ObjectiveTo observe the effects of Dihuang Yinzi （DY） on motor dysfunction in rats with advanced Parkinson's disease （PD） and to investigate the mechanisms by which DY improves advanced PD symptoms through the "gut microbiota-short-chain fatty acids （SCFAs）-inflammation-neuroprotection pathway". MethodsAn advanced PD rat model was induced by rotenone. Rats were divided into a normal group， model group， positive drug group （levodopa， 50 mg·kg^-1）， and DY low-， medium-， and high-dose groups （5.2， 10.4， 20.8 g·kg^-1）. After 7 days of administration， motor function was evaluated using the open-field， pole-climbing， and inclined plate tests. Hematoxylin-eosin （HE） staining was used to observe pathological changes in the substantia nigra and colon， and immunohistochemistry was performed to detect α-Synuclein （α-Syn） and tyrosine hydroxylase （TH） expression in the substantia nigra. Enzyme-linked immunosorbent assay （ELISA） was used to measure levels of dopamine （DA）， 5-hydroxytryptamine （5-HT）， 3，4-dihydroxyphenylacetic acid （DOPAC）， Levodopa， homovanillic acid （HVA）， tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， and interleukin-1β （IL-1β）. Western blot analysis was used to detect the expression of zonula occludens-1 （ZO-1） and occludin. Gut microbiota diversity was analyzed by 16S rRNA sequencing， and gas chromatography （GC） was used to determine the content of SCFAs in colonic contents. ResultsCompared with the normal group， the model group showed significantly decreased movement speed and distance in the open-field test， prolonged pole-climbing time， and reduced retention angle on the inclined plate （P<0.01）， accompanied by increased α-Syn expression （P<0.01） and decreased TH expression （P<0.01） in the brain. Compared with the model group， all DY dose groups improved motor dysfunction in advanced PD rats to varying degrees （P<0.05， P<0.01） and alleviated pathological damage in the brain and colon. High-dose DY significantly reduced α-Syn aggregation in the substantia nigra （P<0.01） and increased TH expression （P<0.01）. ELISA and Western blot results showed that， compared with the normal group， the model group exhibited decreased levels of DA， 5-HT， DOPAC， Levodopa， and HVA in the striatum （P<0.01）， increased levels of TNF-α， IL-6， and IL-1β in the colon and striatum （P<0.01）， and significantly reduced expression of ZO-1 （P<0.05） and occludin in the colon （P<0.01）. Compared with the model group， all DY dose groups increased the levels of DA， 5-HT， DOPAC， Levodopa， and HVA in the striatum to varying degrees （P<0.05， P<0.01）. In the high-dose DY group， the levels of TNF-α， IL-6， and IL-1β in the colon and striatum were reduced （P<0.01）， while the expression of ZO-1 （P<0.05） and occludin in the intestine was increased. The 16S rRNA sequencing results indicated that the relative abundances of Actinobacteriota， Enterobacteriaceae， and Erysipelotrichaceae were increased in the model group， whereas the relative abundances of Bacteroidota， class Clostridia， Lachnospiraceae， and Akkermansia muciniphila were decreased. These changes were effectively reversed after high-dose DY intervention. GC analysis showed that the content of SCFAs in the colonic contents of rats in the model group was decreased （P<0.05， P<0.01）， while after high-dose DY intervention， the levels of acetate， propionate， isobutyrate， and butyrate were significantly increased （P<0.05， P<0.01）. ConclusionDY may exert therapeutic effects in advanced PD by regulating the gut microbiota-SCFAs-inflammation pathway.

