ObjectiveTo explore the material basis of the "interaction of blood stasis and toxin" mechanism in cerebral ischemia-reperfusion injury， as well as the protective role of Huoxue Huayu Jiedu prescription （HXHYJDF） against ferroptosis. MethodsSixty SPF-grade male SD rats were randomly divided into six groups： sham group， model group， deferoxamine （DFO） group （100 mg·kg^-1）， low-dose HXHYJDF group （4.52 g·kg^-1）， medium-dose HXHYJDF group （9.04 g·kg^-1）， and high-dose HXHYJDF group （18.07 g·kg^-1）， with ten rats in each group. Except for the sham group， the other groups were used to replicate the model of focal cerebral ischemia-reperfusion in the middle cerebral artery of rats by the reforming Longa method. Neurological function was assessed at 1^st， 3^rd， 5^th， and 7^th days post-reperfusion using the modified neurological severity scores （m-NSS）. Brain tissue pathology and the morphology of mitochondria were observed using hematoxylin-eosin （HE） staining and transmission electron microscopy. The contents of malondialdehyde （MDA）， glutathione （GSH）， divalent iron ions （Fe²⁺）， and reactive oxygen species （ROS） in the ischemic cerebral tissue were detected using enzyme-linked immunosorbent assay （ELISA）. Immunohistochemistry and Western blot （WB） were used to detect the expression of iron death marker proteins glutathione peroxidase 4 （GPX4）， ferroportin-1 （FPN1）， transferrin receptor protein 1 （TfR1）， and ferritin mitochondrial （FtMt） in brain tissue. ResultsCompared with the sham group， the mNSS score of the model group was significantly increased （P<0.01）. HE staining showed that the number of neurons in the cortex of brain tissue was seriously reduced， and the intercellular space was widened. The nucleus was fragmented， and the cytoplasm was vacuolated. The results of transmission electron microscopy showed that the mitochondria in the cytoplasm contracted and rounded， and the mitochondrial cristae decreased. The matrix was lost and vacuolated， and the density of the mitochondrial bilayer membrane increased. The results of ELISA showed that the content of GSH decreased significantly （P<0.01）， and the contents of MDA， Fe²⁺， and ROS increased significantly （P<0.01）. The results of immunohistochemistry and WB showed that the expression of GPX4 and FPN1 proteins was significantly decreased （P<0.01）， and the expression of FtMt and TfR1 proteins was significantly increased （P<0.01）. Compared with those of the model group， the m-NSS scores of the high-dose and medium-dose HXHYJDF groups began to decrease on the 3^rd and 5^th days， respectively （P<0.05， P<0.01）. The results of HE and transmission electron microscopy showed that the intervention of HXHYJDF improved the pathological changes of neurons and mitochondria. The results of ELISA showed that the content of GSH in the medium-dose and high-dose HXHYJDF groups increased significantly （P<0.01）， and the contents of MDA， Fe²⁺， and ROS decreased significantly （P<0.05， P<0.01）. The content of GSH in the low-dose HXHYJDF group increased significantly （P<0.01）， and the contents of MDA and ROS decreased significantly （P<0.01）. The results of immunohistochemistry showed that the expression of GPX4 and FPN1 in the high-dose HXHYJDF group increased significantly （P<0.01）， and the expression of FtMt and TfR1 decreased significantly （P<0.01）. The expression of GPX4 and FPN1 in the medium-dose HXHYJDF group increased significantly （P<0.05）， and the expression of TfR1 decreased significantly （P<0.01）. WB results showed that the expression levels of FPN1 and GPX4 proteins in the high-dose， medium-dose， and low-dose HXHYJDF groups were significantly up-regulated （P<0.01）， and the expression levels of FtMt and TfR1 proteins were significantly down-regulated （P<0.01）. ConclusionHXHYJDF can significantly improve neurological dysfunction symptoms in rats with cerebral ischemia-reperfusion injury， improve the pathological morphology of the infarcted brain tissue， and protect the brain tissue of rats with cerebral ischemia-reperfusion injury to a certain extent. Neuronal ferroptosis is involved in cerebral ischemia-reperfusion injury， with increased levels of MDA， Fe²⁺， ROS， and TfR1 and decreased levels of FtMt， FPN1， GPX4， and GSH potentially constituting the material basis of the interaction of blood stasis and toxin mechanism in cerebral ischemia-reperfusion injury. HXHYJDF may exert brain-protective effects by regulating iron metabolism-related proteins， promoting the discharge of free iron， reducing brain iron deposition， alleviating oxidative stress， and inhibiting ferroptosis.

