This study investigated the effects and underlying mechanisms of vanillic acid(VA) against cardiac fibrosis(CF) induced by isoproterenol(ISO) in mice. Male C57BL/6J mice were randomly divided into control group, VA group(100 mg·kg~(-1), ig), ISO group(10 mg·kg~(-1), sc), ISO + VA group(10 mg·kg~(-1), sc + 100 mg·kg~(-1), ig), ISO + dynamin-related protein 1(Drp1) inhibitor(Mdivi-1) group(10 mg·kg~(-1), sc + 50 mg·kg~(-1), ip), and ISO + VA + Mdivi-1 group(10 mg·kg~(-1), sc + 100 mg·kg~(-1), ig + 50 mg·kg~(-1), ip). The treatment groups received the corresponding medications once daily for 14 consecutive days. On the day after the last administration, cardiac functions were evaluated, and serum and cardiac tissue samples were collected. These samples were analyzed for serum aspartate aminotransferase(AST), lactate dehydrogenase(LDH), creatine kinase-MB(CK-MB), cardiac troponin I(cTnI), reactive oxygen species(ROS), interleukin(IL)-1β, IL-4, IL-6, IL-10, IL-18, and tumor necrosis factor-α(TNF-α) levels, as well as cardiac tissue catalase(CAT), glutathione(GSH), malondialdehyde(MDA), myeloperoxidase(MPO), superoxide dismutase(SOD), total antioxidant capacity(T-AOC) activities, and cytochrome C levels in mitochondria and cytoplasm. Hematoxylin-eosin, Masson, uranium acetate and lead citrate staining were used to observe morphological and mitochondrial ultrastructural changes in the cardiac tissues, and myocardial injury area and collagen volume fraction were calculated. Flow cytometry was applied to detect the relative content and M1/M2 polarization of cardiac macrophages. The mRNA expression levels of macrophage polarization markers [CD86, CD206, arginase 1(Arg-1), inducible nitric oxide synthase(iNOS)], CF markers [type Ⅰ collagen(Coll Ⅰ), Coll Ⅲ, α-smooth muscle actin(α-SMA)], and cytokines(IL-1β, IL-4, IL-6, IL-10, IL-18, TNF-α) in cardiac tissues were determined by quantitative real-time PCR. Western blot was used to detect the protein expression levels of Coll Ⅰ, Coll Ⅲ, α-SMA, Drp1, p-Drp1, voltage-dependent anion channel(VDAC), hexokinase 1(HK1), NOD-like receptor protein 3(NLRP3), apoptosis-associated speck-like protein(ASC), caspase-1, cleaved-caspase-1, gasdermin D(GSDMD), cleaved N-terminal gasdermin D(GSDMD-N), IL-1β, IL-18, B-cell lymphoma-2(Bcl-2), B-cell lymphoma-xl(Bcl-xl), Bcl-2-associated death promoter(Bad), Bcl-2-associated X protein(Bax), apoptotic protease activating factor-1(Apaf-1), pro-caspase-3, cleaved-caspase-3, pro-caspase-9, cleaved-caspase-9, poly(ADP-ribose) polymerase-1(PARP-1), and cleaved-PARP-1 in cardiac tissues. The results showed that VA significantly improved cardiac function in mice with CF, reduced myocardial injury area and cardiac index, and decreased serum levels of AST, CK-MB, cTnI, LDH, ROS, IL-1β, IL-6, IL-18, and TNF-α. VA also lowered MDA and MPO levels, mRNA expressions of IL-1β, IL-6, IL-18, and TNF-α, and mRNA and protein expressions of Coll Ⅰ, Coll Ⅲ, and α-SMA in cardiac tissues, and increased serum levels of IL-4 and IL-10, cardiac tissue levels of CAT, GSH, SOD, and T-AOC, and mRNA expressions of IL-4 and IL-10. Additionally, VA ameliorated cardiac pathological damage, inhibited myocardial cell apoptosis, inflammatory infiltration, and collagen fiber deposition, reduced collagen volume fraction, and alleviated mitochondrial damage. VA decreased the ratio of F4/80~+CD86~+ M1 cells and the mRNA expressions of CD86 and iNOS in cardiac tissue, and increased the ratio of F4/80~+CD206~+ M2 cells and the mRNA expressions of CD206 and Arg-1. VA also reduced protein expressions of p-Drp1, VDAC, NLRP3, ASC, caspase-1, cleaved-caspase-1, GSDMD, GSDMD-N, IL-1β, IL-18, Bad, Bax, Apaf-1, cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP-1, and cytoplasmic cytochrome C, and increased the expressions of HK1, Bcl-2, Bcl-xl, pro-caspase-3, pro-caspase-9 proteins, as well as the Bcl-2/Bax and Bcl-xl/Bad ratios and mitochondrial cytochrome C content. These results indicate that VA has a significant ameliorative effect on ISO-induced CF in mice, alleviates ISO-induced oxidative damage and inflammatory response, and its mechanism may be closely related to the inhibition of Drp1/HK1/NLRP3 and mitochondrial apoptosis signaling pathways, suppression of myocardial cell inflammatory infiltration and collagen fiber deposition, reduction of collagen volume fraction and CollⅠ, Coll Ⅲ, and α-SMA expressions, thus mitigating CF.
