Objective:To investigate the inhibitory effect of Xiaojianzhong Granule(XJZG)on food allergy(FA)and related mecha-nisms in terms of gut microbiota,zonula occluden-1(ZO-1),and Occludin.Methods:A total of 24 specific pathogen-free female BALB/c mice were randomly divided into normal group,model group,prevention group,and treatment group,with 6 mice in each group.The mice in the prevention group were given XJZG by gavage at a standard dose of 5.85 g/kg/day from 3 days before the first challenge till 4 hours before the last challenge;the mice in the treatment group were given XJZG at the double dose for 3 days based on the allergy score;the mice in the other groups were given an equal volume of distilled water by gavage.At the end of the experiment,al-lergy score and anal temperature were measured;flow cytometry was used to measure eosinophils and mast cells in mesenteric lymph nodes(MLNs);toluidine blue staining was performed for mast cells in jejunal tissue;immunohistochemistry was used to measure the expression of ZO-1 and Occludin;16S rRNA sequencing was per-formed to analyze the microbiota in the intestinal content;high-performance liquid chromatography-mass spectrometry was used to measure the content of short-chain fatty acids(SCFAs)in jejunal lavage fluid.Results:Compared with the model group,the prevention group and the treatment group had significant reductions in al-lergy score(P=0.000,P=0.000),anal temperature(P=0.002,P=0.000),the proportion of eosinophils and mast cells in MLNs(P<0.05),and mast cell infiltration in jejunal tissue(P=0.000,P=0.000).Compared with the normal group,the model group had signifi-cant increases in the relative abundances of Erysipelaceae and Turicibacter,while the prevention group and the treatment group had disappearance of Erysipelaceae and Turicibacter and an increase in the relative abundance of Porphyromonadaceae.Compared with the normal group,the model group had a significant reduction in the content of propionate in jejunal lavage fluid(P=0.014),and compared with the model group,the prevention group had a significant increase in the content of propionate in jejunal lavage fluid(P=0.024),as well as a significant increase in the treatment group(P=0.008).In the model group,the expression of ZO-1 was downregulated(P=0.010),and the expression of Occludin was significantly downregulated(P=0.002),while the expression of ZO-1 and Occludin re-turned to normal levels in the prevention group and the treatment group(P=0.001,P=0.013;P=0.025,P=0.015).Conclusion:XJZG can change the composition and abundance of gut microbiota,increase the concentration of SCFAs,upregulate the expression of ZO-1 and Occludin,promote the repair of intestinal barrier,and inhibit food allergy.

Objective To explore the mechanism of the thioredoxin-interacting protein(TXNIP)/thioredoxin-1(Trx-1)pathway in regulating the polarization of retinal microglia in rats after retinal ischemia-reperfusion(RIR)in rats,and to provide new ideas for the prevention and treatment of retinal ischemia reperfusion injury(RIRI).Methods For-ty-two healthy adult male Sprague-Dawley rats were randomly divided into a Sham group,a RIRI group and a TXNIP siRNA group.The right eye of the rats was experimented.For RIRI and TXNIP siRNA groups,RIRI models were established using the anterior chamber high intraocular pressure method.Rats in the TXNIP siRNA group were given the intravitreal injection of TXNIP siRNA 3 d before modeling.Hematoxylin-eosin(HE)staining was used to analyze retinal histopathologic changes of rats in all groups 24 h after modeling.Immunohistochemical staining of brain-specific homeobox/POU domain proteins 3A(Brn-3a)was made to count the number of retinal ganglion cells(RGCs).The dynamical changes in the number of TXNIP+cells 6 h,24 h,72 h and 7 d after modelling were analyzed through immunohistochemical staining in the RIRI group.The retinal microglia polarization and changes in the expression of TXNIP and Trx-1 proteins in each group were de-tected by double immunofluorescence staining and Western blot 24 h after modeling.Results HE staining results showed that 24 h after modelling,the