ObjectiveTo observe the intervention effect of Fufang Kangjiaolv capsules on anxiety-like behaviors in the rat model of chronic restraint stress （CRS） and explore the mechanism underlying the anti-anxiety effect via the microbiota-gut-brain axis. MethodsRats were assigned into blank， model， positive drug （diazepam， 1 mg·kg^-1）， and low-， medium-， and high-dose （0.75， 1.5， 3 g·kg^-1， respectively） Fufang Kangjiaolv capsules groups. After 14 days of administration， the elevated plus maze test， open field test， light and dark box test， and marble burying test were performed. Hematoxylin-eosin staining was employed to observe the pathological changes in the hippocampus and colon of rats， and Nissl staining was conducted to observe the damage of hippocampal neurons. The gut microbiota was analyzed by 16S rRNA gene sequencing. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was employed to determine the mRNA levels of zonula occludens-1 （ZO-1） and occludin in the colon of rats. The levels of tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， and interleukin-1β （IL-1β） in the colon， serum， and hippocampus were determined by enzyme-linked immunosorbent assay. Western blot was employed to determine the protein levels of ZO-1， occludin， nuclear factor-κB p65 （NF-κB p65） in the colon tissue and NF-κB p65 and brain-derived neurotrophic factor （BDNF） in the hippocampal tissue. ResultsCompared with the blank group， the model group showed reductions in the time and frequency ratio of rats entering the elevated plus maze， the time and frequency of rats entering the central area of the open field， the time of entering the open box， the times of passing through the light and dark box， and the number of unburied beads （P<0.05， P<0.01）. Compared with the model group， Fufang Kangjiaolv capsules ameliorated the anxiety of the model rats to varying degrees， and the high-dose group had the best effect， with increases in the proportions of time and frequency of rats entering the open arm in the elevated plus maze （P<0.05）， the number of rats entering the central area in the open field （P<0.05）， the time of entering the open box， the times of passing through the light and dark boxes， and the number of unburied beads （P<0.01）. Moreover， the high-dose group showed alleviated pathological damage of hippocampal neurons and colon. The results of 16S rRNA gene sequencing showed that the model group had increased relative abundance of Firmicutes， Deferribacterota， Romboutsia， and Phascolarctobacterium， while it had decreased relative abundance of Bavcteroidota and Lactobacillus. The drug administration groups showed increased relative abundance of Bavcteroidota， Bacteroides， norank f norank o Clostridia UCG-014， and Blautia and decreased relative abundance of Firmicutes and Deferribacterota. Compared with the blank group， the model group showed down-regulated protein and mRNA levels of ZO-1 and occludin in the colon （P<0.01）， elevated levels of TNF-α， IL-6， and IL-β in the colon， serum， and hippocampus （P<0.01）， up-regulated protein level of NF-κB p65 in the colon and hippocampus （P<0.01）， and down-regulated protein level of BDNF in the hippocampus （P<0.05）. Compared with the model group， high-dose Fufang Kangjiaolv capsules up-regulated the mRNA levels of ZO-1 and occludin in the colon （P<0.01）， lowered the levels of TNF-α， IL-6， and IL-β in the colon， serum， and hippocampus （P<0.01）， up-regulated the protein levels of ZO-1 （P<0.01） and occludin （P<0.05） in the colon， down-regulated the protein level of NF-κB p65 in the colon and hippocampus （P<0.05）， and up-regulated the protein level of BDNF in the hippocampus. ConclusionFufang Kangjiaolv capsules can reduce the anxiety-like behaviors in the rat model of CRS by regulating the gut microbiota disturbance， up-regulating the expression of tight junction proteins in the colon， repairing intestinal mucosal mechanical barrier， and down-regulating NF-κB/BDNF signaling pathway， thereby reducing peripheral and central inflammation. This study proves the hypothesis that Fufang Kangjiaolv capsules play an anti-anxiety role via the microbiota-gut-brain axis， providing a new idea for further research.

