BACKGROUND:Traditional methods of urinary tract reconstruction are limited by donor scarcity,high complication rates,and suboptimal functional recovery.Tissue engineering strategies offer new directions in this field.Since the urinary tract is mainly composed of muscle tissue,the key is to find suitable seed cells and efficiently induce them to differentiate into smooth muscle cells.Comparative studies on the efficacy of different smooth muscle cell induction regimens are still lacking. OBJECTIVE:To isolate,culture,and identify human urine-derived stem cells,and to compare the effects of two different induction protocols. METHODS:Human urine-derived stem cells were isolated from urine samples of 11 healthy adult volunteers by multiple centrifugations.Surface markers were identified by flow cytometry.The multi-directional differentiation potential of human urine-derived stem cells was verified through osteogenic and adipogenic differentiation.Differentiation was induced by transforming growth factor-β1 or transforming growth factor-β1 combined with platelet derived growth factor for 14 days.Immunofluorescence staining and western blot assay were employed to compare the expression differences of smooth muscle-specific proteins(α-SMA and SM22). RESULTS AND CONCLUSION:(1)Urine-derived stem cells were successfully isolated from the eight urine samples of healthy people.These cells exhibit a"rice grain"-like morphology and possess a robust proliferative capacity.(2)Urine-derived stem cells exhibited high expression of mesenchymal stem cell surface markers(CD73,CD90,and CD44)and extremely low expression of hematopoietic stem cell surface markers(CD34 and CD45).These cells did not express CD19,CD105,and HLA-DR.(3)After osteogenic and adipogenic differentiation,the formation of calcium nodules and lipid droplets was observed,with positive staining results from Alizarin Red S and Oil Red O staining.(4)After 14 days of smooth muscle induction culture,immunofluorescence staining revealed that the smooth muscle differentiation rate of urine-derived stem cells treated with a combination of transforming growth factor-β1 and platelet derived growth factor was significantly higher compared to those treated with transforming growth factor-β1 alone(P<0.005).(5)After 14 days of smooth muscle induction culture,western blot assay further demonstrated that the expression levels of α-SMA and SM22 in the transforming growth factor-β1/platelet derived growth factor group were significantly elevated compared to those in the transforming growth factor-β1 only group(P<0.005).These findings confirm that urine-derived stem cells can be non-invasively isolated using multiple rounds of centrifugation.Compared with transforming growth factor-β1 alone,the combination of transforming growth factor-β1 and platelet derived growth factor can improve the efficiency of inducing urine-derived stem cells to differentiate into smooth muscle cells.

ObjectiveTo investigate the effects and potential mechanisms of morning and evening fasting on postprandial lipid responses, a post hoc analysis based on a crossover randomized controlled trial was conducted to assess the effects of different fasting strategies on postprandial lipid metabolism in community residents in Shanghai. MethodsA total of 23 participants took part in a randomized crossover trial involving two intervention days: morning fasting and evening fasting, with a washout period of 6 days between intervention days. Two-way analysis of variance was used to test the differences in total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and the relative expression of circadian clock genes before and after the next meal under fasting. Wilcoxon rank sum tests were used to analyze the different metabolites between the two groups. Principal component analysis and Orthogonal partial least squares-discriminant analysis were conducted to evaluate the ability of metabolites to differentiate between morning fasting and evening fasting and identify the important differential metabolites. After adjusting for age, sex, and BMI, a partial correlation analysis was performed to identify metabolites associated with plasma lipids. In addition, important metabolites associated with plasma lipids were computed by pathway enrichment analysis. ResultsAfter evening fasting intervention, fasting TG level [(0.37±0.29) vs (0.27±0.18)] mmol·L^-1, fasting and postprandial change values in TC [(2.74±0.47) vs (2.51±0.27)] mmol·L^-1 and LDL-C [(1.32±0.38) vs (0.99±0.27)] mmol·L^-1 were significantly lower than those after morning fasting (P<0.05). While, change values of fasting LDL-C [(0.89±0.37) vs (1.14±0.37)] mmol·L^-1 and TG [(1.14±0.19) vs (1.28±0.17)] mmol·L^-1 were significantly higher than those after morning fasting intervention (P<0.05). After fasting intervention, the relative expression of AMPK, CRY1, CLOCK, MTNR1B, AANAT, and ASMT was correlated with the amount of plasma lipid changes (P<0.05). Specifically, CLOCK and AANAT were upregulated following evening fasting and downregulated after morning fasting. Among the 217 important differential metabolites, 111 were correlated with plasma lipids, and which were primarily enriched in the cysteine and methionine metabolism pathways (P<0.05). ConclusionCompared to morning fasting, evening fasting was more effective in improving postprandial lipid responses, indicating that an evening fasting window during intermittent fasting could be conducive to cardiovascular disease prevention in adults. Meanwhile, it is suggested that morning and evening fasting may affect lipid responses through circadian rhythm oscillations and the cysteine and methionine metabolism pathways.

