OBJECTIVES: To investigate the role of the calcium/calmodulin (CaM)-mediated activation of calcineurin (CN)-nuclear factor of activated T cells (NFAT) signaling pathway in mediating the regulatory effect of hyperforin (HY) on stress-induced depression-like disorder (DP) in mice. METHODS: C57BL/6J mice were randomly divided into control group, DP model group, and hyperforin treatment group (n=15). Behavioral changes of the mice were assessed using open field test (OFT), sucrose preference test (SPT), tail suspension test (TST), light/dark box test (LDB), and novel object suppression test (NSFT). Immunohistochemistry was used to detect tyrosine hydroxylase (TH) expression in the CA1 region of the hippocampus, and serum serotonin (5-HT) and norepinephrine (NA) levels were detected with ELISA. Western blotting was used to analyze the expressions of TNF-α, IL-1β, IL-2, and CN-NFAT pathway proteins. In cultured BV-2 microglial cells with lipopolysaccharide (LPS) stimulation, the effects of hyperforin and CN inhibitor (CNIS) on expressions of ionized calcium-binding adapter molecule 1 (IBA-1), 5-HT, NA, inflammatory cytokines and CN-NFAT pathway proteins were examined using immunofluorescence assay, ELISA or Western blotting. RESULTS: Compared with the control mice, the mice in DP group showed significantly reduced activity in OFT, decreased sucrose consumption in SPT, reduced shuttle crossing in LDB, and lowered food intake in NSFT with significantly increased immobility in TST. The mice with DP showed significantly decreased TH-positive neurons, lowered 5-HT and NA levels, and increased expressions of TNF-α, IL-1β, IL-2 and CaM-CN-NFAT pathway proteins. In cultured BV-2 cells, LPS stimulation strongly increased cellular IBA-1 expression, decreased the levels of neurotransmitters (5-HT and NA), and increased the levels of inflammatory cytokines and CN-NFAT signaling, and these changes were effectively reversed by treatment with hyperforin or CNIS. CONCLUSIONS Hyperforin improves stress-induced depression-like behaviors in mice and activated BV-2 cells by targeting the CN-NFAT signaling pathway.
Animals ; Mice, Inbred C57BL ; Mice ; Microglia/drug effects* ; Depression/etiology* ; Perylene/pharmacology* ; Calcineurin/metabolism* ; NFATC Transcription Factors/metabolism* ; Calcium Signaling/drug effects* ; Stress, Psychological ; Phloroglucinol/pharmacology* ; Signal Transduction ; Male ; Behavior, Animal/drug effects* ; Terpenes

Objective To investigate the effect of ring finger protein 130(RNF130)on myocardial ischemia-reperfusion injury(MI/RI)and its potential mechanism.Methods Male C57BL/6J mice were divided into four groups(n=6):Sham,MI/RI,MI/RI+Vector,and MI/RI+RNF130 overexpression(MI/RI+RNF130OE).Cardiac function was evaluated by echocardiography 24 hours after ischemia-reperfusion.Pathological changes,oxidative damage,and apoptosis in myocardial tissues were observed via IHC,DHE,and TUNEL staining.Protein expression was detected using Western blot,immunofluorescence,and immunohistochemistry.Proteomic analysis was performed to identify downstream proteins regulated by RNF130,and protein-protein interactions were validated by immunoprecipitation(IP)assay.Results Compared with the MI/RI+Vector group,RNF130 overexpression significantly improved cardiac function,as indicated by increased left ventricular ejection fraction(EF)and fractional shortening(FS),reduced myocardial infarction area,and decreased expression of NOX-2 and BAX proteins(P＜0.05).DHE and TUNEL staining showed that RNF130 overexpression alleviated myocardial oxidative damage and apoptosis(P＜0.05).Proteomic analysis and IP assays revealed a significant interaction between RNF130 and PARP1,with PARP1 expression inversely correlated with RNF130.Conclusions RNF130 may mitigate MI/RI injury by regulating the PARP1 ubiquitination pathway,providing a new target for therapeutic intervention.