Animals ; Isoproterenol/adverse effects* ; Male ; Mice ; Signal Transduction/drug effects* ; Vanillic Acid/administration & dosage* ; Dynamins/genetics* ; Mice, Inbred C57BL ; Fibrosis/genetics* ; Apoptosis/drug effects* ; Mitochondria/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Myocardium/metabolism* ; Humans

To improve the quality evaluation system of Hibisci Mutabilis Folium, this study established high performance liquid chromatography(HPLC) fingerprints of Hibisci Mutabilis Folium and evaluated the quality differences of medicinal materials from different places of production by chemometrics. Furthermore, a content measurement method of differential components was established based on quantitative analysis of multi-components by single-marker(QAMS). The fingerprints of 17 batches of Hibisci Mutabilis Folium from different places of production were constructed, with a total of 19 common peaks marked and seven components confirmed. The similarity between the sample fingerprints and the reference fingerprints ranged from 0.890 to 0.974. By utilizing principal component analysis(PCA), hierarchical cluster analysis(HCA), and orthogonal partial least squares-discriminant analysis(OPLS-DA), the chemical patterns of fingerprints were identified. Five components that could be used to evaluate the quality differences of Hibisci Mutabilis Folium were screened, namely peak 6(quercetin 3-O-β-robinobioside), peak 7(rutin), peak 9(kaempferol-3-O-β-robinobioside), peak 10(kaempferol-3-O-rutinoside), and peak 14(tiliroside). The relative correction factors of isoquercitrin, kaempferol-3-O-β-robinobioside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside, and tiliroside were measured with rutin as the internal reference. The QAMS method was established for the content measurement of six flavonoids, and the results showed there was no significant difference compared to the results obtained by an external standard method. In summary, the HPLC fingerprints and QAMS method established in the study, demonstrating stability and accuracy, can provide a reference for the overall quality evaluation of Hibisci Mutabilis Folium.
