female mice were mated and bred to produce the offspring generation, the obtained F1 generation heterozygous mice were continued to be mated. DNA was extracted by clipping the tail tissues of the mice at the age of 2 weeks, the target gene fragment was amplified by PCR, and the genotypic results were determined by agarose gel electrophoresis. The proportion of purebred progeny carrying the CCR2 knockout gene was increased by genetic crosses, the effect of CCR2 knockout in the progeny mice was verified by using Western blot against major immune cells and key organs, and flow cytometry was used to detect whether the knockout of the CCR2 gene had any effect on the function of the immune system by targeting the major immune cells. Results: CCR2 knockout mice were successfully bred and characterized, and three genotypes of F2 generation mice were obtained: CCR2+/+, CCR2+/-, and CCR2-/-. The offspring genotypes were identified by PCR, and Western blot showed extremely low CCR2 protein expression in CCR2 knockout mice. Flow analysis showed that CCR2 knockdown reduced the expression of CD4+T and Th1 cells in mouse spleen-derived T cells, but did not affect macrophage function. Conclusion Correct breeding and identification are important ways to get the pure CCR2 knockout mice, and PCR method for identifying mouse genotypes is simple, fast and reliable.

Objective: To constructCSF1R+/-mice and to analyze their genotypes, so as to provide animal model basis for disease pathological mechanism and drug target. Methods : A linearized targeting vector was designed according to Cre/Loxp system. A Loxp site was inserted upstream of the 5th exon of theCSF1Rgene, and a neomycin resistance box with Loxp sites on both sides was inserted downstream of the 5th exon. The linearized targeting vector was electroporated into embryonic stem cells. The correctly targeted embryonic stem cells were injected into the blastocysts of C57BL/6J mice to obtain chimeric mice, which were bred with Zp3-Cre mice. The newborn mice were numbered 9-14 days after birth and their tails were cut. The DNA of the mice was extracted, and the genotype of the mice was identified by polymerase chain reaction and agarose gel electrophoresis. The expression of CSF1R in mouse macrophages was detected by flow cytometry. The expression of CSF1R in mouse tissues was detected by Western blot. Results: The results of agarose gel electrophoresis showed that 453 bp bands were amplified in wild type mice, and 453 bp and 650 bp bands were amplified in heterozygous mice. The results of flow cytometry showed that the expression of CSF1R in peritoneal macrophages and bone marrow-derived macrophages of CSF1R heterozygous mice was lower than that of WT group(P<0.05). The results of Western blot showed that the expression of CSF1R in spleen, kidney and brain tissue of CSF1R heterozygous group was lower than that of WT group(P<0.05). Conclusion CSF1R+/-mice are successfully constructed, reproduced and identified, which provides an animal model basis for further revealing the potential mechanism of CSF1R in immune regulation.

Objective To verify the safety and effectiveness of a new percutaneous controllable curved plasma radiofrequency instrument for nucleus pulposus ablation. Methods A new percutaneous controllable curved plasma radiofrequency instrument were designed (controllable curved group), and its ablation effect was compared with the currently used straight head non-bendable plasma ablation instrument (non-bendable group) on gross specimens. The ablation instrument was placed through the right intervertebral foramen, and continuous ablation on the same intervertebral disc was conducted for three times. The ablation range and trajectory were recorded, and the temperature changes in the front, back, left, and right of the ablation center during and 15 seconds after ablation were monitored by the inserted temperature probe. Results There were no difference in temperature changes in the front, back, right regions of the ablation center during and 15 seconds after ablation between the two groups. The temperature changes in the left region of the ablation center both during and 15 seconds after 3rd ablation were larger than those in the non-bendable group (P＜0.01). Compared with the non-bendable group, the controllable curved group achieved angle control and larger single ablation area (2.282 5 mm² vs 1.135 8 mm², P＜0.000 1). Conclusions This new percutaneous controllable curved plasma ablation instrument can achieve angle control and ablation on the side opposite to the puncture site, increase ablation volume, and is safe.

