AIM: To identify the primary active components and underlying mechanisms of Yijing Decoction(YJD)in treating early diabetic retinopathy(DR)based on liquid chromatography-mass spectrometry and network pharmacology.METHODS: Active components of YJD were characterized through LC-MS. Components with optimal ADME(absorption, distribution, metabolism, excretion)properties were selected as key bioactive candidates. Network pharmacology approaches were employed to predict YJD-DR therapeutic targets. Protein-protein interaction(PPI)networks, gene ontology(GO)enrichment analysis, and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis were subsequently conducted to predict core targets and networks. Critical targets and pathways were experimentally validated through Western blot.RESULTS: Ten core therapeutic targets were identified, including TNF, Alb, EGFR, STAT3, PTGS2, ESR1, PPAR, MMP9, TLR4, and MAPK. YJD was related to cancer-related signaling, fluid shear stress and atherosclerosis, and neurodegenerative diseases, encompassing key biological processes such as inflammatory response regulation, programmed cell death activation, and enhanced cell migration. Furthermore, Western blot analysis confirmed that YJD significantly inhibited high glucose-induced phosphorylation of STAT3(P-STAT3/STAT3)and ERK(P-ERK/ERK)in rat retinal microvascular endothelial cells.CONCLUSION: This study revealed YJD's pharmacodynamical basis and its multi-component, multi-target, and multi-paths pharmacology. YJD exerts therapeutic effects on DR by coordinately regulating critical signaling pathways and alleviating intraocular inflammation, thus preserving retinal vascular endothelial cells, maintaining blood-retinal barrier integrity, and facilitating retinal neurovascular repair.

ObjectiveTo explore the possible mechanism of Tongfeng Decoction （痛风汤） for preventing and treating acute gouty arthritis. MethodsSixty Wistar male rats were divided into normal group， model group， colchicine group and low-， medium- and high-dose Tongfeng Decoction groups according to the random number table， 10 rats in each group. Tongfeng Decoction of 11.34， 22.68 and 45.36 g/kg were given by gavage to low-， medium- and high-dose Tongfeng Decoction groups， colchicine 3.15×10^-4 g/（kg·d） to the colchicine group， and normal saline 10 ml/（kg·d） to the normal group and model group respectively for 7 consecutive days. After 1 hour of gavage on day 5， rats in all groups except the normal group were modelled as acute gouty arthritis in the ankle joint of the right hind limb with the modified Coderre's method； rats in the normal group were injected with 0.2 ml of normal saline at the same location. The swelling degree of the ankle joint was measured before modelling and after 6 h， 12 h， 24 h and 48 h of modelling， respectively. HE staining was used to observe the histopathological and morphological changes in the synovial tissue of the ankle joint； immunoblotting and RT-qPCR were used to detect NOD-like receptor protein 3 （NLRP3）， apoptosis-associated speck-like protein （ASC）， cysteine aspartate protease 1 （Caspase-1）， and interleukin 1β （IL-1β） protein and mRNA expression levels in ankle synovial tissues， respectively； immunohistochemistry was used to detect the positive expression of Caspase-1 and IL-1β in ankle synovial tissues； ELISA was used to detect the tumour necrosis factor alpha （TNF-α）， IL-1β， and interleukin in serum； immunofluorescence staining was used to observe the formation of neutrophil extracellular traps （NETs） in the synovial tissues of the ankle joints. ResultsCompared with the normal group， rats in the model group showed elevated joint swelling at all time points， significantly increased inflammatory cell infiltration and the number of synovial cell layers in the synovial tissues of the ankle joints， elevated NLRP3， Caspase-1， ASC and IL-1β protein and mRNA expression， elevated positive expression of Caspase-1 and IL-1β， increased formation of NETs， and elevation of TNF-α， IL-6， and IL-1β in serum （P＜0.05）. Compared with the model group， there was an improvement in synovial cell proliferation and inflammatory cell infiltration of the ankle joint in the colchicine group， high- and medium-dose Tongfeng Decoction groups. In the colchicine group and the high-， medium- and low-dose Tongfeng Decoction groups， the degree of joint swelling， positive expression of Caspase-1， IL-1β and formation of NETs in the synovial tissue of the ankle joint， and TNF-α， IL-6 and IL-1β in serum reduced 24 h and 48 h after modelling； in colchicine group and high-dose Tongfeng Decoction group， NLRP3， ASC， Caspase-1， IL-1β protein and mRNA expression in the synovial tissues of ankle joints all reduced （P＜0.05）. The colchicine group and high-dose Tongfeng Decoction group were superior to the low-dose Tongfeng Decoction group in reducing ASC， Caspase-1， IL-1β protein and mRNA expression in the ankle synovial tissues， the positive expression of Caspase-1， as well as TNF-α， IL-6， and IL-1β level in serum （P＜0.05）. ConclusionTongfeng Decoction showed effectiveness for the prevention and treatment of acute gouty arthritis， and its mechanism may be related to the inhibition of the assembly and activation of NLRP3 inflammatory vesicles in ankle synovial tissues， the inhibition of the release of inflammatory factors， and the formation of NETs.

