ObjectiveTo investigate the mechanism by which Shaoyaotang regulates the miRNA-155-mediated suppressor of cytokine signaling 1 （SOCS1）/Janus kinase 1 （JAK1）/signal transducer and activator of transcription 1 （STAT1） signaling pathway and thereby affects macrophage polarization. MethodsThe cell-counting kit-8 （CCK-8） assay was used to detect the effect of drug-containing serum of Shaoyaotang at different concentrations on the viability of RAW 264.7 cells. A cell model of inflammation was established by stimulating RAW264.7 cells with lipopolysaccharide （LPS） at a concentration of 10 mg·L^-1 The modeled cells were assigned by the random number table method into seven groups： LPS-induced M1 polarization （model）， M1+miRNA-155 mimics， M1+miRNA-155 inhibitor， M1+Shaoyaotang-containing serum， M1+miRNA-155 mimics+Shaoyaotang-containing serum， M1+miRNA-155 inhibitor+Shaoyaotang-containing serum， and M1+blank serum. Enzyme-linked immunosorbent assay was employed to measure the levels of inflammatory factors ［tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， and interleukin-1β （IL-1β）］. Immunofluorescence assay was used to detect the expression of macrophage polarization markers ［inducible nitric oxide synthase （iNOS） and macrophage mannose receptor 1 （CD206）］. Real-time PCR was employed to measure the expression of miRNA-155 in cells. Western blot was performed to determine the protein levels of SOCS1， STAT1， and JAK1. ResultsCompared with the LPS-induced M1 polarization （model） group， the M1+miRNA-155 mimics group showed up-regulated expression of miRNA-155， JAK1， STAT1， TNF-α， IL-6， IL-1β， and iNOS （P<0.05） and down-regulated expression of CD206 （P<0.05）. In both the M1+miRNA-155 inhibitor group and the M1+Shaoyaotang-containing serum group， the expression levels of miRNA-155， JAK1， STAT1， TNF-α， IL-6， IL-1β， and iNOS were down-regulated （P<0.05）， while those of SOCS1 and CD206 were up-regulated （P<0.05）. Compared with the M1+miRNA-155 mimics group， the M1+miRNA-155 mimics+Shaoyaotang-containing serum group showed down-regulated expression of miRNA-155， JAK1， STAT1， TNF-α， IL-6， IL-1β， and iNOS （P<0.05） and up-regulated expression of SOCS1 and CD206 （P<0.05）. Compared with the M1+miRNA-155 inhibitor group， the M1+miRNA-155 inhibitor+Shaoyaotang-containing serum group showed down-regulated expression of miRNA-155， JAK1， STAT1， TNF-α， IL-6， IL-1β， and iNOS （P<0.05） and up-regulated expression of SOCS1 and CD206 （P<0.05）. ConclusionShaoyaotang regulates macrophage polarization by modulating miRNA-155 expression and interfering with the SOCS1/JAK1/STAT1 signaling pathway. The findings provide new experimental evidence for the treatment of ulcerative colitis with Shaoyaotang.