OBJECTIVE: To observe the efficacy and safety of moxibustion in treating patients with central obesity of phlegm-dampness constitution. METHODS: A total of 66 patients with central obesity of phlegm-dampness constitution were randomly assigned to a moxibustion group (n=33, 3 cases dropped out) and a sham moxibustion group (n=33, 4 cases dropped out). The moxibustion group received mild moxibustion combined with lifestyle intervention; the moxibustion was applied at Shenque (CV8) and bilateral Zusanli (ST36), 30 min per session, maintaining a local skin temperature of (43±1) ℃. The sham moxibustion group received simulated moxibustion combined with lifestyle intervention; the simulated moxibustion was applied at the same acupoints, with the same session length, but with a maintained skin temperature of (37±1) ℃. Both groups were treated once every other day, three times per week for 8 consecutive weeks. Obesity-related physical indicators (waist circumference, hip circumference, body weight, body fat percentage, body mass index [BMI]), constitution evaluation indicators (phlegm-dampness constitution conversion score, symptom score), the impact of weight on quality of life-lite (IWQOL-Lite), the hospital anxiety and depression scale (HADS), and the incidence of adverse events were measured before and after treatment, and after 4 weeks of follow-up. RESULTS: Compared with before treatment, both groups showed significant reductions in waist circumference, hip circumference, body weight, body fat percentage, BMI, phlegm-dampness constitution conversion score and symptom score, IWQOL-Lite, and both anxiety and depression subscale scores of HADS after treatment and at follow-up (P<0.001). These improvements were significantly greater in the moxibustion group than those in the sham moxibustion group (P<0.001, P<0.01, P<0.05). One patient in the moxibustion group experienced a mild burn that resolved with routine care; the incidence of adverse reactions was 3.0% (1/33) in the moxibustion group and 0% (0/33) in the sham moxibustion group, with no statistically significant difference (P>0.05). CONCLUSION On the basis of lifestyle intervention, moxibustion effectively improves obesity-related physical indicators, enhances quality of life, alleviates anxiety and depression, and improves the phlegm-dampness constitution in patients with central obesity. These benefits persist for at least 4 weeks after treatment.
Humans ; Moxibustion ; Male ; Female ; Middle Aged ; Adult ; Obesity, Abdominal/psychology* ; Acupuncture Points ; Treatment Outcome ; Aged ; Quality of Life ; Young Adult ; Body Mass Index

Objective To study the plasma metabolomics of mice poisoned by different dosage of the combination of diazepam and ethanol,and to reveal the toxicological mechanisms of combined poisoning of diazepam and ethanol.Methods Female Kunming mice were randomly divided into blank group,single and combined poisoning group(n=6),Based on the LD50 of diazepam co-administered with graded ethanol doses,mice in the single-drug and combined groups received oral gavage at 1/2,1,and 2 × LD50.Retro-orbital blood samples(～500 μL)were collected within 24 hours post-administration and analyzed by UPLC-QE-MS technology.Principal component analysis and orthogonal partial least squares discriminant analysis were used to identify differential metabolites and associated metabolic pathways.Results A total of 387 differential metabolites were identified in the combined poisoning group of diazepam and ethanol implicating the key pathways including tryptophan metabolism,phenylalanine metabolism,arginine and proline metabolism,Glycerophospholipid metabolism,phenylalanine,tyrosine and tryptophan biosynthesis.Conclusion Combined diazepam and ethanol poisoning exerts significant systemic effects by disrupting neurotransmitters conduction,exacerbating oxidative stress response and dysregulating energy metabolism.

Objective To study the plasma metabolomics of mice poisoned by different dosage of the combination of diazepam and ethanol,and to reveal the toxicological mechanisms of combined poisoning of diazepam and ethanol.Methods Female Kunming mice were randomly divided into blank group,single and combined poisoning group(n=6),Based on the LD50 of diazepam co-administered with graded ethanol doses,mice in the single-drug and combined groups received oral gavage at 1/2,1,and 2 × LD50.Retro-orbital blood samples(～500 μL)were collected within 24 hours post-administration and analyzed by UPLC-QE-MS technology.Principal component analysis and orthogonal partial least squares discriminant analysis were used to identify differential metabolites and associated metabolic pathways.Results A total of 387 differential metabolites were identified in the combined poisoning group of diazepam and ethanol implicating the key pathways including tryptophan metabolism,phenylalanine metabolism,arginine and proline metabolism,Glycerophospholipid metabolism,phenylalanine,tyrosine and tryptophan biosynthesis.Conclusion Combined diazepam and ethanol poisoning exerts significant systemic effects by disrupting neurotransmitters conduction,exacerbating oxidative stress response and dysregulating energy metabolism.