Background::T-cell-mediated immunity is crucial for the effective clearance of viral infection, but the T-cell-mediated immune responses that are induced by booster doses of inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines in people living with human immunodeficiency virus (PLWH) remain unclear.Methods::Forty-five PLWH who had received antiretroviral therapy (ART) for more than two years and 29 healthy controls (HCs) at Beijing Youan Hospital were enrolled to assess the dynamic changes in T-cell responses between the day before the third vaccine dose (week 0) and 4 or 12 weeks (week 4 or week 12) after receiving the third dose of inactivated SARS-CoV-2 vaccine. Flow cytometry, enzyme-linked immunospot (ELISpot), and multiplex cytokines profiling were used to assess T-cell responses at the three timepoints in this study.Results::The results of the ELISpot and activation-induced marker (AIM) assays showed that SARS-CoV-2-specific T-cell responses were increased in both PLWH and HCs after the third dose of the inactivated SARS-CoV-2 vaccine, and a similar magnitude of immune response was induced against the Omicron (B.1.1.529) variant compared to the wild-type strain. In detail, spike-specific T-cell responses (measured by the ELISpot assay for interferon γ [IFN-γ] release) in both PLWH and HCs significantly increased in week 4, and the spike-specific T-cell responses in HCs were significantly stronger than those in PLWH 4 weeks after the third vaccination. In the AIM assay, spike-specific CD4 + T-cell responses peaked in both PLWH and HCs in week 12. Additionally, significantly higher spike-specific CD8 + T-cell responses were induced in PLWH than in HCs in week 12. In PLWH, the release of the cytokines interleukin-2 (IL-2), tumour necrosis factor-alpha (TNF-α), and IL-22 by peripheral blood mononuclear cells (PBMCs) that were stimulated with spike peptides increased in week 12. In addition, the levels of IL-4 and IL-5 were higher in PLWH than in HCs in week 12. Interestingly, the magnitude of SARS-CoV-2-specific T-cell responses in PLWH was negatively associated with the extent of CD8 + T-cell activation and exhaustion. In addition, positive correlations were observed between the magnitude of spike-specific T-cell responses (determined by measuring IFN-γ release by ELISpot) and the amounts of IL-4, IL-5, IL-2 and IL-17F. Conclusions::Our findings suggested that SARS-CoV-2-specific T-cell responses could be enhanced by the booster dose of inactivated COVID-19 vaccines and further illustrate the importance of additional vaccination for PLWH.