The rabies virus(RABV)that causes rabies mainly attacks the peripheral and central nervous systems.In the later stages of infection,it is scattered in the salivary glands and transmit-ted to other susceptible animals through infectious saliva.To study dispersion of the RABV in the three pairs of salivary gland tissues,the street strain PB4 of the RABV was inoculated into 21-day-old female mice through the hind limb muscles.During the moribund stage of the mice,the sublin-gual gland,submandibular gland and parotid gland were collected,respectively.The TCID50 titer of RABV in the three kinds of glands of the mice and the copy number of the RABV N gene were de-tected,and RABV in different salivary glands was observed by immunofluorescence.The results showed that PB4 was dispersed in all three kinds of salivary glands of the mice,with the largest a-mounts in the parotid gland,followed by the submandibular gland,and the lowest amount in the sublingual gland.Three-month-old dogs were inoculated with PB4 through the cranial cavity,and saliva were collected every 12 h after inoculation.The saliva samples were detected by TCID50 and RT-qPCR.And during the moribund stage of the dogs when the disease occurred,the three pairs of salivary glands were collected.Through the determination of the TCID50 titer,RT-qPCR and immu-nofluorescence detection,it was demonstrated that among the three different salivary glands of the dogs,the largest amount of PB4 was found in the parotid gland and the lowest in the sublingual gland.Our results in mice and dogs clearly proved that the parotid gland was consistently found to exhibit the highest content of street RABV among the three major salivary glands,which could en-rich experimental data for analyzing the dispersion of RABV in the salivary glands and interpreta-tion of the intermittent secretion of saliva in clinically rabid dogs.

The rabies virus(RABV)that causes rabies mainly attacks the peripheral and central nervous systems.In the later stages of infection,it is scattered in the salivary glands and transmit-ted to other susceptible animals through infectious saliva.To study dispersion of the RABV in the three pairs of salivary gland tissues,the street strain PB4 of the RABV was inoculated into 21-day-old female mice through the hind limb muscles.During the moribund stage of the mice,the sublin-gual gland,submandibular gland and parotid gland were collected,respectively.The TCID50 titer of RABV in the three kinds of glands of the mice and the copy number of the RABV N gene were de-tected,and RABV in different salivary glands was observed by immunofluorescence.The results showed that PB4 was dispersed in all three kinds of salivary glands of the mice,with the largest a-mounts in the parotid gland,followed by the submandibular gland,and the lowest amount in the sublingual gland.Three-month-old dogs were inoculated with PB4 through the cranial cavity,and saliva were collected every 12 h after inoculation.The saliva samples were detected by TCID50 and RT-qPCR.And during the moribund stage of the dogs when the disease occurred,the three pairs of salivary glands were collected.Through the determination of the TCID50 titer,RT-qPCR and immu-nofluorescence detection,it was demonstrated that among the three different salivary glands of the dogs,the largest amount of PB4 was found in the parotid gland and the lowest in the sublingual gland.Our results in mice and dogs clearly proved that the parotid gland was consistently found to exhibit the highest content of street RABV among the three major salivary glands,which could en-rich experimental data for analyzing the dispersion of RABV in the salivary glands and interpreta-tion of the intermittent secretion of saliva in clinically rabid dogs.