Objective:To correlate blood lipids and body mass index (BMI) with Helicobacter pylori (Hp) infection. Methods:A total of 303 participants who underwent physical examinations at The 903 Hospital of PLA Joint Logistics Support Force from May 2022 to May 2023 were included in this case-control study. These patients were divided into an Hp-infected group ( n = 97) and a non-Hp-infected group ( n = 206) based on whether they had Hp infection or not. Participants' body height and weight were recorded, and BMI was calculated. The levels of four blood lipid indicators were determined using an automatic biochemical analyzer. The distribution of different BMIs and abnormal statuses of these four blood lipid indicators were compared between the two groups. Spearman correlation analysis was used to analyze the correlation between Hp infection and these four blood lipid indicators. A multivariate logistic regression model was applied to analyze the influential factors for Hp infection. Results:The number of participants who had 24 kg/m 2 ≤ BMI < 28 kg/m 2 [39.17% (38/97)] and the number of participants who had BMI ≥ 28 kg/m 2 [10.31% (10/97)] in the Hp-infected group was significantly higher than those in the non-Hp-infected group [19.90% (41/206) and 2.43% (5/206), χ2 = 12.71, 7.11, P < 0.001, 0.008]. The decrease rate of high-density lipoprotein cholesterol (HDL-C), increase rate of low-density lipoprotein cholesterol (LDL-C), increase rate of triglyceride (TG), and increase rate of total cholesterol (TC) in the Hp-infected group were 23.71% (23/97), 31.96% (31/97), 17.53% (17/97), and 22.68% (22/97), respectively, which were significantly higher than 9.22% (19/206), 11.17% (23/206), 7.28% (15/206), and 8.74% (18/206) in the non-Hp-infected group ( χ2 = 11.59, 19.47, 7.33, 11.19, P = 0.001, < 0.001, 0.007, 0.001). The Spearman analysis showed that Hp infection was linearly positively correlated with BMI, LDL-C, TG, and TC ( r = 0.571, 0.519, 0.473, 0.535, all P < 0.001), while it was linearly negatively correlated with HDL-C ( r = -0.628, P < 0.001). Multivariate logistic regression analysis showed that BMI ≥ 24 kg/m 2, decreased HDL-C, increased LDL-C, elevated TG, and elevated TC are independent risk factors for Hp infection. Conclusion:Blood lipids and BMI are closely associated with Hp infection, and abnormal blood lipids and elevated BMI are independent risk factors for Hp infection.

Objective To construct myeloid-specific Spi1 gene knockout mice and analyze their genotypes,so as to provide animal model basis for the study of pathological mechanism of diseases and drug targets.Methods Ac-cording to the principle of CRISPR/Cas9 technology and Cre/LoxP system,sgRNA and Donor vectors were de-signed and constructed.The transcript of Exon 2(Exon 2)was used as the knockout region,and Loxp elements were placed on both sides of Exon 2.Cas9 protein,sgRNA and Donor vector were mixed and microinjected into the fertilized eggs of C57BL/6J mice,the fertilized eggs were transplanted into the uterus of C57BL/6J pregnant female mice,and F0 generation was obtained after 19～20 days.Positive F0 mice were mated with C57BL/6J mice to ob-tain stable F1 Spi1flox/+mice.Spi1flox/+mice of F1 generation were selfed to obtain Spi1flox/flox mice.Spi1flox/flox mated with Lyz2-Cre+mice to obtain Spi1flox/+/Lyz2-Cre+mice,and then mated with Spi1flox/flox,the Spi1flox/flox/Lyz2-Cre+mice were myeloid-specific Spi1 gene knockout(KO)mice.Spi1flox/flox/Lyz2-cre-mice were used as wild-type(WT)mice.DNA of WT and KO mice was extracted,and the genotypes were identified by agarose gel electro-phoresis after PCR amplification.Western blot was used to detect the expression of spleen focus forming virus provi-ral integration oncogene,Spi-1/purine rich box-1(PU.1)in immune cells of WT and KO mice.Results The results of PCR identification showed that the genotype of mice with only 220 bp amplified by flox primer was Spi1flox/flox homozygote,and the genotype of mice with 700 bp amplified by Lyz2-Cre primer was Lyz2-Cre+.Western blot showed that compared with WT group,the protein PU.1 was not expressed in bone marrow-derived macropha-ges(BMDMs)and peritoneal macrophages(PM)in KO group(P<0.01).There was no significant difference of statistics in the expression level of PU.1 in T cells between KO mice and WT mice.The results of PCR and West-ern blot showed that myeloid-specific Spi1 KO mice were successfully constructed.Conclusion The myeloid-spe-cific Spi1 gene KO mice are successfully constructed and identified,which provides animal model basis for further revealing the potential mechanism of PU.1 inimmune regulation.