BACKGROUND: T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domains (TIGIT), an inhibitory receptor expressed on T cells, plays a dysfunctional role in antiviral infection and antitumor activity. However, it is unknown whether TIGIT expression on T cells influences the immunological effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inactivated vaccines. METHODS: Forty-five people living with HIV (PLWH) on antiretroviral therapy (ART) for more than two years and 31 healthy controls (HCs), all received a third dose of a SARS-CoV-2 inactivated vaccine, were enrolled in this study. The amounts, activation, proportion of cell subsets, and magnitude of the SARS-CoV-2-specific immune response of TIGIT + CD4 + and TIGIT + CD8 + T cells were investigated before the third dose but 6 months after the second vaccine dose (0W), 4 weeks (4W) and 12 weeks (12W) after the third dose. RESULTS: Compared to that in HCs, the frequency of TIGIT + CD8 + T cells in the peripheral blood of PLWH increased at 12W after the third dose of the inactivated vaccine, and the immune activation of TIGIT + CD8 + T cells also increased. A decrease in the ratio of both T naïve (T N ) and central memory (T CM ) cells among TIGIT + CD8 + T cells and an increase in the ratio of the effector memory (T EM ) subpopulation were observed at 12W in PLWH. Interestingly, particularly at 12W, a higher proportion of TIGIT + CD8 + T cells expressing CD137 and CD69 simultaneously was observed in HCs than in PLWH based on the activation-induced marker assay. Compared with 0W, SARS-CoV-2-specific TIGIT + CD8 + T-cell responses in PLWH were not enhanced at 12W but were enhanced in HCs. Additionally, at all time points, the SARS-CoV-2-specific responses of TIGIT + CD8 + T cells in PLWH were significantly weaker than those of TIGIT - CD8 + T cells. However, in HCs, the difference in the SARS-CoV-2-specific responses induced between TIGIT + CD8 + T cells and TIGIT - CD8 + T cells was insignificant at 4W and 12W, except at 0W. CONCLUSIONS TIGIT expression on CD8 + T cells may hinder the T-cell immune response to a booster dose of an inactivated SARS-CoV-2 vaccine, suggesting weakened resistance to SARS-CoV-2 infection, especially in PLWH. Furthermore, TIGIT may be used as a potential target to increase the production of SARS-CoV-2-specific CD8 + T cells, thereby enhancing the effectiveness of vaccination.
Humans ; Antibodies, Viral ; CD8-Positive T-Lymphocytes ; COVID-19/complications* ; COVID-19 Vaccines/immunology* ; HIV Infections/complications* ; Receptors, Immunologic ; SARS-CoV-2

Objective To investigate the effects of fluoride on protein oxidative damage in rat plasma by measuring oxidative stress levels,advanced glycation end products (AGEs) and advanced oxidation protein products (AOPP).Methods Eighty SPF male 3-week-old Wistar rats weighing (82.34 ± 10.60) g were randomly divided into 4 groups,20 rats in each group.The control group drank distilled water,and the fluoride groups drank distilled water with fluoride concentrations of 25,50 and 100 mg/L,respectively.Rats were allowed to eat and drink freely,and they were sacrificed at 1 month and 3 month,respectively,and samples such as urine,femur and peripheral blood were collected for experiments.Fluoride contents in urine and bone were detected by ion selective electrode method,the superoxide dismutase (SOD) activity was detected by hydroxylamine method,malondialdehyde (MDA) content was detected by thiobarbituric acid (TBA) method,and AGEs and AOPP contents were detected by enzyme linked immunosorbent assay (ELISA).Results For 1 month and 3 months,compared urinary fluoride contents (mg/L:2.088 + 0.638,9.170 ± 2.865,20.094 ± 8.186,54.866 ± 2.866;2.202 ± 1.282,9.112 ± 2.364,21.854 ±8.325,52.513 ± 16.211),and bone fluoride contents (mg/kg:324.985 ± 127.094,846.148 ± 331.861,1 886.601 ±250.140,2 420.971 ± 135.883;417.591 ± 88.324,1 582.243 ± 347.975,2 163.519 ± 614.932,2 755.434 ±265.370) in control group and fluoride concentrations of 25,50 and 100 mg/L groups,the differences were statistically significant (F =88.379,29.225;87.440,33.998,P ＜ 0.05).For 1 month and 3 months,compared SOD activity (U/ml:32.469 ± 5.674,35.931 ± 2.262,36.746 ± 3.994,38.042 ± 4.632;31.027 ± 4.147,30.777 ±4.791,34.148 ± 1.755,36.585 ± 2.860) and AGEs contents (μg/L:26.977 ± 5.285,33.303 ± 6.226,28.021 ±5.946,34.117 ± 6.706;35.681 ± 3.802,33.651 ± 7.214,28.114 ± 4.660,24.330 ± 3.581) in control group and fluoride concentrations of 25,50 and 100 mg/L groups,the differences were statistically significant (F =2.896,5.780;3.565,10.195,P ＜ 0.05).By factorial design anova,there was an interaction between the exposure concentration and exposure time of fluorine and the content of AGEs (F =8.957,P ＜ 0.01).Conclusion Excessive fluoride can affect urinary,bone fluoride contents,SOD activity,AGEs content,suggesting that excessive fluoride may regulate protein expression through direct and indirect oxidative damage pathways,which leading to fluorosis.