Objective To investigate the effect of ring finger protein 130(RNF130)on myocardial ischemia-reperfusion injury(MI/RI)and its potential mechanism.Methods Male C57BL/6J mice were divided into four groups(n=6):Sham,MI/RI,MI/RI+Vector,and MI/RI+RNF130 overexpression(MI/RI+RNF130OE).Cardiac function was evaluated by echocardiography 24 hours after ischemia-reperfusion.Pathological changes,oxidative damage,and apoptosis in myocardial tissues were observed via IHC,DHE,and TUNEL staining.Protein expression was detected using Western blot,immunofluorescence,and immunohistochemistry.Proteomic analysis was performed to identify downstream proteins regulated by RNF130,and protein-protein interactions were validated by immunoprecipitation(IP)assay.Results Compared with the MI/RI+Vector group,RNF130 overexpression significantly improved cardiac function,as indicated by increased left ventricular ejection fraction(EF)and fractional shortening(FS),reduced myocardial infarction area,and decreased expression of NOX-2 and BAX proteins(P＜0.05).DHE and TUNEL staining showed that RNF130 overexpression alleviated myocardial oxidative damage and apoptosis(P＜0.05).Proteomic analysis and IP assays revealed a significant interaction between RNF130 and PARP1,with PARP1 expression inversely correlated with RNF130.Conclusions RNF130 may mitigate MI/RI injury by regulating the PARP1 ubiquitination pathway,providing a new target for therapeutic intervention.

Objective To investigate the effect and mechanism of Congrong Shujing Granules on promoting microglial polarization and inhibiting neuroinflammation through the nucleotide-binding oligomeric domain-like receptor protein 3(NLRP3)/Caspase-1 signaling pathway.Methods Twenty Sprague-Dawley rats were assigned to the blank serum and Congrong Shujing Granules containing serum groups using random number table method,with 10 rats in each group.Rats in the Congrong Shujing Granules containing serum group received intragastric administration of Congrong Shujing Granules(2.57 g/kg)and the rats in the blank serum group received intragastric administration of physiological saline of equal volume.Blank serum and Congrong Shujing Granules containing serum were prepared separately.Mouse microglia cells BV-2 were cultured in vitro,and the optimal concentration of 1-methyl-4-phenylpyridine(MPP+)and optimal volume fraction of Congrong Shujing Granules containing serum were selected by the CCK-8 assay and immunofluorescence staining.And the NLRP3 inhibitor MCC950 was used as a postive control.Cells were divided into the blank serum group(10%blank serum),model group(10%blank serum+500 μmol/L MPP+),Congrong Shujing Granules containing serum group(10%Congrong Shujing Granules containing serum+500 μmol/L MPP+),and MCC950 group(10%blank serum+10 μmol/L MCC950+500 μmol/L MPP+),and intervened separately.After 14 h of intervention,morphological changes in BV-2 cells were observed.The contents of interleukin(IL)-1β,tumor necrosis factor-α(TNF-α),IL-6,and IL-4 were detected by an enzyme-linked immunosorbent assay.The mRNA expressions of differentiation cluster 86(CD86),inducible nitric oxide synthase(iNOS),CD206,and arginase 1(Arg1)were detected by real-time fluorescence PCR.The expressions of CD86,Arg1,Ionized calcium binding adaptor molecule 1(Iba1),and NLRP3 were detected by immunofluorescence staining.The expression of iNOS,Arg1,TNF-α,IL-6,IL-4,NLRP3,pro-cysteinyl aspartate specific proteinase 1(pro-Caspase-1),and Caspase-1 proteins was detected by Western blotting.Results Iba1 activation and expression increased under the MPP+(12 h,500 μmol/L)intervention(P<0.05),and cell viability was not affected.There was no