Chromatography, High Pressure Liquid/methods* ; Drugs, Chinese Herbal/chemistry* ; Quality Control ; Principal Component Analysis

OBJECTIVE: To evaluate the dynamic changes of glucocorticoid (GC) dose and the feasibility of GC discontinuation in rheumatoid arthritis (RA) patients under the background of Chinese medicine (CM). METHODS: This multicenter retrospective cohort study included 1,196 RA patients enrolled in the China Rheumatoid Arthritis Registry of Patients with Chinese Medicine (CERTAIN) from September 1, 2019 to December 4, 2023, who initiated GC therapy. Participants were divided into the Western medicine (WM) and integrative medicine (IM, combination of CM and WM) groups based on medication regimen. Follow-up was performed at least every 3 months to assess dynamic changes in GC dose. Changes in GC dose were analyzed by generalized estimator equation, the probability of GC discontinuation was assessed using Kaplan-Meier curve, and predictors of GC discontinuation were analyzed by Cox regression. Patients with <12 months of follow-up were excluded for the sensitivity analysis. RESULTS: Among 1,196 patients (85.4% female; median age 56.4 years), 880 (73.6%) received IM. Over a median 12-month follow-up, 34.3% (410 cases) discontinued GC, with significantly higher rates in the IM group (40.8% vs. 16.1% in WM; P<0.05). GC dose declined progressively, with IM patients demonstrating faster reductions (median 3.75 mg vs. 5.00 mg in WM at 12 months; P<0.05). Multivariate Cox analysis identified age <60 years [P<0.001, hazard ratios (HR)=2.142, 95% confidence interval (CI): 1.523-3.012], IM therapy (P=0.001, HR=2.175, 95% CI: 1.369-3.456), baseline GC dose ⩽7.5 mg (P=0.003, HR=1.637, 95% CI: 1.177-2.275), and absence of non-steroidal anti-inflammatory drugs use (P=0.001, HR=2.546, 95% CI: 1.432-4.527) as significant predictors of GC discontinuation. Sensitivity analysis (545 cases) confirmed these findings. CONCLUSIONS RA patients receiving CM face difficulties in following guideline-recommended GC discontinuation protocols. IM can promote GC discontinuation and is a promising strategy to reduce GC dependency in RA management. (Trial registration: ClinicalTrials.gov, No. NCT05219214).
Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Arthritis, Rheumatoid/drug therapy* ; Glucocorticoids/therapeutic use* ; Medicine, Chinese Traditional ; Retrospective Studies

Objective To investigate the correlation between single nucleotide polymorphisms(SNPs)of human leukocyte antigen(HLA)genes and the clinicopathological features and postoperative prognosis of primary hepatocellular carcinoma(HCC).Methods A retrospective analysis was conducted on 328 patients with primary HCC who underwent surgery and had complete clinical data at Yantai Yuhuangding Hospital Affiliated to Qingdao University Medical College from January 2014 to September 2022.The genotypes of HLA-DRB1 rs2647073,rs3997872 and HLA-DQB1 rs1049055 loci were detected in all patients using polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)technology.Binary logistic regression was employed to examine the association between SNPs in the HLA gene and the clinicopathological features of primary HCC.Age,gender,drinking history,and HBV/HCV infection history were used as stratification factors for stratified analysis.Univariate Cox regression was used to analyze the correlation between HLA gene SNPs and postoperative prognosis,and the Kaplan-Meier survival curve of patients with primary HCC after surgery was drawn.Multivariate Cox regression was utilized to evaluate the independent risk factors affecting the postoperative prognosis of patients with primary HCC.Results Binary logistic regression analysis showed that rs2647073 CC genotype patients had a higher risk of tumor diameter≥5 cm than AA genotype patients(P=0.005).Patients with TT genotype of rs3997872 had a higher risk of vascular invasion than those with AA genotype(P=0.003).Stratified analysis showed that age≥60 years,history of HBV/HCV infection,female,male,no drinking history,and drinking history,HLA-DRB1 rs2647073 CC genotype had a higher risk of tumor diameter≥5 cm than AA+AC genotype(P<0.05).In patients≥60 years old,the TT genotype of HLA-DRB1 rs3997872 had a higher risk of vascular invasion than the AA+AT genotype(P<0.001).Moreover,regardless of whether the patients were male or female,with or without a drinking history,and with or without a history of HBV/HCV infection,patients with the TT genotype of HLA-DRB1 rs3997872 had a higher risk of vascular invasion than those with the AA+AT genotype(P<0.05).Patients with TT genotype of HLA-DRB1 rs3997872 had a poorer postoperative prognosis than those with the AT+AA genotype(P<0.001).HLA-DRB1 rs3997872 SNP(P=0.019),older age(P<0.001),history of HBV/HCV infection(P<0.001),capsular invasion(P=0.005),vascular invasion(P=0.018),advanced BCLC stage(P<0.001),non-radical surgery(P=0.024),and higher PS score(P=0.023)were independent risk factors for the postoperative prognosis of HCC patients.Conclusion The SNPs of rs2647073 and rs3997872 in HLA-DRB1 gene are associated with the clinicopathological features of primary HCC,and the SNP of rs3997872 is associated with the postoperative prognosis of patients with HCC.