Objective To investigate the effects of fluoride exposure dose and exposure time on the expression of 5-methylcytosine (5-mC) in blood,liver,kidney and brain of rats;and to understand whether there is a difference in the effects of fluoride on DNA methylation levels in different tissues.Methods Eighty three-week old SPF male Wistar rats were randomly divided into four groups according to body weight [(82.34 ± 10.60) g],with 20 rats in each group.The rats of control group drank distilled water and the fluoride group's drank distilled water containing 25,50 and 100 mg/L of F ion,respectively.Rats were sacrificed after fed for 1 month and 3 months (n =10),and peripheral blood and tissue samples were collected.The incidence of dental fluorosis was observed in rats.Bone and urine fluoride content was detected by ion selective electrode method.The content of 5-mC in blood,liver,kidney and brain was detected by enzyme-linked immunosorbent assay (ELISA).The independent and interactive effects of fluoride exposure dose and exposure time on 5-mC in rat peripheral blood and different tissues were analyzed by factorial design anova.Results After feeding for 1 month and 3 months,all rats in the fluoride group had dental fluorosis with different severities,while none dental fluorosis was found in the control groups.Fluoride exposure dose and exposure time had a main effect on bone fluoride contents [1 month:(324.985 + 127.094),(846.148 ± 331.861),(1 886.601 + 250.140),(2 420.971 + 135.883) mg/kg;3 months:(417.591 ± 88.324),(1 582.243 ± 347.975),(2 163.519 ± 614.932),(2 755.434 ± 265.370)mg/kg;F =96.692,13.077,P ＜ 0.01],respectively,but there was no interaction effect (F =2.013,P ＞ 0.05);fluoride exposure dose had a main effect on urinary fluoride contents (F =62.358,P ＜ 0.01),the exposure time had no effect on it (F =0.862,P ＞ 0.05),and there was no interaction effect (F =0.081,P ＞ 0.05).Fluoride exposure dose had a main effect on the 5-mC content in the blood (F =8.446,P ＜ 0.01),the exposure time had no effect on it (F =0.095,P ＞0.05),and there had an interaction effect (F =4.676,P ＜ 0.01).Fluoride exposure dose and exposure time had a main effect on the 5-mC content in the liver,respectively (F =4.737,7.064,P ＜ 0.01 or ＜ 0.05),and an interaction effect was exist (F =8.302,P ＜ 0.01).Fluoride exposure time had a main effect on the 5-mC content in the kidney (F =6.340,P ＜ 0.05),the exposure dose had no effect on it (F =0.140,P ＞ 0.05),and there was no interaction effect (F =1.269,P ＞ 0.05).Fluoride exposure dose and exposure time had no effect on 5-mC content in the brain (F =0.633,2.065,P ＞ 0.05).Conclusion Fluoride exposure dose and exposure time have the different effect on the levels of 5-mC in blood,liver,kidney and brain,suggesting that there may be differences in the effects of fluoride on DNA methylation levels in different tissues.

OBJECTIVETo investigate RANK-RANKL expression in the kidneys of a rat model of puromycin aminonucleoside nephropathy (PAN).

METHODSThirty-six SD rats were randomly divided into PAN model group and normal control group. PAN was induced by a single intravenous injection of 100 mg/kg puromycin aminonucleoside. Serum creatinine and 24-hour urinary protein were measured on days 3, 7, and 14 after the injection, and renal pathologies were assessed with optical and immune transmission electron microscopy. The expression of RANK and RANKL in the kidneys was examined using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting.

RESULTSThe PAN model rats showed massive proteinuria and elevated serum creatinine on day 3, which peaked on day 7. RANK-RANKL protein and mRNA expressions in PAN model group was higher than those in the control group. In the PAN rats, RANK was expressed mainly on the top cell membrane and in the cytoplasm of renal podocytes with a significantly increased expression level compared with that in the control group.

CONCLUSIONThe PAN rat model shows aberrant RANK and RANKL expressions in the podocytes, indicating their contribution to podocyte injury in PAN.