ObjectiveTo understand the relative bioavailability （RBA） of cadmium in different aquatic products. Based on the consideration of the gender differences and the relative bioavailability of cadmium in different foods， the physiologically based toxicokinetic （PBTK） model of cadmium was further optimized and verified. The correlation between internal and external exposure in quantitative risk assessment of food safety was optimized， and the provisional tolerable daily intake （PTDI） value of cadmium was derived. MethodsThe relative bioavailability of cadmium in different aquatic products was determined in four-week-old Balb/c female mice. The PBTK model of cadmium metabolism was optimized by ABC-MCMC method using combined internal and external exposure data of cadmium in Shanghai residents， and the PTDI was calculated accordingly. ResultsExcept for scallops and squid， the RBA of aquatic samples was less than 1， indicating that the absorption rate of cadmium in aquatic products was lower than that of cadmium chloride. The higher RBA of squid and scallop may be due to the presence of cadmium in the visceral organs， which is conducive to cadmium absorption and its higher concentration of cadmium. Frying at the temperature less than 160 ℃ reduced cadmium absorption but may increase cadmium absorption at the temperature greater than 160 ℃. The optimized model parameters converged well and the model could reasonably estimate urinary cadmium level according to the external exposure of cadmium. The PTDI value was0.466 4 μg·（kg·day）^-1 according to the optimized single-chamber model. ConclusionThe relative bioavailability of cadmium in different foods varies greatly， except for squid and scallops， RBA is less than 1， and cooking processing will affect the RBA of food. The construction of the PBTK model did not only consider the effects of gender differences on cadmium metabolism， but also included the relative bioavailability data to optimize and adjust the correlation coefficient of absorption rate. Compared with the model without RBA adjustment， the adjusted model has enhanced the ability to predict urinary cadmium level， which provides a new， more accurate method for the risk assessment of food safety.

To investigate the effect of mitochondrial division inhibitor 1(Mdivi-1)on imiquimod(IMQ)-induced psoriasis-like skin inflammation in mice and its mechanism,female 8-week-old C57BU6 mice were recruited and randomly divided into control group,IMQ model group,IMQ+Mdivi-1 experiment group.IMQ was used to induce the psoriasis-like skin inflammation model in mice.The mice in the experiment group were injected intraperitoneally(i.p.)with Mdivi-1,and the mice in the control group and model group were injected with the same volume of solvent.The mice were sacrificed on the 7th day for sampling.Psoriasis area and severity index(PASI)score was used to evaluate the severity of skin lesions in each group;the reactive oxygen species(R0S)content in skin tissue was detected by fluorescence staining of frozen section;HE staining was used to observe the histomorphologic change of skin lesions;immunohistochemical staining was used to detect the expression of dynamin-related protein 1(Drp1)in the skin of mice;Western blot was used to detect the protein levels of Drp1,NLRP3 and IL-1β in the skin tissues of mice in each group;and the expressions of IL-17A and IL-18 in mouse serum were detected by ELISA.Data showed that the model group had typical psoriatic lesions such as erythema,scale and thickening,and the Mdivi-1 group demonstrated obvious reduction of the lesions.The PASI score of the experiment group was significantly lower than that of the model group.HE staining indicated that the epidermal thickness of the back skin in the treatment group was significantly lower than that in the model group,and Munro microabscess was significantly reduced.R0S fluorescence staining indicated that ROS content in the experiment group was significantly lower than that in the model group;immunohistochemical results showed that the expression of Drp1 protein in the experiment group was significantly lower than that in the model group;Western blot results showed that the expression levels of Drp1,NLRP3 and IL-1 β in the experiment group were significantly lower than those in the model group;ELISA results indicated that the expressions of IL-17A and IL-18 in serum of mice in the experiment group were lower than those in the model group.Taken together,Mdivi-1 can reduce mitochondrial damage and ROS production by inhibiting the expression of Drp1,thereby reducing the production of NLRP3 inflammasome,down-regulating IL-1 β,IL-18 and IL-17A,and alleviating the IMQ-induced psoriasis-like skin inflammation in mice.