ObjectiveMicrotube associated protein-2 （MAP-2）， alpha-tubulin （α-tubulin）， and synaptophysin （SYP） are important proteins in neuronal signal communication. This paper observed the effects of modified Zhigancao Tang on the expression of serum α-Synuclein （α-Syn） and its oligomers， MAP-2， α-tubulin， and SYP of patients with liver and kidney deficiency of Parkinson's disease （PD）， analyzed their correlation， and evaluated the therapeutic effect of modified Zhigancao Tang in patients with liver and kidney deficiency of PD based on α-Syn transmission pathway mediated by neuronal communication in vivo. MethodsA total of 60 patients with PD who met the inclusion criteria were randomly divided into a treatment group （30 cases） and a control group （30 cases）. Both groups were treated on the basis of PD medicine， and the treatment group was treated with modified Zhigancao Tang. Both groups were treated for 12 weeks. The changes in UPDRS score， TCM syndrome score， and expression of serum α-Syn and its oligomers， MAP-2， α-tubulin， and SYP were observed before and after 12 weeks of treatment in each group. The correlation between the above-mentioned serum biological indexes and the levels of serum α-Syn and its oligomers was analyzed. ResultsAfter treatment， the TCM syndrome score， UPDRS score， UPDRS-Ⅱ score， and UPDRS-Ⅲ score of the treatment group were significantly decreased （P<0.05， P<0.01）. The UPDRS score， UPDRS-Ⅱ score， and UPDRS-Ⅲ scores in the treatment group were significantly decreased compared with those in the control group after treatment （P<0.05）. After treatment， the total effective rate of the control group was 63.3% （19/30）， and that of the treatment group was 86.7% （26/30）. The clinical effect of the observation group was better than the control group （Z=-2.03， P<0.05）. The total effective rate of the observation group was better than that of the control group， and the difference was statistically significant （χ²=5.136， P<0.05）. After treatment， the oligomer level of serum α-Syn and MAP-2 level in the treatment group were significantly decreased （P<0.05， P<0.01）. The levels of serum α-Syn and its oligomers， as well as α-tubulin in the treatment group， were significantly decreased compared with those in the control group after treatment （P<0.05， P<0.01）. Serum α-Syn was correlated with serum MAP-2 and α-Syn oligomer in patients with PD （P<0.05， P<0.01） but not correlated with serum SYP . Serum α-Syn oligomers of patients with PD were correlated with serum MAP-2 and α-tubulin （P<0.05， P<0.01） but not correlated with serum SYP level. Serum SYP of patients with PD was correlated with serum MAP-2 （P<0.05）. ConclusionModified Zhigancao Tang has a therapeutic effect on patients with liver and kidney deficiency of PD by inhibiting the production of α-Syn oligomers and intervening α-Syn microtubule transport pathway in vivo.

Objective To analyze the role and mechanism of Xiaoqinglong decoction in alleviating the pathological process of pulmonary arterial hypertension(PAH),and to observe the effect of Xiaoqinglong decoction on endothelial-mesenchymal transition(EMT)in human pulmonary arterial endothelial cell(HPAEC)and the involvement of the Toll-like receptor/nuclear factor-κB/hypoxia-inducible factor-1α(TLR/NF-κB/HIF-1α)pathway in this mechanism.Methods Thirty-six male Sprague Dawley(SD)rats and HPAEC were randomly divided into control group,model group,Xiaoqinglong decoction plus Earthworm group,Bosentan tablet group,dimethyl sulfoxide(DMSO)group,and monophosphoryl lipid A(MPL)group.PAH rat models and HPAEC models were established by hypoxic exposure.The Xiaoqinglong decoction plus Earthworm group received intragastric administration Xiaoqinglong decoction plus Earthworm(5 g·kg-1·d-1)or cultured with 10%corresponding drug serum,the Bosentan group received Bosentan(100 mg·kg-1·d-1)by gavage or cultured with 10%corresponding drug serum,the MPL group received 1 μg MPL,and the DMSO group received an equivalent volume of the DMSO and corn oil mixed solvent as a negative control for the MPL group.The hemodynamic parameters,including mean pulmonary arterial pressure(mPAP),right ventricular systolic pressure(RVSP),and the maximum rate of right ventricular pressure(+dp/dt max),were measured via right heart catheterization.After euthanasia,lung and heart tissues were collected to assess the right ventricular hypertrophy index(RVHI);hematoxylin-eosin(HE)staining was used to observe the degree of right ventricular cardiomyocyte