statistically significant effect on cell viability after treatment with 10%Congrong Shujing Granules containing serum alone or in combination with MPP+(P>0.05).Compared to the blank serum group,BV-2 cells in the model group showed multiple branches and protruded in the shape of an amoeba.The contents of IL-1β,TNF-α,and IL-6 increased,while the contents of IL-4 decreased.The mRNA expressions of CD86 and iNOS increased,while mRNA expressions of CD206 and Arg1 decreased.The mean fluorescence intensity of CD86,Iba1,and NLRP3 increased,while the mean fluorescence intensity of Arg1 decreased.The protein expressions of iNOS,TNF-α,IL-6,NLRP3,pro-Caspase-1,and Caspase-1 increased,while the protein expressions of Arg1,IL-4 decreased,P<0.05.Compared to the model group,the Congrong Shujing Granules containing serum group and MCC950 group showed a decrease in the branch of cell protrusions,reduced cell activation,decreased levels of IL-1β,TNF-α,and IL-6,increased levels of IL-4,decreased expression of CD86 and iNOS mRNA,increased expression of CD206 mRNA,the decreased mean fluorescence intensity of CD86,Iba1,and NLRP3,the increased mean fluorescence intensity of Arg1,decreased expression of iNOS,TNF-α,IL-6,NLRP3,pro-Caspase-1,and Caspase-1 proteins,and increased expression of Arg1 and IL-4 proteins,P<0.05.Conclusion Congrong Shujing Granules containing serum may alleviate the MPP+-induced neuroinflammatory response by inhibiting the NLRP3/Caspase-1 signaling pathway to regulate M1/M2 phenotype polarization of microglia.

Objective To investigate the effect and mechanism of Congrong Shujing Granules on promoting microglial polarization and inhibiting neuroinflammation through the nucleotide-binding oligomeric domain-like receptor protein 3(NLRP3)/Caspase-1 signaling pathway.Methods Twenty Sprague-Dawley rats were assigned to the blank serum and Congrong Shujing Granules containing serum groups using random number table method,with 10 rats in each group.Rats in the Congrong Shujing Granules containing serum group received intragastric administration of Congrong Shujing Granules(2.57 g/kg)and the rats in the blank serum group received intragastric administration of physiological saline of equal volume.Blank serum and Congrong Shujing Granules containing serum were prepared separately.Mouse microglia cells BV-2 were cultured in vitro,and the optimal concentration of 1-methyl-4-phenylpyridine(MPP+)and optimal volume fraction of Congrong Shujing Granules containing serum were selected by the CCK-8 assay and immunofluorescence staining.And the NLRP3 inhibitor MCC950 was used as a postive control.Cells were divided into the blank serum group(10%blank serum),model group(10%blank serum+500 μmol/L MPP+),Congrong Shujing Granules containing serum group(10%Congrong Shujing Granules containing serum+500 μmol/L MPP+),and MCC950 group(10%blank serum+10 μmol/L MCC950+500 μmol/L MPP+),and intervened separately.After 14 h of intervention,morphological changes in BV-2 cells were observed.The contents of interleukin(IL)-1β,tumor necrosis factor-α(TNF-α),IL-6,and IL-4 were detected by an enzyme-linked immunosorbent assay.The mRNA expressions of differentiation cluster 86(CD86),inducible nitric oxide synthase(iNOS),CD206,and arginase 1(Arg1)were detected by real-time fluorescence PCR.The expressions of CD86,Arg1,Ionized calcium binding adaptor molecule 1(Iba1),and NLRP3 were detected by immunofluorescence staining.The expression of iNOS,Arg1,TNF-α,IL-6,IL-4,NLRP3,pro-cysteinyl aspartate specific proteinase 1(pro-Caspase-1),and Caspase-1 proteins was detected by Western blotting.Results Iba1 activation and expression increased under the MPP+(12 h,500 