Animals ; Creatinine ; blood ; Female ; Kidney ; drug effects ; metabolism ; Kidney Diseases ; chemically induced ; metabolism ; pathology ; Male ; Podocytes ; drug effects ; metabolism ; Proteinuria ; pathology ; Puromycin Aminonucleoside ; adverse effects ; RANK Ligand ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor Activator of Nuclear Factor-kappa B ; metabolism

Objective To investigate RANK-RANKL expression in the kidneys of a rat model of puromycin aminonucleoside nephropathy (PAN). Methods Thirty-six SD rats were randomly divided into PAN model group and normal control group. PAN was induced by a single intravenous injection of 100 mg/kg puromycin aminonucleoside. Serum creatinine and 24-hour urinary protein were measured on days 3, 7, and 14 after the injection, and renal pathologies were assessed with optical and immune transmission electron microscopy. The expression of RANK and RANKL in the kidneys was examined using reverse transcription-ploymerase chain reaction (RT-PCR) and Western blotting. Results The PAN model rats showed massive proteinuria and elevated serum creatinine on day 3, which peaked on day 7. RANK-RANKL protein and mRNA expressions in PAN model group was higher than those in the control group. In the PAN rats, RANK was expressed mainly on the top cell membrane and in the cytoplasm of renal podocytes with a significantly increased expression level compared with that in the control group. Conclusion The PAN rat model shows aberrant RANK and RANKL expressions in the podocytes, indicating their contribution to podocyte injury in PAN.

Objective To investigate RANK-RANKL expression in the kidneys of a rat model of puromycin aminonucleoside nephropathy (PAN). Methods Thirty-six SD rats were randomly divided into PAN model group and normal control group. PAN was induced by a single intravenous injection of 100 mg/kg puromycin aminonucleoside. Serum creatinine and 24-hour urinary protein were measured on days 3, 7, and 14 after the injection, and renal pathologies were assessed with optical and immune transmission electron microscopy. The expression of RANK and RANKL in the kidneys was examined using reverse transcription-ploymerase chain reaction (RT-PCR) and Western blotting. Results The PAN model rats showed massive proteinuria and elevated serum creatinine on day 3, which peaked on day 7. RANK-RANKL protein and mRNA expressions in PAN model group was higher than those in the control group. In the PAN rats, RANK was expressed mainly on the top cell membrane and in the cytoplasm of renal podocytes with a significantly increased expression level compared with that in the control group. Conclusion The PAN rat model shows aberrant RANK and RANKL expressions in the podocytes, indicating their contribution to podocyte injury in PAN.

OBJECTIVETo explore the influences of di-(2-ethylhexyl)phthalate (DEHP) and its principle metabolite mono(2-ethylhexyl)phthalate (MEHP) on spermatogenic cell apoptosis in young male Wistar rats.

METHODSNinety-eight 2-week-old male Wistar rats were randomly divided into 14 equal groups to receive daily intragastric administration of 0.2 ml/kg normal saline for 3 weeks (normal control), 100 mg/kg cyclophosphamide (CTX) for 1 week (positive control), 100, 200, and 300 mg/kg DEHP or MEHP for 1 week, or 100 mg/kg DEHP or MEHP for 1, 2, and 3 weeks. After the treatments, the pathological changes of the testicular tissues were examined, spermatogenic cell apoptosis was detected, and serum sex hormones levels were measured using TUNEL assay or radioimmunoassays.

RESULTSCTX, DEHP, and MEHP all caused shrinkage, development retardation and quantitative reduction of spermatogenic cells with and mitochondrial swelling vacuolar changes. The damage of spermatogenic cells increased significantly with the increment of DEHP and MEHP doses and exposure time. Both DEHP and MEHP treatments resulted in significantly increased cell apoptosis index (AI) in close correlation with the exposure doses and duration (P<0.01). DEHP and MEHP treatments also significantly increased serum levels of follicle stimulating hormone and luteinizing hormone and decreased testosterone levels in a dose- and time-dependent manner (P<0.05).

CONCLUSIONDEHP and MEHP can induce obvious apoptosis of spermatogenic cells in young male rats with a dose- and time-dependent effect.

Animals ; Apoptosis ; drug effects ; Diethylhexyl Phthalate ; analogs & derivatives ; toxicity ; Dose-Response Relationship, Drug ; Environmental Exposure ; Male ; Rats ; Rats, Wistar ; Spermatozoa ; cytology ; drug effects