ObjectiveTo investigate the potential role and underlying mechanisms of Shaoyaotang in intervening macrophage glycolytic reprogramming in ulcerative colitis （UC）. MethodsForty-eight C57BL/6 mice were randomly divided into six groups： Normal control group， model group， mesalazine group （0.39 g·kg^-1）， Shaoyaotang group （15.54 g·kg^-1）， 2-deoxy-D-glucose （2-DG） group （glycolysis inhibitor， 100 mg·kg^-1）， and 2-DG + Shaoyaotang combined group （100 mg·kg^-1+15.54 g·kg^-1）. Except for the normal control group， mice in the other five groups were induced to establish UC models using dextran sulfate sodium （DSS）. The normal control group was administered pure water via intragastric gavage， while the other groups received intragastric gavage of mesalazine solution， intragastric gavage of Shaoyaotang， and the 2-DG group was treated with 2-DG via intraperitoneal injection. After 7 consecutive days of treatment， colonic tissues were extracted. Hematoxylin and eosin （HE） staining was performed to evaluate histopathological changes and tissue injury in the colon. Enzyme-linked immunosorbent assay （ELISA） was used to detect the expression of interleukin-10 （IL-10） and tumor necrosis factor-α （TNF-α） in colonic tissues. Western blot analysis was employed to determine the expression levels of hypoxia-inducible factor-1α （HIF-1α）， glucose transporter （GLUT1）， lactate dehydrogenase A （LDHA）， pyruvate kinase M2 （PKM2）， and 6-phosphofructo-2-kinase/fructose-2，6-biphosphatase 3 （PFKFB3） in colonic tissues. Immunofluorescence was conducted to detect the expression of CD206 and inducible nitric oxide synthase （iNOS） in colonic tissues. Liquid chromatography-mass spectrometry （LC-MS） was utilized to measure lactate and citrate levels in colonic tissues. ResultsCompared with the normal control group， mice in the model group exhibited a significant increase in disease activity index （DAI） scores， accompanied by colonic mucosal congestion， edema， and inflammatory cell infiltration， significantly elevated expression of the inflammatory cytokine TNF-α （P<0.05）， significantly decreased IL-10 expression （P<0.05）， significantly increased levels of HIF-1α， GLUT1， LDHA， PKM2， and PFKFB3 in colonic tissues （P<0.05）， markedly elevated iNOS expression （P<0.05）， significantly decreased CD206 expression （P<0.05）， and significantly elevated lactate and citrate levels in colonic tissues （P<0.05）. In contrast to the model group， the Shaoyaotang group， inhibitor group， and Shaoyaotang combined with inhibitor group demonstrated amelioration of mucosal injury in colonic tissues， markely decreased expression levels of the inflammatory cytokine TNF-α （P<0.05）， elevated IL-10 expression levels， significantly decreased expression of HIF-1α， GLUT1， LDHA， PKM2， and PFKFB3 （P<0.05）， markedly reduced iNOS expression levels （P<0.05）， significantly increased CD206 expression （P<0.05） and significantly decreased lactate and citrate levels （P<0.05）. ConclusionShaoyaotang ameliorates symptoms of DSS-induced UC in mice， and its therapeutic mechanism may be associated with regulating macrophage glycolytic reprogramming via modulation of the HIF-1α signaling pathway.

ObjectiveTo investigate the role of microRNA-155-5p （miR-155-5p） in ulcerative colitis （UC） and study the molecular mechanism of Shaoyaotang in the treatment of UC by regulating miR-155-5p. MethodsForty-eight SPF-grade male C57BL/6 mice were selected and assigned via the random number table method into 6 groups （n=8）： A blank control group， a model group， a mesalazine （0.39 g·kg^-1） group， a Shaoyaotang （31.08 g·kg^-1） group， a Janus kinase 1 （JAK1） inhibitor （baricitinib， 10 mg·kg^-1） group， and a Shaoyaotang combined with inhibitor （baricitinib 10 mg·kg^-1 + Shaoyaotang 31.08 g·kg^-1） group. After successful modeling of UC by gavage of 3% dextran sulphate sodium solution， each group received corresponding drug intervention for 7 days. Shaoyaotang and mesalazine were administered by gavage， and baricitinib by intraperitoneal injection. Twenty-four hours after the last administration， mice were anesthetized by intraperitoneal injection of pentobarbital sodium， and blood was collected for determination of white blood cell count and erythrocyte sedimentation rate （ESR）. Mice were then sacrificed for measurement of colon length. Hematoxylin-eosin staining was used to observe colonic pathological changes and perform pathological scoring. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was employed to determine the relative expression of miR-155-5p in the colonic tissue， and Western blot was used to determine the protein levels of JAK1， phosphorylated JAK1 （p-JAK1）， suppressor of cytokine signaling 1 （SOCS1）， signal transducer and activator of transcription 1 （STAT1）， and phosphorylated STAT1 （p-STAT1）. ResultsCompared with the blank control group， the model group showed increased disease activity index （DAI） score and pathological score， shortened colon， upregulated relative expression of miR-155-5p and protein levels of p-JAK1 and p-STAT1， downregulated protein level of SOCS1 in the colonic tissue， prolonged time of erythrocyte sedimentation， and increased white blood cell count （P<0.01）. Compared with the model group， all drug-treated groups exhibited improvements in the above indicators （P<0.01）. Moreover， the Shaoyaotang group showed better therapeutic effects than the mesalazine group in regulating miR-155-5p expression， related protein levels， DAI score， and colonic pathological score （P<0.01）. ConclusionShaoyaotang may downregulate miR-155-5p to relieve its inhibition on SOCS1， thereby suppressing the excessive activation of the JAK1/STAT1 signaling pathway and ultimately alleviating intestinal inflammatory damage.