hypertrophy and to calculate the average intima-media thickness(IMT)in small pulmonary arteries;Western blotting was used to detect the protein expression levels of proliferating cell nuclear antigen(PCNA),CD68,TLR4,NF-κB,HIF-1α,vascular endothelial cadherin,and vimentin;cell counting kit-8(CCK-8),Transwell,and scratch assays were used to observe cell proliferation and migration;Enzyme-linked immunosorbent assay(ELISA)was used to measure the levels of interleukins(IL-8,IL-6),tumor necrosis factor-α(TNF-α),endothelin-1(ET-1),and nitric oxide(NO).Results Compared with the model group,the Xiaoqinglong decoction plus Earthworm group showed significant reductions in mPAP,RVSP,RVHI,and IMT in PAH rats[mPAP(mmHg,1 mmHg≈0.133 kPa):22.17±1.94 vs.42.00±4.90,RVSP(mmHg):34.67±3.20 vs.52.83±3.76,RVHI:0.402±0.057 vs.0.822±0.101,IMT:(37.85±2.49)%vs.(62.06±4.52)%,all P<0.05],and a significant increase in+dP/dT max(mmHg/s:2 730.83±137.89 vs.1 718.33±148.36,P<0.05).Western blotting and ELISA results showed that compared with the model group,the Xiaoqinglong decoction plus Earthworm group had significantly lower protein expression of PCNA and CD68 in lung tissue,and levels of inflammatory factors(IL-6,IL-8,TNF-α)in rat serum[lung tissue:PCNA protein expression(PCNA/GAPDH)was 1.56±0.08 vs.2.20±0.26,CD68 protein expression(CD68/GAPDH):1.46±0.09 vs.2.60±0.23;serum:IL-8(ng/L)was 39.67±6.28 vs.149.17±7.49,IL-6(ng/L):81.00±6.63 vs.211.00±25.31,TNF-α(ng/L):213.17±24.86 vs.799.50±43.51,all P<0.05].In vitro experiments,compared with the model group,Xiaoqinglong decoction plus Earthworm inhibited abnormal proliferation(A value:2.052±0.087 vs.2.242±0.057,P<0.05)and migration[number of migrating cells(per field):101.33±12.01 vs.226.67±17.56,P<0.05]of HPAEC,and reversed the EMT process,manifested as upregulation of vascular endothelial cadherin protein expression levels(vascular endothelial cadherin/GAPDH:0.39±0.06 vs.0.12±0.03,P<0.05)and downregulation of vimentin protein expression(vimentin/GAPDH:4.96±0.33 vs.7.89±0.44,P<0.05).Western blotting results indicated that compared with the model group,the protein expression levels of TLR4,the ratio of phosphorylated p65 to total p65,and HIF-1α in both lung tissue and HPAEC were significantly reduced in the Xiaoqinglong decoction plus Earthworm group[lung tissue:TLR4 protein expression(TLR4/GAPDH)was 3.13±0.20 vs.4.38±0.30,p-p65/p65 ratio:7.11±0.81 vs.12.73±1.80,HIF-1α protein expression(HIF-1α/GAPDH):2.37±0.32 vs.4.45±0.34;HPAEC:TLR4 protein expression(TLR4/GAPDH)was 1.42±0.03 vs.2.43±0.05,p-p65/p65 ratio:6.01±1.84 vs.11.28±1.06,HIF-1α protein expression(HIF-1α/GAPDH)was 3.24±0.17 vs.5.50±0.44,all P<0.05],accompanied by upregulated vascular endothelial cadherin protein expression(vascular endothelial cadherin/GAPDH:0.66±0.03 vs.0.49±0.03,P<0.05)and downregulated vimentin protein expression(vimentin/GAPDH:1.81±0.12 vs.2.47±0.10,P<0.05),indicating that Xiaoqinglong decoction plus Earthworm inhibits the EMT process in endothelial cells by suppressing the activation of the TLR/NF-κB/HIF-1α pathway.Experiments with a TLR agonist further confirmed that activation of the TLR pathway reverses the protective effects of Xiaoqinglong decoction plus Earthworm,as shown by the MPL group compared to the DMSO group having significantly increased protein expression of the p-p65/p65 ratio and HIF-1α[p-p65/p65 ratio:2.17±0.35 vs.1.08±0.14,HIF-1α/GAPDH:3.96±0.25 vs.1.03±0.10,both P<0.05],further decreased vascular endothelial cadherin protein expression(vascular endothelial cadherin/GAPDH:0.66±0.04 vs.0.99±0.02,P<0.05),further increased vimentin protein expression(vimentin/GAPDH:1.53±0.12 vs.0.93±0.07,P<0.05),along with enhanced cell migration capacity[number of migrating cells(per field):176.67±17.50 vs.107.00±11.14;cell migration rate in scratch assay:(34.32±2.82)%vs.(22.71±2.49)%,both P<0.05]and increased proliferation activity(48 hours A value:2.156±0.044 vs.1.810±0.088,P<0.05).Conclusions Xiaoqinglong decoction combined with Pheretima not only significantly reduces pulmonary artery pressure,improves cardiac function and mitigates pulmonary vascular fibrosis in PAH rats,but also alleviates pulmonary vascular remodeling by inhibiting inflammatory responses and EMT.It can further decrease the content of ET-1,increase the level of NO,and ameliorate vascular stenosis.This result further indicates that exterior-relieving medicines exert a significant dilating and supporting effect on the narrowed meridians and collaterals.