At present,some new tongue manifestations with distinct characteristics and high clinical value have not yet gained widespread consensus,which is unfavorable to their research and promotion.This paper representatively explores four types of tongue texture manifestations(liver gall line,bulging vein tongue,concave tip tongue,convex surface tongue)and two types of tongue coating manifestations(cracked tongue coating,white saliva line)among them.It is found that the liver gall line manifests as dark stagnation of various forms on the tongue surface,indicating liver qi stagnation and blood stasis as well as heat-toxin accumulation;the bulging vein tongue is characterized by clearly raised blood vessels on the tongue surface,suggesting blood stasis due to yang deficiency or cold congelation;the concave tip tongue is characterized by a depressed tip,indicating an abnormal tongue frenulum or malnutrition of heart,lung,and kidney;the convex surface tongue features a broad and thick tongue body with an overall"convex"shape,which is observed in healthy individuals or suggests qi movement disorder and spleen and stomach stagnation;the cracked tongue coating is characterized by cracks only on the tongue coating,indicating qi deficiency with disordered fluid or yin consumption due to intense heat;the white saliva line presents as strip-like white foam bands on the tongue margin,suggesting liver qi stagnation and dampness stagnancy due to spleen deficiency.No consensus exists regarding issues such as naming and description,reflecting the necessity of expanding and standardizing the theory of tongue diagnosis.This paper aims to emphasize the value and standardization of such new tongue manifestations,enrich the theoretical system of tongue diagnosis in traditional Chinese medicine,and provide novel insights and reference basis for clinical syndrome differentiation.

Depression is closely associated with a neuro-endocrine-immune(NEI)system imbalance.The"excessive vitality leading to restraint and counter-regulation(Kang Hai Cheng Zhi)"theory elucidates the self-regulating mechanism for maintaining dynamic equilibrium in the body,and serves as an importance principle guiding treatment formulation and medication selection.Based on the correlation between NEI system imbalance and the traditional Chinese medicine pathogenesis of depression,and integrating the"Kang Hai Cheng Zhi"theory,the author posits that the pathogenesis of depression lies in overactive liver invading spleen,earth dampness impeding wood′s ascendancy,and disharmony between body and mind,as well as imbalance in storage and discharge functions of liver and kidney,disharmony between Yin and Yang,and disrupted counter-regulation.This dosely aligns with two key pathological methanisms at the micro level:microglial-limbic system homeostatic imbalance and hypothalamic-pituitary-adrenal axis-inflammatory circuit dysregulation.Clinically,the treatment principle for depression adheres to supporting the counter-regulation to restrain excess,with herbal interventions using strategies such as restraining wood to support earth,dredging earth to unblock wood,and harmonizing pivotal functions,as well as nourishing water to nurture wood,warming kidney to tonify liver,and relieving depression to calm the spirit.These approaches aim to regulate the liver,spleen,and kidney,embodying the core therapeutic tenet of"striving for equilibrium,"thereby restoring the body′s self-regulating capability.

To establish a highly specific,sensitive,and efficient method for detecting antibodies a-gainst the Erns protein of classical swine fever virus(CSFV),and to distinguish CSFV vaccine strains from wild strains infections in combination with the E2 subunit vaccine.The purified Erns protein of the CSFV expressed by baculovirus was conjugated to carboxylated magnetic beads as a solid-phase carrier and horseradish peroxidase(HRP),separately.A double-antigen sandwich chemiluminescent enzyme immunoassay(CLEIA)was developed by optimizing various reaction parameters using a fully automated chemiluminescence analyzer.This method was then applied to quantitatively detect Erns protein antibodies in sera from pigs infected with prevalent strains and those immunized with the CSFV E2 subunit vaccine and challenged with field strains.The results showed that the optimal conditions for coupling protein-to-magnetic bead were as follows:coupling buffer pH of 8.0,a protein coupling amount of 2.5 mg/g,blocking solution of 10％BSA,serum sample volume of 20 μL.The optimal dilution of enzyme-labeled antigen was at 1:500 with a one-step reaction time of 15 minutes.The cutoff value of the established CLEIA method for detecting CSFV Erns protein antibodies was 5.83 U/mL and a diagnostic sensitivity of 1:128.No cross-reac-tivity was observed with positive sera against African swine fever virus,pseudorabies virus,porcine circovirus type 2,porcine epidemic diarrhea virus,porcine reproductive and respiratory syndrome virus,or porcine gastroenteritis virus.Additionally,the method yielded negative results with sera from pigs immunized with the E2 subunit vaccine.In repeatability tests,the intra-assay coefficient of variation(CV)ranged from 0.77％to 11.56％,and the inter-assay CV ranged from 10.30％to 14.55％,both below 15％.The positive and negative concordance rates with a commercial CSFV Erns protein antibody detection kit were 95.24％and 92.71％,separately,with an overall concord-ance rate of 93.23％.The double-antigen sandwich chemiluminescence method established in this study exhibits high sensitivity,excellent repeatability,and suitability for automated detection,making it applicable for serological differentiation between CSFV E2 subunit vaccination and infec-tion with prevalent strains.