Background: Epimedii Folium, first recorded in the Shennong’s Classic of Materia Medica (Shen Nong Ben Cao Jing), is a traditional Chinese medicine (TCM) known for its effects of “benefiting Qi and strengthening the heart.” Icariside II (ICS II) is one of the main active components of Epimedii Folium, possessing cardiovascular protective and anti-inflammatory properties. However, the potential mechanisms of ICS II on myocardial ischemia (MI) remain unclear. Objective: The aim of the study was to investigate the effects and preliminary molecular mechanisms of ICS II in treating isoproterenolinduced MI in rats. Methods: A rat model of MI was established by subcutaneous injection of isoproterenol. Electrocardiography, echocardiography, myocardial enzymes analysis, heart weight index, triphenyltetrazolium chloride staining, histopathology, TUNEL staining, RT-qPCR, and Western blot were employed to evaluate the effects and preliminary molecular mechanisms of ICS II on MI rats. Results: Pharmacodynamic studies suggested that ICS II inhibited ST-segment elevation in electrocardiograms, improved cardiac function, reduced heart weight index and myocardial enzyme levels, decreased myocardial infarct size, alleviated cardiac histological damage, and inhibited apoptosis, thereby exerting cardioprotective effects in MI rats. Further studies revealed that ICS II may partially inhibit the expression of NLRP3/Caspase-1 axis-related targets at both protein and mRNA levels. Conclusions: Our findings indicate that ICS II exerts anti-MI effects, and its preliminary molecular mechanisms may be related to inhibiting the activation of the NLRP3/Caspase-1 axis to alleviate inflammatory responses.

Background: Epimedii Folium, first recorded in the Shennong’s Classic of Materia Medica (Shen Nong Ben Cao Jing), is a traditional Chinese medicine (TCM) known for its effects of “benefiting Qi and strengthening the heart.” Icariside II (ICS II) is one of the main active components of Epimedii Folium, possessing cardiovascular protective and anti-inflammatory properties. However, the potential mechanisms of ICS II on myocardial ischemia (MI) remain unclear. Objective: The aim of the study was to investigate the effects and preliminary molecular mechanisms of ICS II in treating isoproterenolinduced MI in rats. Methods: A rat model of MI was established by subcutaneous injection of isoproterenol. Electrocardiography, echocardiography, myocardial enzymes analysis, heart weight index, triphenyltetrazolium chloride staining, histopathology, TUNEL staining, RT-qPCR, and Western blot were employed to evaluate the effects and preliminary molecular mechanisms of ICS II on MI rats. Results: Pharmacodynamic studies suggested that ICS II inhibited ST-segment elevation in electrocardiograms, improved cardiac function, reduced heart weight index and myocardial enzyme levels, decreased myocardial infarct size, alleviated cardiac histological damage, and inhibited apoptosis, thereby exerting cardioprotective effects in MI rats. Further studies revealed that ICS II may partially inhibit the expression of NLRP3/Caspase-1 axis-related targets at both protein and mRNA levels. Conclusions: Our findings indicate that ICS II exerts anti-MI effects, and its preliminary molecular mechanisms may be related to inhibiting the activation of the NLRP3/Caspase-1 axis to alleviate inflammatory responses.

Background: Epimedii Folium, first recorded in the Shennong’s Classic of Materia Medica (Shen Nong Ben Cao Jing), is a traditional Chinese medicine (TCM) known for its effects of “benefiting Qi and strengthening the heart.” Icariside II (ICS II) is one of the main active components of Epimedii Folium, possessing cardiovascular protective and anti-inflammatory properties. However, the potential mechanisms of ICS II on myocardial ischemia (MI) remain unclear. Objective: The aim of the study was to investigate the effects and preliminary molecular mechanisms of ICS II in treating isoproterenolinduced MI in rats. Methods: A rat model of MI was established by subcutaneous injection of isoproterenol. Electrocardiography, echocardiography, myocardial enzymes analysis, heart weight index, triphenyltetrazolium chloride staining, histopathology, TUNEL staining, RT-qPCR, and Western blot were employed to evaluate the effects and preliminary molecular mechanisms of ICS II on MI rats. Results: Pharmacodynamic studies suggested that ICS II inhibited ST-segment elevation in electrocardiograms, improved cardiac function, reduced heart weight index and myocardial enzyme levels, decreased myocardial infarct size, alleviated cardiac histological damage, and inhibited apoptosis, thereby exerting cardioprotective effects in MI rats. Further studies revealed that ICS II may partially inhibit the expression of NLRP3/Caspase-1 axis-related targets at both protein and mRNA levels. Conclusions: Our findings indicate that ICS II exerts anti-MI effects, and its preliminary molecular mechanisms may be related to inhibiting the activation of the NLRP3/Caspase-1 axis to alleviate inflammatory responses.