Objective:To investigate the effect of low-dose ketamine on neuroinflammation and microcirculation in mice with traumatic brain injury (TBI).Methods:Sixty adult male C57BL/6 mice, weighing 22-28 g, were randomly divided into sham-operated group, TBI group, Sham+ketamine group, and TBI+ketamine group ( n=15). A controlled cortical impingement (CCI) method was used to establish TBI models in the later 2 groups. Sham+ketamine group and TBI+ketamine group were intraperitoneally injected with 30 mg/kg ketamine once daily for 3 d at 30 min after TBI; sham-operated group and TBI group were intraperitoneally injected same amount of saline at the same time points. Cerebral cortical blood flow in 6 mice from each group was measured by laser speckle contrast imaging (LSCI) before, immediately after, 30 min after, 1 d after and 3 d after modeling, respectively. Three d after modeling, immunohistochemical staining and immunofluorescent double label staining were used to detect the nuclear translocation of microglia markers, ionized calcin-antibody-1 (Iba-1) and nuclear factor (NF)-κB p65 in damaged cortical brain tissues in 6 mice from each group. The remaining 3 mice in each group were sacrificed and tissue plasma was extracted 3 d after modeling; levels of NF-κB p65, phosphorylated (p)-NF-κB p65, p-IκB and inducible nitric oxide synthase (iNOS) in cortical brain tissues were detected by Western blotting. Expressions of tumor necrosis factor-α (TNF-α), interleukin-1-β (IL-1β) and interleukin-6 (IL-6), iNOS, reactive oxygen species (ROS) and reactive nitrogen species (RNS) in cortical brain tissues were detected by ELISA. Results:LSCI indicated that, 3 d after modeling, relative blood flow in local cerebral microcirculation of TBI+ketamine group was significantly increased compared with that of TBI group ( P<0.05). Immunohistochemical staining indicated that compared with the sham-operated group and Sham+ketamine group, the TBI group and TBI+ketamine group had significantly increased number of Iba-1 positive cells in the cerebral cortex ( P<0.05); compared with the TBI group, the TBI+ketamine group had significantly decreased number of Iba-1 positive cells ( P<0.05). ELISA indicated that compared with the sham-operated group and Sham+ketamine group, the TBI group and TBI+ketamine group had significantly increased expressions of TNF-α, IL-1β, IL-6, iNOS, ROS and RNS in damaged cortical brain tissues ( P<0.05); compared with the TBI group, the TBI+ ketamine group had significantly decreased expressions of TNF-α, IL-1β, IL-6, iNOS, ROS and RNS in damaged cortical brain tissues ( P<0.05). Immunofluorescent double label staining indicated obviously inhibited NF-κB p65 nuclear translocation in TBI+ketamine group when it was compared with TBI group. Western blotting indicated that compared with the sham-operated group and Sham+ketamine group, the TBI+ketamine group had significantly increased iNOS, NF-κB p65, p-NF-κB p65 and P-IκB protein expressions in damaged cortical brain tissues ( P<0.05); compared with the TBI group, the TBI+ketamine group had significantly decreased protein expressions of iNOS, NF-κB p65, p-NF-κB p65 and p-IκB in damaged cortical brain tissues ( P<0.05). Conclusion:Low-dose ketamine reduces neuroinflammation and improves cerebral microcirculatory blood flow after open TBI, whose mechanism may be related to inhibition of microglia NF-κB/iNOS pathway.