ObjectiveTo investigate the role of the Toll-like receptor 4 （TLR4）/myeloid differentiation factor 88 （MyD88）/nuclear factor-κB （NF-κB） signaling pathway in intestinal mucosal barrier damage in ulcerative colitis， as well as the intervention mechanism of Shaoyaotang. MethodsSixty SD rats were allocated into a blank group， a model group， a mesalazine （0.42 g·kg^-1） group， and low-， medium-， and high-dose （11.1， 22.2， 44.4 g·kg^-1， respectively） Shaoyaotang groups. A model of ulcerative colitis was induced by 2，4，6-trinitrobenzenesulfonic acid （TNBS）. After successful modeling， rats were administrated with corresponding agents via gavage for 7 days. Changes in colon length and colon weight were observed. Hematoxylin-eosin staining was performed to examine the pathological changes of the colon， and immunohistochemistry was employed to detect the expression of the inflammatory cytokine interleukin-8 （IL-8）， cyclooxygenase-2 （COX-2）， junction adhesion molecule-1 （JAM-1）， and claudin-1 in the colon. Western blot analysis was performed to determine the protein levels of TLR4， MyD88， and NF-κB in the colon. ResultsCompared with the blank group， the model group showed elevated DAI score （P<0.01）， reduced colon length and colon weight （P<0.01）， down-regulated protein levels of JAM-1 and claudin-1 （P<0.01）， and up-regulated protein levels of IL-8， COX-2， TLR4， MyD88， and NF-κB p65 （P<0.01） in the colon tissue. Compared with the model group， each treatment group showed decreased DAI score （P<0.05， P<0.01）， increased colon length and colon weight （P<0.05， P<0.01）， up-regulated protein levels of JAM-1 and claudin-1 （P<0.01）， and down-regulated protein levels of IL-8， COX-2， TLR4， MyD88， and NF-κB p65 （P<0.01） in the colon tissue. ConclusionShaoyaotang alleviates intestinal inflammation and intestinal mucosal damage to protect intestinal barrier integrity by regulating the TLR4/MyD88/NF-κB signaling pathway.