retinal cells were disordered and the inner retinal layer was thickened and swelled in RIRI and TXNIP siRNA groups,compared with those in the Sham group(all P＜0.05).Immunohistochemical staining results of Brn-3a showed that 24 h after modeling,the number of Brn-3a+cells in RIRI and TXNIP siRNA groups significantly decreased,compared with that in the Sham group(both P＜0.05).The number of Brn-3a+cells in the TXNIP siRNA group was signifi-cantly higher than that in the RIRI group(P＜0.05).Immunohistochemical staining results of TXNIP at different time points after modeling showed that the expression of TXNIP+proteins started to increase 6 h after modeling.The TXNIP+protein level reached a peak at 24 h and then decreased gradually.Western blot results revealed that 24 h after modeling,RIRI and TXNIP siRNA groups had significantly higher TXNIP levels and significantly lower Trx-1 levels than the Sham group(all P＜0.05).Compared with those in the RIRI group,the expression of TXNIP proteins was significantly lower and the expression of Trx-1 proteins was significantly higher in the TXNIP siRNA group(both P＜0.05).Double immunofluores-cence staining showed that 24 h after modeling,Iba1+/CD206+cells were significantly more and Iba1+/CD16+cells were significantly less in the TXNIP siRNA group than those in the RIRI group(both P＜0.05).RIRI and TXNIP siRNA groups had significantly more Ibal+/TXNIP+cells and significantly less Iba1+/Trx-1+cells than the Sham group(both P＜0.05).The number of Iba1+/TXNIP+cells was significantly lower and the number of Iba1+/Trx-1+cells was significantly higher in the TXNIP siRNA group than those in the RIRI group(both P＜0.05).Conclusion RIR activates the TXNIP/Trx-1 path-way to induce the activation of retinal microglia and regulate the polarization of microglia,thereby resulting in RIRI in rats.

Objective:To investigate the effects of melatonin(Mel)on inflammatory damage in the retina of rats with oxygen-induced retinopathy(OIR)and the molecular mechanisms.Methods:Healthy neonatal SD rats were di-vided into the sham group(Sham),the model group(OIR),and the melatonin treatment group(OIR+Mel).The OIR model was induced by alternating 50%/10%oxygen concentration exposure for 14 d.The OIR+Mel group was in-jected intraperitoneally with 10 mg-kg-1 melatonin.Hematoxylin-eosin(HE)staining was used to observe the morpho-logical changes in the retinal tissue;immunohistochemical staining was used to detect the expression of retinal cleaved-caspase-1 and IL-1β proteins;and immunofluorescence staining was used to detect the expression of cGAS-STING-NL-RP3 signaling molecules and gasdermin(GSDMD)in the microglia of the retina.Results:HE staining results showed that compared with the Sham group,the retinal cells in the OIR group were disorganized and the thickness of the inner retina was significantly thinner,and the retinal cells in the OIR+Mel group were more neatly arranged compared with those in the OIR group(P<0.05).Immunohistochemical staining results showed that the number of cleaved-caspase-1+and IL-1β+cells in the retina of rats in the OIR group increased significantly compared with that in the Sham group,and the number of cleaved-caspase-1+and IL-1β+cells in the retina of rats in the OIR+Mel group decreased signifi-cantly compared with that of the OIR group(P<0.05).Immunofluorescence staining results showed that the number of cGAS+,STING+and NLRP3+cells in the retina of rats in the OIR group increased significantly compared with that in the Sham group,and the number of cGAS+,STING+and NLRP3+cells in the retina of rats in the OIR+Mel group de-creased significantly compared with that in the OIR group(P<0.05);The number of Iba-1+/N-GSDMD+cells in-creased significantly in the OIR group compared with the Sham group,whereas the number of Iba-1+/N-GSDMD+cells in the OIR+Mel group was significantly less than that in the OIR group,but still more than that in the Sham group(P<0.05).Conclusion:Mel inhibits the pyroptosis of retinal microglia,thus attenuates retinal inflammatory injury in OIR rats,and its mechanism may be related to the cGAS-STING-NLRP3 signaling pathway.