μmol/L)intervention(P<0.05),and cell viability was not affected.There was no statistically significant effect on cell viability after treatment with 10%Congrong Shujing Granules containing serum alone or in combination with MPP+(P>0.05).Compared to the blank serum group,BV-2 cells in the model group showed multiple branches and protruded in the shape of an amoeba.The contents of IL-1β,TNF-α,and IL-6 increased,while the contents of IL-4 decreased.The mRNA expressions of CD86 and iNOS increased,while mRNA expressions of CD206 and Arg1 decreased.The mean fluorescence intensity of CD86,Iba1,and NLRP3 increased,while the mean fluorescence intensity of Arg1 decreased.The protein expressions of iNOS,TNF-α,IL-6,NLRP3,pro-Caspase-1,and Caspase-1 increased,while the protein expressions of Arg1,IL-4 decreased,P<0.05.Compared to the model group,the Congrong Shujing Granules containing serum group and MCC950 group showed a decrease in the branch of cell protrusions,reduced cell activation,decreased levels of IL-1β,TNF-α,and IL-6,increased levels of IL-4,decreased expression of CD86 and iNOS mRNA,increased expression of CD206 mRNA,the decreased mean fluorescence intensity of CD86,Iba1,and NLRP3,the increased mean fluorescence intensity of Arg1,decreased expression of iNOS,TNF-α,IL-6,NLRP3,pro-Caspase-1,and Caspase-1 proteins,and increased expression of Arg1 and IL-4 proteins,P<0.05.Conclusion Congrong Shujing Granules containing serum may alleviate the MPP+-induced neuroinflammatory response by inhibiting the NLRP3/Caspase-1 signaling pathway to regulate M1/M2 phenotype polarization of microglia.

Objective:To analyze the clinical efficacy of bone transport with unilateral external fixation in the treatment of massive tibial bone defects.Methods:A retrospective study was conducted to review the 21 patients with massive tibial bone defects who had been treated by bone transport with unilateral external fixation from February 2017 to January 2022 at Department of Trauma Orthopedics, Ganzhou People's Hospital. There were 14 males and 7 females with a mean age of (46.3 ± 11.3) years. Causes for bone defects: trauma ( n=5), resection of bone non-union ( n=9), resection of infected bone ( n=6) and resection of bone tumor ( n=1). The mean bone defect length was (8.3 ± 1.7) cm. Bone transport started from 10 to 12 days after operation, with a speed of 1 mm/d, and was completed in 4 times. X-ray films were reviewed every 2 weeks. The bone union time, external fixation time (EFT), external fixation index (EFI), docking site situation and complications were recorded. The clinical efficacy was assessed by Paley score. Results:All patients were followed-up for a mean time of (13.5 ± 5.5) months. The mineralization of regenerated bone was good. The bone union time was (9.6 ± 2.2) months, the EFT (10.3 ± 4.0) months, and the EFI (1.3 ± 0.4) months/cm. All docking sites got united. The docking sites were cleaned in 14 patients, of whom simple compression with external fixation was performed in 5 and bone grafts at the docking sites in 9. Postoperative nail tract infection was observed in 6 cases, tibial alignment deviation in 1 case, foot drop deformity in 5 cases, horseshoe varus foot deformity in 1 case, toe flexion deformity in 3 cases, and refracture after removing the external fixation in 1 case. By the Paley score, the bony outcomes were rated as excellent in 16 and as good in 5 cases. The functional outcomes were excellent in 10, good in 7, and acceptable in 4.Conclusion:Bone transport with unilateral external fixation is an effective treatment for massive tibial bone defects, showing advantages of easy operation and convenient carry.