ObjectiveTo investigate the effect of Shaoyaotang on T helper cell 17/regulatory T lymphocyte（Th17/Treg） cell balance in ulcerative colitis and decipher the intervention mechanism based on glucose metabolism reprogramming. MethodsThe mouse model of ulcerative colitis was established by the dextran sulfate sodium （DSS） method. Forty-eight C57BL/6 mice were randomly allocated into normal， model， Western drug control （mesalazine， 0.39 g·kg^-1·d^-1）， Shaoyaotang （15.54 g·kg^-1·d^-1）， inhibitor （2-deoxy-D-glucose， 2-DG， 100 mg·kg^-1·d^-1）， and inhibitor （2-DG， 100 mg·kg^-1·d^-1） + Shaoyaotang （15.54 g·kg^-1·d^-1） groups. Mice were administrated with the corresponding drugs by gavage for 7 days. The general conditions and the colon injury degree were observed 24 h after the last administration. The expression of interleukin （IL）-10 and IL-17 in the colon tissue was detected by immunohistochemical staining. Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） were performed to determine the protein and mRNA levels， respectively， of hypoxia-inducing factor-1α （HIF-1α）， lactate dehydrogenase （LDHA）， and hexokinase 2 （HK2） in the colon tissue. Th17/Treg cell differentiation was detected by flow cytometry. Enzyme-linked immunosorbent assay was employed to measure the levels of lactic acid and glucose in the colon tissue and IL-10， IL-17， and IL-6 in the serum. ResultsCompared with the normal group， the model group showed decreases in body weight and disease activity index （DAI） （P<0.05）， elevations in levels of HIF-1α， LDHA， HK2， IL-17， IL-6， Th17 cells， lactic acid， and glucose in the colon tissue （P<0.05）， and declines in the levels of of IL-10 and Treg cells （P<0.05）. Compared with the model group， the drug administration groups showed increases in body weight and DAI （P<0.05）， declines in levels of HIF-1α， LDHA， HK2， IL-17， IL-6， Th17 cells， lactic acid， and glucose in the colon tissue （P<0.05）， and rises in levels of IL-10 and Treg cells （P<0.05）. Shaoyaotang+2-DG group had the most obvious effect. ConclusionShaoyaotang can relieve diarrhea and bloody stool in mice with ulcerative colitis by restoring the Th17/Treg cell balance via regulation of glucose metabolism reprogramming， thus playing a role in the treatment of ulcerative colitis.

ObjectiveTo investigate the effect of Shaoyaotang on T helper cell 17/regulatory T lymphocyte（Th17/Treg） cell balance in ulcerative colitis and decipher the intervention mechanism based on glucose metabolism reprogramming. MethodsThe mouse model of ulcerative colitis was established by the dextran sulfate sodium （DSS） method. Forty-eight C57BL/6 mice were randomly allocated into normal， model， Western drug control （mesalazine， 0.39 g·kg^-1·d^-1）， Shaoyaotang （15.54 g·kg^-1·d^-1）， inhibitor （2-deoxy-D-glucose， 2-DG， 100 mg·kg^-1·d^-1）， and inhibitor （2-DG， 100 mg·kg^-1·d^-1） + Shaoyaotang （15.54 g·kg^-1·d^-1） groups. Mice were administrated with the corresponding drugs by gavage for 7 days. The general conditions and the colon injury degree were observed 24 h after the last administration. The expression of interleukin （IL）-10 and IL-17 in the colon tissue was detected by immunohistochemical staining. Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） were performed to determine the protein and mRNA levels， respectively， of hypoxia-inducing factor-1α （HIF-1α）， lactate dehydrogenase （LDHA）， and hexokinase 2 （HK2） in the colon tissue. Th17/Treg cell differentiation was detected by flow cytometry. Enzyme-linked immunosorbent assay was employed to measure the levels of lactic acid and glucose in the colon tissue and IL-10， IL-17， and IL-6 in the serum. ResultsCompared with the normal group， the model group showed decreases in body weight and disease activity index （DAI） （P<0.05）， elevations in levels of HIF-1α， LDHA， HK2， IL-17， IL-6， Th17 cells， lactic acid， and glucose in the colon tissue （P<0.05）， and declines in the levels of of IL-10 and Treg cells （P<0.05）. Compared with the model group， the drug administration groups showed increases in body weight and DAI （P<0.05）， declines in levels of HIF-1α， LDHA， HK2， IL-17， IL-6， Th17 cells， lactic acid， and glucose in the colon tissue （P<0.05）， and rises in levels of IL-10 and Treg cells （P<0.05）. Shaoyaotang+2-DG group had the most obvious effect. ConclusionShaoyaotang can relieve diarrhea and bloody stool in mice with ulcerative colitis by restoring the Th17/Treg cell balance via regulation of glucose metabolism reprogramming， thus playing a role in the treatment of ulcerative colitis.