Hepatitis B virus (HBV) infection is a major global public health problem. There will be about 257 million chronic HBV-infected patients worldwide, according to the World Health Organization's estimation by 2025. Currently, the main drugs for the treatment of chronic HBV infection are nucleos(t)ide analogues and pegylated interferon (PEG-IFN). However, previous research results show that whether it is nucleos(t)ide analogues and PEG-IFN-α monotherapy or combination therapy, or sequential combination therapy, the rate of hepatitis B surface antigen seroconversion in patients is low, and there is still a high risk of disease progression after a certain course of antiviral treatment. Therefore, to achieve the ambitious target of "eliminating viral hepatitis by 2030" set by the World Health Organization and help more hepatitis B patients achieve functional cure, a large number of new drugs have been developed and entered clinical trials. This paper summarizes and reviews the types, safety, and efficacy of new drugs to further promote advancements in the field of treatment of chronic HBV infection.

Microbial communities such as those residing in the human gut are highly diverse and complex, and many with important implications for health and diseases. The effects and functions of these microbial communities are determined not only by their species compositions and diversities but also by the dynamic intra- and inter-cellular states at the transcriptional level. Powerful and scalable technologies capable of acquiring single-microbe-resolution RNA sequencing information in order to achieve a comprehensive understanding of complex microbial communities together with their hosts are therefore utterly needed. Here we report the development and utilization of a droplet-based smRNA-seq (single-microbe RNA sequencing) method capable of identifying large species varieties in human samples, which we name smRandom-seq2. Together with a triple-module computational pipeline designed for the bacteria and bacteriophage sequencing data by smRandom-seq2 in four human gut samples, we established a single-cell level bacterial transcriptional landscape of human gut microbiome, which included 29,742 single microbes and 329 unique species. Distinct adaptive response states among species in Prevotella and Roseburia genera and intrinsic adaptive strategy heterogeneity in Phascolarctobacterium succinatutens were uncovered. Additionally, we identified hundreds of novel host-phage transcriptional activity associations in the human gut microbiome. Our results indicated that smRandom-seq2 is a high-throughput and high-resolution smRNA-seq technique that is highly adaptable to complex microbial communities in real-world situations and promises new perspectives in the understanding of human microbiomes.
Humans ; Gastrointestinal Microbiome/genetics* ; Bacteriophages/physiology* ; High-Throughput Nucleotide Sequencing ; Sequence Analysis, RNA/methods* ; Bacteria/virology*

AIM:To investigate the effect of plasmin on epithelial sodium channel γ-subunit(γ-ENaC)acti-vation in mouse M-1 cells and the role of nuclear factor-κB(NF-κB).METHODS:Mouse M-1 cells were divided into three experimental parts.In the first part,the cells were divided into a control group,a plasmin group,and a plasmin+BAY 11-7082(NF-κB inhibitor)group to explore the effects of plasmin on inflammatory factors and γ-ENaC.In the sec-ond part,the cells were divided into a control group,a BAY 11-7082 group and a BAY 11-7082+plasmin group.M-1 cells were pretreated with BAY 11-7082 to explore the role of NF-κB in the regulation of γ-ENaC expression by plasmin.In the third part,the cells were divided into a control group,a plasmin group and an interleukin-6(IL-6)group.M-1 cells were treated with plasmin or IL-6 to explore the regulatory effect of IL-6 downstream of NF-κB on γ-ENaC.After drug interven-tion,the levels of IL-6,tumor necrosis factor-α(TNF-α),monocyte chemoattractant protein-1(MCP-1)and IL-1β in cell culture supernatants were detected by ELISA.The protein levels of phosphorylated inhibitor of nuclear factor-κB α(p-IκBα)/IκBα,p-NF-κB p65/NF-κB p65,and γ-ENaC was detected by Western blot.The distribution of γ-ENaC on the membrane was detected by immunofluorescence staining.RESULTS:Compared with control group,the levels of IL-6,TNF-α,MCP-1 and IL-1β in the supernatants of cells treated with 10 mg/L plasmin for 24 h were increased(n=3,P<0.01),the protein levels of p-IκBα/IκBα,p-NF-κB p65/NF-κB p65 and γ-ENaC were increased(n=5,P<0.01),and the distribution of γ-ENaC on the membrane was increased.Compared with plasmin group,the levels of IL-6,TNF-α,MCP-1,IL-1β,p-IκBα/IκBα,p-NF-κB p65/NF-κB p65 and γ-ENaC,and the γ-ENaC distribution on the membrane were lowered in plasmin and BAY 11-7082 coculture group(n=3,P<0.05 or P<0.01).Compared with control group,the ratio of p-NF-κB p65/NF-κB p65 decreased in BAY 11-7082 group(n=5,P<0.05),whereas there was no significant change in γ-ENaC.After stimulation with plasmin,the levels of p-IκBα/IκBα,p-NF-κB p65/NF-κB p65 and γ-ENaC in-creased(n=5,P<0.05 or P<0.01),and the γ-ENaC distribution on the membrane increased.Treatment with IL-6,downstream of NF-κB,increased the protein expression of γ-ENaC(n=5,P<0.05)and the distribution of γ-ENaC on the membrane after 24 h of intervention.CONCLUSION:The regulation of γ-ENaC expression by plasmin in mouse M-1 cells is related to the abnormal increase in inflammatory factors after NF-κB activation.