Objective:To investigate the effects of melatonin(Mel)on inflammatory damage in the retina of rats with oxygen-induced retinopathy(OIR)and the molecular mechanisms.Methods:Healthy neonatal SD rats were di-vided into the sham group(Sham),the model group(OIR),and the melatonin treatment group(OIR+Mel).The OIR model was induced by alternating 50%/10%oxygen concentration exposure for 14 d.The OIR+Mel group was in-jected intraperitoneally with 10 mg-kg-1 melatonin.Hematoxylin-eosin(HE)staining was used to observe the morpho-logical changes in the retinal tissue;immunohistochemical staining was used to detect the expression of retinal cleaved-caspase-1 and IL-1β proteins;and immunofluorescence staining was used to detect the expression of cGAS-STING-NL-RP3 signaling molecules and gasdermin(GSDMD)in the microglia of the retina.Results:HE staining results showed that compared with the Sham group,the retinal cells in the OIR group were disorganized and the thickness of the inner retina was significantly thinner,and the retinal cells in the OIR+Mel group were more neatly arranged compared with those in the OIR group(P<0.05).Immunohistochemical staining results showed that the number of cleaved-caspase-1+and IL-1β+cells in the retina of rats in the OIR group increased significantly compared with that in the Sham group,and the number of cleaved-caspase-1+and IL-1β+cells in the retina of rats in the OIR+Mel group decreased signifi-cantly compared with that of the OIR group(P<0.05).Immunofluorescence staining results showed that the number of cGAS+,STING+and NLRP3+cells in the retina of rats in the OIR group increased significantly compared with that in the Sham group,and the number of cGAS+,STING+and NLRP3+cells in the retina of rats in the OIR+Mel group de-creased significantly compared with that in the OIR group(P<0.05);The number of Iba-1+/N-GSDMD+cells in-creased significantly in the OIR group compared with the Sham group,whereas the number of Iba-1+/N-GSDMD+cells in the OIR+Mel group was significantly less than that in the OIR group,but still more than that in the Sham group(P<0.05).Conclusion:Mel inhibits the pyroptosis of retinal microglia,thus attenuates retinal inflammatory injury in OIR rats,and its mechanism may be related to the cGAS-STING-NLRP3 signaling pathway.

Objective To explore the mechanism of the thioredoxin-interacting protein(TXNIP)/thioredoxin-1(Trx-1)pathway in regulating the polarization of retinal microglia in rats after retinal ischemia-reperfusion(RIR)in rats,and to provide new ideas for the prevention and treatment of retinal ischemia reperfusion injury(RIRI).Methods For-ty-two healthy adult male Sprague-Dawley rats were randomly divided into a Sham group,a RIRI group and a TXNIP siRNA group.The right eye of the rats was experimented.For RIRI and TXNIP siRNA groups,RIRI models were established using the anterior chamber high intraocular pressure method.Rats in the TXNIP siRNA group were given the intravitreal injection of TXNIP siRNA 3 d before modeling.Hematoxylin-eosin(HE)staining was used to analyze retinal histopathologic changes of rats in all groups 24 h after modeling.Immunohistochemical staining of brain-specific homeobox/POU domain proteins 3A(Brn-3a)was made to count the number of retinal ganglion cells(RGCs).The dynamical changes in the number of TXNIP+cells 6 h,24 h,72 h and 7 d after modelling were analyzed through immunohistochemical staining in the RIRI group.The retinal microglia polarization and changes in the expression of TXNIP and Trx-1 proteins in each group were de-tected by double immunofluorescence staining and Western blot 24 h after modeling.Results HE staining results showed that 24 h after modelling,the retinal cells were disordered and the inner retinal layer was thickened and swelled in RIRI and TXNIP siRNA groups,compared with those in the Sham group(all P＜0.05).Immunohistochemical staining results of Brn-3a showed that 24 h after modeling,the number of Brn-3a+cells in RIRI and TXNIP siRNA groups significantly decreased,compared with that in the Sham group(both P＜0.05).The number of Brn-3a+cells in the TXNIP siRNA group was signifi-cantly higher than that in the RIRI group(P＜0.05).Immunohistochemical staining results of TXNIP at different time points after modeling showed that the expression of TXNIP+proteins started to increase 6 h after modeling.The TXNIP+protein level reached a peak at 24 h and then decreased gradually.Western blot results revealed that 24 h after modeling,RIRI and TXNIP siRNA groups had significantly higher TXNIP levels and significantly lower Trx-1 levels than the Sham group(all P＜0.05).Compared with those in the RIRI group,the expression of TXNIP proteins was significantly lower and the expression of Trx-1 proteins was significantly higher in the TXNIP siRNA group(both P＜0.05).Double immunofluores-cence staining showed that 24 h after modeling,Iba1+/CD206+cells were significantly more and Iba1+/CD16+cells were significantly less in the TXNIP siRNA group than those in the RIRI group(both P＜0.05).RIRI and TXNIP siRNA groups had significantly more Ibal+/TXNIP+cells and significantly less Iba1+/Trx-1+cells than the Sham group(both P＜0.05).The number of Iba1+/TXNIP+cells was significantly lower and the number of Iba1+/Trx-1+cells was significantly higher in the TXNIP siRNA group than those in the RIRI group(both P＜0.05).Conclusion RIR activates the TXNIP/Trx-1 path-way to induce the activation of retinal microglia and regulate the polarization of microglia,thereby resulting in RIRI in rats.