Objective To study the effects of geniposide(Gen)on the injury of human neuroblastoma cell SH-SY5Y induced by oxygen glucose deprivation/reoxygenation(OGD/R)and its mechanism.Methods SH-SY5Y cells were divided into control group,OGD/R group,low medium and high concentration groups of Gen(12.5,25,50 μmol·L-1).Except the control group,OGD/R injury models were established in other groups,and different concentrations of Gen were used for intervention.Cell proliferation activity was detected by CCK-8 assay.The levels of lactate dehydrogenase(LDH),superoxide dismutase(SOD),malonaldehyde(MDA)and glutathione(GSH)in cell supernatant were detected by biochemical method.The intracellular Fe2+ level was detected by colorimetry.The level of reactive oxygen species(ROS)in cells was detected by flow cytometry.The protein expression levels of glutathione peroxidase 4(GPX4),solute carrier family 7 member 11(SLC7A11)and transferrin receptor 1(TFR1)in cells were detected by Western blot.Results Compared with the control group,LDH release rate,MDA content,Fe2+ level,ROS level and TFR1 expression level in OGD/R group were significantly increased(P<0.05),while cell proliferation activity,SOD activity,GSH level,SLC7A11 and GPX4 expression levels were significantly decreased(P<0.05).Compared with the OGD/R group,the LDH release rate,MDA content,Fe2+ level,ROS level and TFR1 expression level of cells in each concentration group of Gen decreased significantly(P<0.05),while cell proliferation activity,SOD activity,GSH level,SLC7A11 and GPX4 expression levels increased significantly(P<0.05),especially in H-Gen group.Conclusion Gen can inhibit oxidative stress and ferroptosis of SH-SY5Y cells induced by OGD/R,and its mechanism may be related to the activation of SLC7A11/GPX4 signal pathway.

Objective The objective of this study was to establish a mouse model of allergic rhinoconjunctivitis and investigate the role of the NLRP3 signaling pathway in allergic rhinoconjunctivitis.Methods Thirty-three female C57 mice(SPF)were randomLy divided into 3 groups:the control group,the experimental group,and the NLRP3-/-group.On days 0,4,7,14,and 21,the experimental group and NLRP3-/-group received a 0.2 mL intraperitoneal injection of medicine containing OVA(100 μg)and adjuvant Al(OH)3(4 mg),respectively.After an interval of 3 days,each eye and nose were dosed with 10 μL of 5%OVA for five consecutive days a week to induce allergic symptoms.During sensitization and excitation stages,the control group was replaced with an equiva-lent amount of PBS.Ocular and nasal symptoms were observed and scored.The levels of OVA-specific IgE,IL-4,IL-17,and IL-18 in serum were measured using ELISA,while changes in palpebral conjunctiva and nasal mucosa were assessed by hematoxylin-eosin staining.The expression of NLRP3 mRNA in conjunctival tissue and nasal mucosa was determined using real-time PCR analysis.Statistical analysis was performed using SPSS17.0 software with P<0.05 considered as statistically significant difference.Results The experimental group and NLRP3-/-group exhibited induced nasal and ocular allergic symptoms.In the experimental group,the duration of nasal allergy symptoms was(10.500±1.080)days,while the duration of eye allergy symptoms was(20.300±2.058)days.In the NLRP3-/-group,the duration of nasal allergy symptoms was(13.400±1.955)days,and for eye allergy symp-toms it was(20.900±2.132)days.The duration of nasal allergies in the NLRP3-/-group significantly exceeded that in the experimental group(P<0.05),whereas there were no significant differences observed in eye allergy durations between these two groups(P>0.05).Levels of OVA-specific IgE,IL-4,and IL-17 were significantly higher in both the experimental and NLRP3-/-groups compared to those in the control group(P<0.05).Additionally,serum IL-18 content increased significantly in the experimental group when compared with both control and NLRP3-/-groups(P<0.05).Conjunctival tissue lesions as well as nasal mucosa damage were evident in both experimental and NLRP3-/-groups.mRNA expression levels of NLRP3 within conjunctival tissue and nasal mucosa from the experimental group showed a significant increase when compared to those from both control and NLRP3-/-groups(P<0.05).Conclusion Allergic rhinoconjunctivitis pathogenesis is influenced by various factors;however,the involvement of NLPR3 signaling pathway promotes its development.