Objective To explore the value of dual-energy CT Bone Marrow Edema in quantitatively evaluating the timing of rib fracture evolution.Methods Sixty patients with thoracic trauma were scanned by dual-energy CT.Using CT Bone Marrow Edema technique,bone marrow CT values were standardized and quantified in both the Bone Marrow Edema area at the rib fracture site and in normal areas 1 cm on both sides of the fracture.The increments of standardized CT values for Bone Marrow Edema and VNCa were obtained for three stages of healing.The numerical variables were statistically described,and both the standardized CT value increments and VNCa standardized CT value increments were compared between the three stages and between each pair of stages.Diagnostic efficacy for stages with significant differences was evaluated using the area under the receiver operating characteristic(ROC)curve(AUC),and Cut-offvalues were calculated.Results There were significant differences in standardized CT value increments and VNCa standardized CT value increments across the three stages of Bone Marrow Edema(H=10.788,p=0.005;F=115.787,p=0.000).The standardized CT value increment of Bone Marrow Edema showed significant differences between the cartilaginous callus stage(fibrous callus stage)and the bony callus-remodeling stage(H=54.958,p=0.003),while the other pairwise comparisons showed no statistical significance(H=-25.603,p=0.183;H=29.354,p=0.113,respectively).However,the VNCa standardized CT value increments showed statistical significance across all three pairwise comparisons(all p=0.000).The ROC curve for differentiating the cartilaginous callus stage(fibrous callus stage)from the bony callus-remodeling stage using Bone Marrow Edema standardized CT value increment had an AUC of 0.652,with a Cut-offvalue of 81.575 Hu.The ROC curve for distinguishing the hematoma inflammation stage from the cartilaginous callus stage(fibrous callus stage)using VNCa standardized CT value increment had an AUC of 0.668,with a Cut-offvalue of 55.700 Hu.The ROC curve for differentiating the cartilaginous callus stage(fibrous callus stage)from the bony callus-remodeling stage using VNCa standardized CT value increment had an AUC of 0.905,with a Cut-offvalue of 37.625 Hu.Conclusion Dual-energy CT Bone Marrow Edema can quantitatively evaluate the timing of rib fracture evolution,and the differences in standardized CT value increments at different stages can provide a theoretical basis for forensic identification of fractures at different time periods.The Cut-offvalues of standardized CT value increments can,to some extent,predict the time stage of a fracture,providing quantitative evidence for forensic experts in the identification of rib fractures.