Objective:Cerebral ischemia reperfusion injury(CIRI)can cause damage to distant organs,such as the gastrointestinal tract,through the gut-brain axis.Melatonin is known to play a neuroprotective role by activating specific receptor pathways.However,the changes of melatonin receptors in the gastrointestinal tract after brain injury and their relationship with intestinal injury are still unclear.Methods:Twenty-four male Sprague-Dawley rats were randomly di-vided into the Sham group and CIRI group.The CIRI model was prepared by Zea-Longa method.The jejunum,ileum,and colon tissues of the rats were collected 2 h after ischemia and 24 h after reperfusion.The damage of intestinal and brain tissues was observed by using 2,3,5-triphenyl tetrazolium chloride(TTC)and HE staining.The positive expres-sion of zonula occludens-1(ZO-1)and Claudin5 was observed by immunofluorescence staining.The melatonin receptor 1(MT1)and melatonin receptor 2(MT2)expression was detected by reverse transcription-quantitative polymerase chain reaction(RT-qPCR),immunofluorescence staining and Western Blot.The correlation between the melatonin receptors and intestinal tight junction proteins was analyzed by general linear regression.Results:TTC staining showed that the infarct size of rats in the CIRI group was significantly higher than that in the Sham group.HE staining showed that intestinal villi was broken and shortened,and goblet cells were reduced in the CIRI group.The results of immuno-fluorescence staining revealed that the positive expression of ZO-1 and Claudin5 in the intestinal tissues of rats in the CIRI group was significantly lower than that in the Sham group(P＜0.05).Compared with the Sham group,the RT-qPCR revealed a significantly lower expression of MT1 and MT2 mRNA in the CIRI group(P＜0.05),and the decrease in colon tissue was the most obvious.The results of immunofluorescence staining and WB also showed that the expression of MTl and MT2 in the CIRI group was significantly lower than that in the Sham group(P＜0.05).A gen-eral linear regression analysis revealed the difference in mean fluorescence intensities of MT1+and MT2+between the Sham group and the CIRI group were positively correlated with the difference of ZO-1+and Claudin5+between the two groups.Conclusion:After CIRI,the expression of both MT1 and MT2 receptors in various intestinal segments was de-creased,and the decrease in colon tissue was the most significant.It is suggested that the poor recovery of stroke may be related to the decrease of melatonin receptor.

Objective:Using T2-weighted imaging(T2WI),diffusion-weighted imaging(DWI),pulsed gradient spin echo(PGSE)multiparametric magnetic resonance imaging combined with various histopathological techniques to observe the effects of reperfusion after different times of cerebral ischemia(CIRI)on the cytotoxic edema of brain tis-sues in rats.This provides a solid foundation for the establishment of ischemic stroke models and clinical diagnosis and treatment.Methods:Healthy adult male Sprague Dawley rats were randomly allocated to four groups:the sham-opera-ted(Sham)group,the ischaemia 60 min reperfusion(IR-60 min)group,the IR-120 min group,and the IR-180 min group.CIRI rat model was prepared by modified Zea Longa method.Laser doppler flowmetry combined with hematoxy-lin-eosin(HE)staining was employed to identify the model.Multiparametric magnetic resonance imaging,combined with water content of brain,immunofluorescence staining of aquaporin-4(AQP4),and Western Blot were performed to observe cytotoxic edema of brain tissues and tumour necrosis factor alpha(TNF-α),interleukin 1β(IL-1β)in brain tissue of rats in each group.Results:HE staining and determination of brain tissue water content prove that as ischemia time prolongs,the degree of cerebral cortex edema on the ischemic side increases.The T2WI results showed that the injury in the IR-60 min group began to affect the cerebral cortex,the injury in the IR-120 min group had completely affected the cerebral cortex,and the injury in the IR-180 min group was the most severe(P＜0.05),with a significant shift of the midline of the brain towards the opposite side.The relative apparent diffusion coefficient and water exchange time of the cerebral cortex on the ischemic side of the rats were found to be significantly lower than those of the sham group,with an declining trend(P＜0.05).Additionally,the values of cell membrane permeability of the cerebral cor-tex on the ischemic side of the rats were observed to be significantly higher than those of the sham group in all groups(P＜0.05).The expression of AQP4,IL-1 β and TNF-α in the cerebral cortex of ischemic side was significantly higher than that in the sham group,and showed an upward trend(P＜0.05).Conclusion:Reperfusion following 60,120 and,180 minutes of middle cerebral artery ischemia in rats can result in brain damage.Prolongation of ischemia time has been shown to exacerbate the toxic edema of brain cells and brain damage.Ischemia of 120 minutes reperfusion has been identified as the optimal modeling time for ischemic stroke.Multiparametric MRI can be utilized to monitor the mi-crostructural changes of brain tissue in rats following CIRI in vivo,providing a foundation for the visualization of early clinical diagnosis and treatment.