To establish a highly specific,sensitive,and efficient method for detecting antibodies a-gainst the Erns protein of classical swine fever virus(CSFV),and to distinguish CSFV vaccine strains from wild strains infections in combination with the E2 subunit vaccine.The purified Erns protein of the CSFV expressed by baculovirus was conjugated to carboxylated magnetic beads as a solid-phase carrier and horseradish peroxidase(HRP),separately.A double-antigen sandwich chemiluminescent enzyme immunoassay(CLEIA)was developed by optimizing various reaction parameters using a fully automated chemiluminescence analyzer.This method was then applied to quantitatively detect Erns protein antibodies in sera from pigs infected with prevalent strains and those immunized with the CSFV E2 subunit vaccine and challenged with field strains.The results showed that the optimal conditions for coupling protein-to-magnetic bead were as follows:coupling buffer pH of 8.0,a protein coupling amount of 2.5 mg/g,blocking solution of 10％BSA,serum sample volume of 20 μL.The optimal dilution of enzyme-labeled antigen was at 1:500 with a one-step reaction time of 15 minutes.The cutoff value of the established CLEIA method for detecting CSFV Erns protein antibodies was 5.83 U/mL and a diagnostic sensitivity of 1:128.No cross-reac-tivity was observed with positive sera against African swine fever virus,pseudorabies virus,porcine circovirus type 2,porcine epidemic diarrhea virus,porcine reproductive and respiratory syndrome virus,or porcine gastroenteritis virus.Additionally,the method yielded negative results with sera from pigs immunized with the E2 subunit vaccine.In repeatability tests,the intra-assay coefficient of variation(CV)ranged from 0.77％to 11.56％,and the inter-assay CV ranged from 10.30％to 14.55％,both below 15％.The positive and negative concordance rates with a commercial CSFV Erns protein antibody detection kit were 95.24％and 92.71％,separately,with an overall concord-ance rate of 93.23％.The double-antigen sandwich chemiluminescence method established in this study exhibits high sensitivity,excellent repeatability,and suitability for automated detection,making it applicable for serological differentiation between CSFV E2 subunit vaccination and infec-tion with prevalent strains.

At present,some new tongue manifestations with distinct characteristics and high clinical value have not yet gained widespread consensus,which is unfavorable to their research and promotion.This paper representatively explores four types of tongue texture manifestations(liver gall line,bulging vein tongue,concave tip tongue,convex surface tongue)and two types of tongue coating manifestations(cracked tongue coating,white saliva line)among them.It is found that the liver gall line manifests as dark stagnation of various forms on the tongue surface,indicating liver qi stagnation and blood stasis as well as heat-toxin accumulation;the bulging vein tongue is characterized by clearly raised blood vessels on the tongue surface,suggesting blood stasis due to yang deficiency or cold congelation;the concave tip tongue is characterized by a depressed tip,indicating an abnormal tongue frenulum or malnutrition of heart,lung,and kidney;the convex surface tongue features a broad and thick tongue body with an overall"convex"shape,which is observed in healthy individuals or suggests qi movement disorder and spleen and stomach stagnation;the cracked tongue coating is characterized by cracks only on the tongue coating,indicating qi deficiency with disordered fluid or yin consumption due to intense heat;the white saliva line presents as strip-like white foam bands on the tongue margin,suggesting liver qi stagnation and dampness stagnancy due to spleen deficiency.No consensus exists regarding issues such as naming and description,reflecting the necessity of expanding and standardizing the theory of tongue diagnosis.This paper aims to emphasize the value and standardization of such new tongue manifestations,enrich the theoretical system of tongue diagnosis in traditional Chinese medicine,and provide novel insights and reference basis for clinical syndrome differentiation.