Objective To explore the value of dual-energy CT Bone Marrow Edema in quantitatively evaluating the timing of rib fracture evolution.Methods Sixty patients with thoracic trauma were scanned by dual-energy CT.Using CT Bone Marrow Edema technique,bone marrow CT values were standardized and quantified in both the Bone Marrow Edema area at the rib fracture site and in normal areas 1 cm on both sides of the fracture.The increments of standardized CT values for Bone Marrow Edema and VNCa were obtained for three stages of healing.The numerical variables were statistically described,and both the standardized CT value increments and VNCa standardized CT value increments were compared between the three stages and between each pair of stages.Diagnostic efficacy for stages with significant differences was evaluated using the area under the receiver operating characteristic(ROC)curve(AUC),and Cut-offvalues were calculated.Results There were significant differences in standardized CT value increments and VNCa standardized CT value increments across the three stages of Bone Marrow Edema(H=10.788,p=0.005;F=115.787,p=0.000).The standardized CT value increment of Bone Marrow Edema showed significant differences between the cartilaginous callus stage(fibrous callus stage)and the bony callus-remodeling stage(H=54.958,p=0.003),while the other pairwise comparisons showed no statistical significance(H=-25.603,p=0.183;H=29.354,p=0.113,respectively).However,the VNCa standardized CT value increments showed statistical significance across all three pairwise comparisons(all p=0.000).The ROC curve for differentiating the cartilaginous callus stage(fibrous callus stage)from the bony callus-remodeling stage using Bone Marrow Edema standardized CT value increment had an AUC of 0.652,with a Cut-offvalue of 81.575 Hu.The ROC curve for distinguishing the hematoma inflammation stage from the cartilaginous callus stage(fibrous callus stage)using VNCa standardized CT value increment had an AUC of 0.668,with a Cut-offvalue of 55.700 Hu.The ROC curve for differentiating the cartilaginous callus stage(fibrous callus stage)from the bony callus-remodeling stage using VNCa standardized CT value increment had an AUC of 0.905,with a Cut-offvalue of 37.625 Hu.Conclusion Dual-energy CT Bone Marrow Edema can quantitatively evaluate the timing of rib fracture evolution,and the differences in standardized CT value increments at different stages can provide a theoretical basis for forensic identification of fractures at different time periods.The Cut-offvalues of standardized CT value increments can,to some extent,predict the time stage of a fracture,providing quantitative evidence for forensic experts in the identification of rib fractures.

Objective To analyze the role and mechanism of Xiaoqinglong decoction in alleviating the pathological process of pulmonary arterial hypertension(PAH),and to observe the effect of Xiaoqinglong decoction on endothelial-mesenchymal transition(EMT)in human pulmonary arterial endothelial cell(HPAEC)and the involvement of the Toll-like receptor/nuclear factor-κB/hypoxia-inducible factor-1α(TLR/NF-κB/HIF-1α)pathway in this mechanism.Methods Thirty-six male Sprague Dawley(SD)rats and HPAEC were randomly divided into control group,model group,Xiaoqinglong decoction plus Earthworm group,Bosentan tablet group,dimethyl sulfoxide(DMSO)group,and monophosphoryl lipid A(MPL)group.PAH rat models and HPAEC models were established by hypoxic exposure.The Xiaoqinglong decoction plus Earthworm group received intragastric administration Xiaoqinglong decoction plus Earthworm(5 g·kg-1·d-1)or cultured with 10%corresponding drug serum,the Bosentan group received Bosentan(100 mg·kg-1·d-1)by gavage or cultured with 10%corresponding drug serum,the MPL group received 1 μg MPL,and the DMSO group received an equivalent volume of the DMSO and corn oil mixed solvent as a negative control for the MPL group.The hemodynamic parameters,including mean pulmonary arterial pressure(mPAP),right ventricular systolic pressure(RVSP),and the maximum rate of right ventricular pressure(+dp/dt max),were measured via right heart catheterization.After euthanasia,lung and heart tissues were collected to assess the right ventricular hypertrophy index(RVHI);hematoxylin-eosin(HE)staining was used to observe the degree of right ventricular cardiomyocyte hypertrophy and to calculate the average intima-media thickness(IMT)in