Objective To evaluate the regulatory role of azithromycin-induced persistent Chlamydia trachomatis (Ct) infection in the apoptosis of Hela229 cells.Methods Hela229 cells were firstly co-cultured with Ct for 22 hours,and then cultured with Dulbecco's modified Eagle's medium (DMEM) containing 0.08 mg/L azithromycin for 26 hours to establish a cell model of persistent Ct infection (persistent infection group).These infected Hela229 cells cultured with azithromycin-free DMEM served as a cell model of acute Ct infection (acute infection group).After 48-hour infection with Ct,azithromycin was removed,and infected Hela229 cells in the above 2 groups were successively cultured with DMEM for the resurgence of Ct.Immunofluorescence assay and electron microscopy were performed to verify the persistent Ct infection model.The Hela229 cells in the persistent infection group and acute infection group as well as uninfected Hela229 cells (control group) were treated with staurosporine (STS) for 4 hours to induce the apoptosis,and then cell apoptosis was detected by Hoechst 33258 staining,annexin V/propidium iodide staining and flow cytometry.Results After the treatment with azithromycin,atypical inclusions with aberrant reticulate bodies appeared in the Ct-infected cells.After removing azithromycin,cells were cultured until 96 hours after infection,and infectious elementary bodies reappeared in the Ct inclusions.After the treatment with STS,Hoechst staining showed that there was loose chromatin in the persistently infected cells,while chromatin condensation was observed in the uninfected cells.After 24-hour infection with Ct and 4-hour induction with STS,the apoptosis rate was significantly higher in the persistent infection group (45.567％ ± 2.631％) than in the acute infection group (38.567％ ± 1.701％,t =2.686,P =0.028),but significantly lower in the persistent infection group than in the uninfected group (69.800％ ± 2.835％,t =8.187,P ＜ 0.001).After 48-hour infection with Ct and 4-hour induction with STS,there was a significant difference in the apoptosis rate between the persistent infection group (46.700％ ± 5.257％) and acute infection group (61.767％ ± 1.815％,t =5.781,P ＜ 0.001),as well as between the persistent infection group and the uninfected group (68.667％ ± 3.156％,t =7.421,P ＜ 0.001).Conclusion This study showed that azithromycin-induced persistent Ct infection regulated the apoptosis of host cells,and this effect lasted 48 hours.

Objective To explore the feasibility of tibial nerve motor branches transfer to the deep fibular nerve in an anatomical study.Methods Twenty-three sides lower limbs from 12 adult cadavers which preserved in Formalin were used for dissection of the tibial nerve and its all motor branches,and the proximal deep and superficial fibular nerve.Experimental measurement were performed for the parameters of each branch such as length,diameter,the location of original point relative to the level of the fibular head.The diameter of proximal part of the deep fibular nerve was measured simultaneously.Finally,the length from original point of each branch to the fibular neck was also measured during simulation of nerve transfer procedure.Results The average length of motor branches to the flexor digitorum longus muscle,to the flexor hallucis longus muscle and the superficial branches to the soleus muscle were (95.70 ± 13.40)mm,(96.90± 13.60)mm and (73.60 ± 12.00)mm respectively.Their average diameter were (0.63 ± 0.16)mm,(0.65 ±0.20)mm and ( 1.56 ± 0.26)mm respectively.The average diameter of proximal deep fibular nerve was (2.54± 0.26)mm.Based on length,branches to the flexor digitorum longus muscle and flexor hallucis longus muscle were adequate for direct nerve transfer to the deep fibular nerve in all specimens without interpositional grafr.And in 22 specimens (95.7 percent),the superficial branches to the soleus muscle were long enough to directly transfer.Other branches of the tibial nerve were not adequate for direct nerve transfer Conclusion This study confirmed the anatomical feasibility of using motor branches from tibial nerve for direct transfer to restore the deep fibular nerve.The superficial branches to soleus muscle were the best donor nerve if considering the branches,length,diameter and the difficulty of surgical procedures.

Objective To study the mechanism of heat transfer by simulating a complete freezing-thawing process of biological tissue.Methods A numerical model of phase change heat transfer in biological tissue was developed with consideration of the difference of thermophysical properties for normal biological tissue and tumor.The biological tissue was assumed as a porous media.The different thermophysical properties between tissue framework and tissue fluid(as water in tissue,etc) were considered.An apparent heat capacity method was applied to solve the phase change heat transfer problem.Results It was showed that the temperature of biological tissue decreased more quickly during the freezing process when the initial cryoprobe temperature was lower and the cooling-rate of cryoprobe was faster.The temperature of biological tissue increased more quickly with faster warming-rate of cryoprobe in the thawing process.It was also showed that the porosity,blood perfusion rate and metabolic heat generation of the biological tissue had effects on tissue freezing temperature in biological tissue.Conclusion To study the thermal process of biological tissue in cryosurgery will be very helpful for further application in cryosurgery.