Objective:The neurovascular unit(NVU)damage in rats with cerebral ischemia-reperfusion injury(CI-RI)was evaluated based on multimodal MRI combined with multiple histological techniques to provide visual evidence for clinical assessment of CIRI.Methods:28 healthy male Sprague-Dawley rats were randomly divided into sham-oper-ated(Sham)group and CIRI group.At 24 h after modeling,rats underwent head T2-weighted imaging,diffusion-weighted imaging,water extraction with phase-contrast arterial spin labeling magnetic resonance imaging(MRI),and cerebral blood bleeding oxygen monitoring.Hematoxylin-eosin(HE)staining was used to observe the morphological changes of nerve cells in cerebral cortex.Immunofluorescence staining and transmission electron microscopy were used to observe the changes of NVU after CIRI.The endothelial tight junction proteins(claudin-5 and ZO-1)and matrix metalloproteinase-9(MMP-9)were detected by immunohistochemical staining and Western Blot analysis,to evaluate blood-brain-barrier(BBB)disruption in CIRI rats.Results:The MRI results showed that the brain tissue of the CIRI group was edematous,and the blood perfusion significantly decreased and the BBB permeability significantly increased compared with that of the Sham group(P＜0.01).The results of cerebral blood hemoglobin oxygen monitoring showed that the hemoglobin oxygen saturation of the cerebral cortex on the injured side was significantly lower than that of the Sham group(P＜0.05).HE staining showed the neuronal cells of the injured cerebral cortex were disordered and the nuclei shrank.Immunofluorescence staining results showed the number of NeuN and CD31 positive cells in the cerebral cortex of the injured side was significantly lower than that in the Sham group(P＜0.01),and the number of TUNEL positive cells in the cerebral cortex and striatum was significantly higher than that in the Sham group(P＜0.05).Transmission electron microscopy revealed the tight junctions of cerebral cortical endothelial cells were damaged,neuro-nal nucleus contraction,and astrocyte body edema.Immunohistochemical staining showed that the number of claudin-5 and ZO-1 positive cells in the cerebral cortex on the injured side was significantly lower than that in the Sham group,and the expression of MMP-9 was significantly higher than that in the Sham group(P＜0.01).Conclusion:CIRI re-sults in NVU injury,BBB disruption,and exacerbation of brain damage in rats.Multimodal MRI combined with various histopathological techniques effectively assesses NVU injury after CIRI.