Depression is closely associated with a neuro-endocrine-immune(NEI)system imbalance.The"excessive vitality leading to restraint and counter-regulation(Kang Hai Cheng Zhi)"theory elucidates the self-regulating mechanism for maintaining dynamic equilibrium in the body,and serves as an importance principle guiding treatment formulation and medication selection.Based on the correlation between NEI system imbalance and the traditional Chinese medicine pathogenesis of depression,and integrating the"Kang Hai Cheng Zhi"theory,the author posits that the pathogenesis of depression lies in overactive liver invading spleen,earth dampness impeding wood′s ascendancy,and disharmony between body and mind,as well as imbalance in storage and discharge functions of liver and kidney,disharmony between Yin and Yang,and disrupted counter-regulation.This dosely aligns with two key pathological methanisms at the micro level:microglial-limbic system homeostatic imbalance and hypothalamic-pituitary-adrenal axis-inflammatory circuit dysregulation.Clinically,the treatment principle for depression adheres to supporting the counter-regulation to restrain excess,with herbal interventions using strategies such as restraining wood to support earth,dredging earth to unblock wood,and harmonizing pivotal functions,as well as nourishing water to nurture wood,warming kidney to tonify liver,and relieving depression to calm the spirit.These approaches aim to regulate the liver,spleen,and kidney,embodying the core therapeutic tenet of"striving for equilibrium,"thereby restoring the body′s self-regulating capability.

Objective: To explore the differences in the gut microbiota of primary school students with different levels of sugar sweetened beverage intake, so as to provide scientific evidence for better identification of health risks in children and the development of targeted health policies. Methods: In June 2022, a total of 192 healthy primary school students from Chengdu were selected using a stratified cluster random sampling method. The sugar sweetened beverage intake was assessed through a dietary frequency questionnaire. Based on the median daily sugar sweetened beverage intake, primary school students were categorized into a low intake group ( n =96) and a high intake group ( n =96). The gut microbiota in fresh fecal samples from the two groups of primary school students was analyzed using 16S rRNA high throughput sequencing, and the diversity and community structure differences in the gut microbiota were compared. Results: Children in the low intake group had a sugar sweetened beverage intake of (21.3±1.6) mL/d, while the high intake group had an intake of (269.6±37.3) mL/d. Diversity analysis results showed that there were no statistically significant differences between the low intake and the high intake group in terms of α diversity metrics: Observed_otus index [298.50 (259.75, 342.25), 305.50 (244.25, 367.75)], Goods_coverage index [1.00 (1.00, 1.00), 1.00 (1.00, 1.00)], Chao index [304.18 (260.75, 348.78), 305.88 (245.68, 370.88)], Shannon index [5.88 (5.29, 6.45), 5.71 (4.89, 6.28)] and Simpson index [0.95 (0.91, 0.97), 0.94 (0.88, 0.97)] ( Z =-0.64, -0.76, -0.54, -1.76, -1.67, P >0.05). Furthermore, no statistically significant difference was observed in β diversity between the two groups ( R 2=0.006, P >0.05). At the genus level, the abundance of Blautia [0.033 (0.018, 0.055)] and Fusicatenibacter [0.009 (0.005, 0.015)] were higher in the low intake group compared to the high intake group [0.024 (0.013, 0.041),0.006 (0.003, 0.011)]and differences were statistically significant ( Z =-2.52, -2.81, P <0.05). LEfSe analysis highlighted intergroup differences primarily in Blautia, Fusicatenibacter and Sarcina( LDA= 3.56,3.12,3.53, P <0.05). Conclusions There is no significant difference in the diversity and overall structure of the gut microbiota in primary school students with different levels of sugar sweetened beverage intake. However, there are species variations at the genus level. The information can serve as a scientific basis for identifying health risks in primary school students and formulating targeted health strategies.