Objective: To evaluate the effects of sevoflurane on microglial polarization after traumatic brain injury (TBI) in rats. Methods: Seventy-two healthy adult male Sprague-Dawley rats, weighing 230-250 g, were divided into 3 groups (n=24 each) using a random number table method: sham operation group (group Sham), group TBI, and TBI plus sevoflurane anesthesia group (group TBI+ Sevo). TBI models were established by using Feeney′s method in TBI and TBI+ Sevo groups, 30 min later 2.4% sevoflurane was inhaled for 1 h once a day for 3 consecutive days in TBI+ Sevo group, while pure oxygen was inhaled instead in Sham and TBI groups.At 1, 3, 7 and 14 days after establishing the model, 6 rats were selected, the neurological function was evaluated with the modified neurologic severity score (mNSS), and tail venous blood samples were taken for determination of tumor necrosis factor-a (TNF-α), interleukin-1beta (IL-1β) and IL-6 concentrations by enzyme-linked immunosorbent assay.The rats were then sacrificed, the limbic cortical tissues of brain contusion lesion were taken for determination of cell apoptosis (by TUNEL) and expression of microglial marker Iba-1, microglial M1 phenotypic marker CD86 and microglial M2 phenotypic marker CD206 (by Western blot). Results: Compared with group Sham, the mNSS score, apoptosis rate of cortical cells, expression of Iba-1, CD86 and CD206, and concentrations of serum TNF-α, IL-1β and IL-6 were significantly increased in TBI and TBI+ Sevo groups (P<0.05). Compared with TBI group, the mNSS score, apoptosis rate of cortical cells, expression of Iba-1 and CD86 and concentrations of serum TNF-α, IL-1β and IL-6 were significantly decreased, and the expression of CD206 was up-regulated in TBI+ Sevo group (P<0.05). Conclusion The mechanism by which sevoflurane anesthesia reduces TBI may be related to promoting microglial polarization and inhibiting systemic inflammatory response in rats.

Riedel's thyroiditis (RT) is a rare form of thyroid disease,and the rate of misdiagnosis is high due to the low incidence of RT and the lack of understanding of the disease by clinicians.There is currently no standard and guidance for the diagnosis and treatment of RT.Improving the understanding of RT is of great significance for standardizing diagnosis and treatment and improving the life quality of patients.

Objective To observe the clinical effect of tongluozhitong capsule combined with carbamazepine on the treatment of trigeminal neuralgia.Methods94 patients with trigeminal neuralgia from January 2012 to May 2014 in Dongyang people's hospital were randomized double-blindly divided into the control group and the observation group, 47 cases in each group.The control group received carbamazepine treatment, and the observation group received tongluozhitong capsule combined with carbamazepine.VAS score, the effect and adverse reaction were recorded and analyzed before treatment, atwo weeks and four weeks after.Recurrence was followed-up a half and one year after treatment.Results①VAS scores in the observation group 2 weeks and 4 weeks after treatment were (3.78±0.44), (2.01±0.23) points separately, which were lower than those in the control group (5.96±0.53), (4.02±0.38) points separately, and the differences were statistically significant (P<0.05).②The total effective rate in the observation group 4 weeks after treatment was 95.74%, which significantly higher than that in the control group 78.72%, and the difference was statistically significant (P<0.05).③ The adverse reactions in the observation group 4 weeks after treatment was 21.28%, 27.66% in the control group, the difference was not significant;④ The recurrence rate in the observation group six months and one year after treatment were 6.38% and 10.64%, which significantly lower than those in the control group 23.40% and 29.79%, and the differences were statistically significant (P<0.05).ConclusionIt can effectively relieve pain, reduce the recurrence rate, and will not increase the adverse reactions which tongluozhitong capsule combined with carbamazepine were used on the treatment of trigeminal neuralgia.It is a safe and effective treatment program.