Objective:Using T2-weighted imaging(T2WI),diffusion-weighted imaging(DWI),pulsed gradient spin echo(PGSE)multiparametric magnetic resonance imaging combined with various histopathological techniques to observe the effects of reperfusion after different times of cerebral ischemia(CIRI)on the cytotoxic edema of brain tis-sues in rats.This provides a solid foundation for the establishment of ischemic stroke models and clinical diagnosis and treatment.Methods:Healthy adult male Sprague Dawley rats were randomly allocated to four groups:the sham-opera-ted(Sham)group,the ischaemia 60 min reperfusion(IR-60 min)group,the IR-120 min group,and the IR-180 min group.CIRI rat model was prepared by modified Zea Longa method.Laser doppler flowmetry combined with hematoxy-lin-eosin(HE)staining was employed to identify the model.Multiparametric magnetic resonance imaging,combined with water content of brain,immunofluorescence staining of aquaporin-4(AQP4),and Western Blot were performed to observe cytotoxic edema of brain tissues and tumour necrosis factor alpha(TNF-α),interleukin 1β(IL-1β)in brain tissue of rats in each group.Results:HE staining and determination of brain tissue water content prove that as ischemia time prolongs,the degree of cerebral cortex edema on the ischemic side increases.The T2WI results showed that the injury in the IR-60 min group began to affect the cerebral cortex,the injury in the IR-120 min group had completely affected the cerebral cortex,and the injury in the IR-180 min group was the most severe(P＜0.05),with a significant shift of the midline of the brain towards the opposite side.The relative apparent diffusion coefficient and water exchange time of the cerebral cortex on the ischemic side of the rats were found to be significantly lower than those of the sham group,with an declining trend(P＜0.05).Additionally,the values of cell membrane permeability of the cerebral cor-tex on the ischemic side of the rats were observed to be significantly higher than those of the sham group in all groups(P＜0.05).The expression of AQP4,IL-1 β and TNF-α in the cerebral cortex of ischemic side was significantly higher than that in the sham group,and showed an upward trend(P＜0.05).Conclusion:Reperfusion following 60,120 and,180 minutes of middle cerebral artery ischemia in rats can result in brain damage.Prolongation of ischemia time has been shown to exacerbate the toxic edema of brain cells and brain damage.Ischemia of 120 minutes reperfusion has been identified as the optimal modeling time for ischemic stroke.Multiparametric MRI can be utilized to monitor the mi-crostructural changes of brain tissue in rats following CIRI in vivo,providing a foundation for the visualization of early clinical diagnosis and treatment.

Objective:The neurovascular unit(NVU)damage in rats with cerebral ischemia-reperfusion injury(CI-RI)was evaluated based on multimodal MRI combined with multiple histological techniques to provide visual evidence for clinical assessment of CIRI.Methods:28 healthy male Sprague-Dawley rats were randomly divided into sham-oper-ated(Sham)group and CIRI group.At 24 h after modeling,rats underwent head T2-weighted imaging,diffusion-weighted imaging,water extraction with phase-contrast arterial spin labeling magnetic resonance imaging(MRI),and cerebral blood bleeding oxygen monitoring.Hematoxylin-eosin(HE)staining was used to observe the morphological changes of nerve cells in cerebral cortex.Immunofluorescence staining and transmission electron microscopy were used to observe the changes of NVU after CIRI.The endothelial tight junction proteins(claudin-5 and ZO-1)and matrix metalloproteinase-9(MMP-9)were detected by immunohistochemical staining and Western Blot analysis,to evaluate blood-brain-barrier(BBB)disruption in CIRI rats.Results:The MRI results showed that the brain tissue of the CIRI group was edematous,and the blood perfusion significantly decreased and the BBB permeability significantly increased compared with that of the Sham group(P＜0.01).The results of cerebral blood hemoglobin oxygen monitoring showed that the hemoglobin oxygen saturation of the cerebral cortex on the injured side was significantly lower than that of the Sham group(P＜0.05).HE staining showed the neuronal cells of the injured cerebral cortex were disordered and the nuclei shrank.Immunofluorescence staining results showed the number of NeuN and CD31 positive cells in the cerebral cortex of the injured side was significantly lower than that in the Sham group(P＜0.01),and the number of TUNEL positive cells in the cerebral cortex and striatum was significantly higher than that in the Sham group(P＜0.05).Transmission electron microscopy revealed the tight junctions of cerebral cortical endothelial cells were damaged,neuro-nal nucleus contraction,and astrocyte body edema.Immunohistochemical staining showed that the number of claudin-5 and ZO-1 positive cells in the cerebral cortex on the injured side was significantly lower than that in the Sham group,and the expression of MMP-9 was significantly higher than that in the Sham group(P＜0.01).Conclusion:CIRI re-sults in NVU injury,BBB disruption,and exacerbation of brain damage in rats.Multimodal MRI combined with various histopathological techniques effectively assesses NVU injury after CIRI.