small pulmonary arteries;Western blotting was used to detect the protein expression levels of proliferating cell nuclear antigen(PCNA),CD68,TLR4,NF-κB,HIF-1α,vascular endothelial cadherin,and vimentin;cell counting kit-8(CCK-8),Transwell,and scratch assays were used to observe cell proliferation and migration;Enzyme-linked immunosorbent assay(ELISA)was used to measure the levels of interleukins(IL-8,IL-6),tumor necrosis factor-α(TNF-α),endothelin-1(ET-1),and nitric oxide(NO).Results Compared with the model group,the Xiaoqinglong decoction plus Earthworm group showed significant reductions in mPAP,RVSP,RVHI,and IMT in PAH rats[mPAP(mmHg,1 mmHg≈0.133 kPa):22.17±1.94 vs.42.00±4.90,RVSP(mmHg):34.67±3.20 vs.52.83±3.76,RVHI:0.402±0.057 vs.0.822±0.101,IMT:(37.85±2.49)%vs.(62.06±4.52)%,all P<0.05],and a significant increase in+dP/dT max(mmHg/s:2 730.83±137.89 vs.1 718.33±148.36,P<0.05).Western blotting and ELISA results showed that compared with the model group,the Xiaoqinglong decoction plus Earthworm group had significantly lower protein expression of PCNA and CD68 in lung tissue,and levels of inflammatory factors(IL-6,IL-8,TNF-α)in rat serum[lung tissue:PCNA protein expression(PCNA/GAPDH)was 1.56±0.08 vs.2.20±0.26,CD68 protein expression(CD68/GAPDH):1.46±0.09 vs.2.60±0.23;serum:IL-8(ng/L)was 39.67±6.28 vs.149.17±7.49,IL-6(ng/L):81.00±6.63 vs.211.00±25.31,TNF-α(ng/L):213.17±24.86 vs.799.50±43.51,all P<0.05].In vitro experiments,compared with the model group,Xiaoqinglong decoction plus Earthworm inhibited abnormal proliferation(A value:2.052±0.087 vs.2.242±0.057,P<0.05)and migration[number of migrating cells(per field):101.33±12.01 vs.226.67±17.56,P<0.05]of HPAEC,and reversed the EMT process,manifested as upregulation of vascular endothelial cadherin protein expression levels(vascular endothelial cadherin/GAPDH:0.39±0.06 vs.0.12±0.03,P<0.05)and downregulation of vimentin protein expression(vimentin/GAPDH:4.96±0.33 vs.7.89±0.44,P<0.05).Western blotting results indicated that compared with the model group,the protein expression levels of TLR4,the ratio of phosphorylated p65 to total p65,and HIF-1α in both lung tissue and HPAEC were significantly reduced in the Xiaoqinglong decoction plus Earthworm group[lung tissue:TLR4 protein expression(TLR4/GAPDH)was 3.13±0.20 vs.4.38±0.30,p-p65/p65 ratio:7.11±0.81 vs.12.73±1.80,HIF-1α protein expression(HIF-1α/GAPDH):2.37±0.32 vs.4.45±0.34;HPAEC:TLR4 protein expression(TLR4/GAPDH)was 1.42±0.03 vs.2.43±0.05,p-p65/p65 ratio:6.01±1.84 vs.11.28±1.06,HIF-1α protein expression(HIF-1α/GAPDH)was 3.24±0.17 vs.5.50±0.44,all P<0.05],accompanied by upregulated vascular endothelial cadherin protein expression(vascular endothelial cadherin/GAPDH:0.66±0.03 vs.0.49±0.03,P<0.05)and downregulated vimentin protein expression(vimentin/GAPDH:1.81±0.12 vs.2.47±0.10,P<0.05),indicating that Xiaoqinglong decoction plus Earthworm inhibits the EMT process in endothelial cells by suppressing the activation of the TLR/NF-κB/HIF-1α pathway.Experiments with a TLR agonist further confirmed that activation of the TLR pathway reverses the protective effects of Xiaoqinglong decoction plus Earthworm,as shown by the MPL group compared to the DMSO group having significantly increased protein expression of the p-p65/p65 ratio and HIF-1α[p-p65/p65 ratio:2.17±0.35 vs.1.08±0.14,HIF-1α/GAPDH:3.96±0.25 vs.1.03±0.10,both P<0.05],further decreased vascular endothelial cadherin protein expression(vascular endothelial cadherin/GAPDH:0.66±0.04 vs.0.99±0.02,P<0.05),further increased vimentin protein expression(vimentin/GAPDH:1.53±0.12 vs.0.93±0.07,P<0.05),along with enhanced cell migration capacity[number of migrating cells(per field):176.67±17.50 vs.107.00±11.14;cell migration rate in scratch assay:(34.32±2.82)%vs.(22.71±2.49)%,both P<0.05]and increased proliferation activity(48 hours A value:2.156±0.044 vs.1.810±0.088,P<0.05).Conclusions Xiaoqinglong decoction combined with Pheretima not only significantly reduces pulmonary artery pressure,improves cardiac function and mitigates pulmonary vascular fibrosis in PAH rats,but also alleviates pulmonary vascular remodeling by inhibiting inflammatory responses and EMT.It can further decrease the content of ET-1,increase the level of NO,and ameliorate vascular stenosis.This result further indicates that exterior-relieving medicines exert a significant dilating and supporting effect on the narrowed meridians and collaterals.