OBJECTIVE: To explore the effect of chitosan (CS) hydrogel loaded with tendon-derived stem cells (TDSCs; hereinafter referred to as TDSCs/CS hydrogel) on tendon-to-bone healing after rotator cuff repair in rabbits. METHODS: TDSCs were isolated from the rotator cuff tissue of 3 adult New Zealand white rabbits by Henderson step-by-step enzymatic digestion method and identified by multidirectional differentiation and flow cytometry. The 3rd generation TDSCs were encapsulated in CS to construct TDSCs/CS hydrogel. The cell counting kit 8 (CCK-8) assay was used to detect the proliferation of TDSCs in the hydrogel after 1-5 days of culture in vitro, and cell compatibility of TDSCs/CS hydrogel was evaluated by using TDSCs alone as control. Another 36 adult New Zealand white rabbits were randomly divided into 3 groups ( n=12): rotator cuff repair group (control group), rotator cuff repair+CS hydrogel injection group (CS group), and rotator cuff repair+TDSCs/CS hydrogel injection group (TDSCs/CS group). After establishing the rotator cuff repair models, the corresponding hydrogel was injected into the tendon-to-bone interface in the CS group and TDSCs/CS group, and no other treatment was performed in the control group. The general condition of the animals was observed after operation. At 4 and 8 weeks, real-time quantitative PCR (qPCR) was used to detect the relative expressions of tendon forming related genes (tenomodulin, scleraxis), chondrogenesis related genes (aggrecan, sex determining region Y-related high mobility group-box gene 9), and osteogenesis related genes (alkaline phosphatase, Runt-related transcription factor 2) at the tendon-to-bone interface. At 8 weeks, HE and Masson staining were used to observe the histological changes, and the biomechanical test was used to evaluate the ultimate load and the failure site of the repaired rotator cuff to evaluate the tendon-to-bone healing and biomechanical properties. RESULTS: CCK-8 assay showed that the CS hydrogel could promote the proliferation of TDSCs ( P<0.05). qPCR results showed that the expressions of tendon-to-bone interface related genes were significantly higher in the TDSCs/CS group than in the CS group and control group at 4 and 8 weeks after operation ( P<0.05). Moreover, the expressions of tendon-to-bone interface related genes at 8 weeks after operation were significantly higher than those at 4 weeks after operation in the TDSCs/CS group ( P<0.05). Histological staining showed the clear cartilage tissue and dense and orderly collagen formation at the tendon-to-bone interface in the TDSCs/CS group. The results of semi-quantitative analysis showed that compared with the control group, the number of cells, the proportion of collagen fiber orientation, and the histological score in the TDSCs/CS group increased, the vascularity decreased, showing significant differences ( P<0.05); compared with the CS group, the proportion of collagen fiber orientation and the histological score in the TDSCs/CS group significantly increased ( P<0.05), while there was no significant difference in the number of cells and vascularity ( P>0.05). All samples in biomechanical testing failed at the repair site during the testing process. The ultimate load of the TDSCs/CS group was significantly higher than that of the control group ( P<0.05), but there was no significant difference compared to the CS group ( P>0.05). CONCLUSION TDSCs/CS hydrogel can induce cartilage regeneration to promote rotator cuff tendon-to-bone healing.