In recent years， as the incidence of ulcerative colitis （UC） is growing， intestinal mucosal injury has garnered increasing attention， and it is characterized by high recurrence， risk of inflammation-cancer transformation， and difficulty in repair. Intestinal mucosal injury in UC is centered on persistent inflammation and barrier dysfunction， with its pathological mechanisms involving endoplasmic reticulum stress （ERS）-mediated changes such as abnormal apoptosis， abnormal autophagy， and inflammatory responses. ERS induces apoptosis of intestinal epithelial cells， disrupts tight junction proteins， and exacerbates inflammatory responses through pathways such as protein kinase R-like endoplasmic reticulum kinase （PERK）， inositol-requiring enzyme 1 alpha （IRE1α）， and activating transcription factor 6 （ATF6）， ultimately causing intestinal mucosal injury. Traditional Chinese medicine （TCM） has a long history of research on UC. The theory of sores depending on spleen-earth holds that spleen deficiency is the fundamental cause of UC， while pathological products such as dampness-turbidity and blood stasis are the secondary manifestations. Dysfunction of the spleen-earth leads to insufficient production and transformation of Qi and blood， malnutrition of the intestinal mucosa， and invasion of external pathogens. In the active phase of UC， spleen deficiency is often accompanied by excessive pathogenic factors such as dampness-heat and heat-toxin， leading to acute intestinal mucosal damage. In the remission phase， however， it is mainly characterized by spleen deficiency and healthy Qi deficiency， accompanied by residual pathogens， resulting in weak intestinal mucosal repair. Studies have shown that the endoplasmic reticulum， as a key site for protein synthesis and folding， has functions highly similar to the TCM concept of the spleen governing transportation and transformation. From a TCM perspective， the endoplasmic reticulum can be regarded as the carrier of spleen transportation， and ERS is a microcosmic manifestation of spleen dysfunction， leading to intestinal mucosal injury. ERS impairs the structure and function of the endoplasmic reticulum， induces the generation of abnormal Qi， and triggers pathological changes， making inflammation difficult to be reduced and causing the aggravation of ERS， forming a vicious cycle of spleen deficiency-pathological products-intestinal injury. TCM has unique advantages in regulating ERS to prevent and treat intestinal mucosal injury. According to the theory of sores depending on spleen-earth and the modern medical understanding of ERS， this paper delves into the TCM and Western medicine pathogenesis of intestinal mucosal injury in UC. Furthermore， this paper discusses the roles of TCM active components and compound formulas in reducing intestinal mucosal injury in UC by regulating ERS under the guidance of the treatment principles of invigorating the spleen and replenishing Qi as the key and dispelling dampness and removing blood stasis as the supplementation， aiming to provide new ideas and methods for the prevention and treatment of UC.

Mycolic acids (MAs), i.e. 2-alkyl, 3-hydroxy long-chain fatty acids, are the hallmark of the cell envelope of Mycobacterium tuberculosis and are related with antibiotic resistance and host immune escape. Nowadays, they've become hot target of new anti-tuberculosis drugs. There are two main methods to detect MAs, ¹⁴C metabolic labeling thin-layer chromatography (TLC) and liquid chromatograph mass spectrometer (LC-MS). However, the user qualification of ¹⁴C or the lack of standards for LC-MS hampered the easy use of this method. TLC is a common way to analyze chemical substance and can be used to analyze MAs. In this study, we used tetrabutylammonium hydroxide and methyl iodide to hydrolyze and formylate MAs from mycobacterium cell wall. Subsequently, we used diethyl ether to extract methyl mycolate. By this method, we can easily extract and analyze MA in regular biological labs. The results demonstrated that this method could be used to compare MAs of different mycobacterium in different growth phases, MAs of mycobacteria treated by anti-tuberculosis drugs or MAs of mycobacterium mutants. Therefore, we can use this method as an initial validation for the changes of MAs in researches such as new drug screening without using radioisotope or when the standards are not available.
Mycolic Acids/metabolism* ; Chromatography, Thin Layer ; Mycobacterium tuberculosis ; Fatty Acids ; Antitubercular Agents/pharmacology*