Rabbits ; Animals ; Rotator Cuff/surgery* ; Chitosan ; Hydrogels ; Rotator Cuff Injuries/surgery* ; Wound Healing ; Tendons/surgery* ; Collagen ; Stem Cells ; Biomechanical Phenomena

Objective To investigate the effect of Xiao Chaihu Decoction and Xiangsha Liujunzi Decoction on the changes of gastric mucosal pathological score and gastrointestinal hormones in patients with chronic atrophic gastritis(CAG)of liver stagnation and spleen deficiency type.Methods A total of 156 cases of CAG patients with liver stagnation and spleen deficiency syndrome were randomly divided into a control group and an observation group,78 cases in each group.The control group was treated with conventional western medicine,and the observation group was treated with Xiao Chaihu Decoction and Xiangsha Liujunzi Decoction on the basis of treatment for the control group.The course of treatment covered four weeks.The changes in the scores of traditional Chinese medicine(TCM)symptoms such as epigastric distention and pain,poor appetite,loose stools,limb weakness,belching and acid regurgitation,gastric mucosal pathological scores and serum levels of gastrointestinal hormones of motilin(MTL)and gastrin(GAS)in the two groups were observed before and after treatment.The negative conversion rate of Helicobacter pylori(Hp)in the two groups was compared,and the clinical efficacy and safety of the two groups were evaluated.Results(1)After four weeks of treatment,the total effective rate of the observation group was 93.59％(73/78),which was significantly higher than 82.05％(64/78)of the control group,and the difference between the two groups was statistically significant(P＜0.05).(2)After treatment,the scores of TCM symptoms such as epigastric distention and pain,poor appetite,loose stool,limb weakness,belching and acid regurgitation in the two groups were significantly decreased compared with those before treatment(P＜0.05),and the decrease in the observation group was significantly superior to that in the control group(P＜0.05).(3)After treatment,the pathological scores of gastric mucosa in the two groups were significantly decreased when compared with those before treatment(P＜0.05),and the decrease in the observation group was more significant than that in the control group(P＜0.01).(4)After four weeks of treatment,the negative conversion rate of Hp in the observation group was 91.03％(71/78),which was significantly higher than that in the control group(75.64％,59/78),and the difference between the two groups was statistically significant(P＜0.05).(5)After treatment,the level of serum GAS in the two groups was significantly decreased(P＜0.05)and the serum MTL level was significantly increased compared with that before treatment(P＜0.05);the decrease of serum GAS level and the increase of serum MTL level in the observation group were significantly superior to those in the control group(P＜0.05).(6)There were no obvious abnormalities in the routine test of blood,urine,stool,kidney function,and liver function,electrocardiograph and other safety indicators during the treatment of the two groups of patients,no adverse reactions such as dizziness,rash and chest distress occurred either,with high safety.Conclusion Xiao Chaihu Decoction combined with Xiangsha Liujunzi Decoction exerts a significant therapeutic effect on GAS of liver stagnation and spleen deficiency type,which can effectively relieve the clinical symptoms,improve the pathological changes of gastric mucosa and promote Hp negative conversion.The therapeutic mechanism may be related to the regulation of gastrointestinal hormone levels.

Objective:To screen interleukin(IL)-1β secretion-related membrane transporters by macrophage experiment in vitro and conventional knockout mice.Methods:THP-1 cell line was differentiated to obtain human THP-1-derived macrophages,and the primary macrophages were obtained from human peripheral blood.FVB wild-type mice with the same sex and age were used as the controls of MRP1 knockout mice.The macrophages in abdominal cavity and bone marrow of mice were cultivated.The cells were treated with ABCC1/MRP1,ABCG2/BCRP,ABCB1/P-gp,OATP1B1,and MATE transporter inhibitors,then stimulated by lipopolysaccharide and adenosine triphosphate.The secretion level of IL-iβ was detected by ELISA,Western blot,and immunofluorescence.Results:After inhibiting ABCC1/MRP1 transporter,the secretion of IL-1β decreased significantly,while inhibition of the other 4 transporters had no effect.In animal experiment,the level of IL-1 β secreted by macrophages in bone marrow of MRP1 knockout mice was significantly lower than control group(P＜0.05).Conclusion:ABCC1/MRP1 transporter is a newly discovered IL-1β secretion pathway,which is expected to become a new target for solving clinical problems such as cytokine release syndrome.