Objective To evaluate the clinical efficacy of"Tongdu Tiaoshen"acupuncture combined with hand function rehabilitation training in patients with hand dysfunction following cerebral infarction.Methods A total of 104 patients were randomly assigned to either the observation group(receiving"Tongdu Tiaoshen"acupunc-ture combined with hand function rehabilitation training)or the control group(undergoing hand function rehabilita-tion training alone),with 52 patients in each group.Both groups underwent a 4-week treatment regimen.The following outcome measures were assessed pre-and post-treatment:Modified Ashworth Scale(MAS),Modified Lindmark Scale(MLS),Brunnstrom Motor Function Score(BMS)for upper limbs,Fugl-Meyer Assessment(FMA)of upper limb motor function,Modified Barthel Index(MBI),and grip strength(GS).Adverse events were docu-mented,and the clinical efficacy in each group was evaluated by calculating the effective rate.Results After excluding dropouts,a total of 94 cases were included in the final statistical analysis,with 48 cases assigned to the observation group and 46 to the control group.Both groups demonstrated significant improvements in MLS,MAS,BMS,FMA,MBI,and GS scores compared to pre-treatment levels,with all differences being statistically significant(P<0.05).Following treatment,intergroup comparisons also revealed statistically significant differences in all mea-sured indicators(P<0.05).The total effective rate was higher in the observation group(91.7%)than in the control group(82.6%),and the difference in efficacy grades between the two groups was statistically significant(P<0.05).Conclusions"Tongdu Tiaoshen"acupuncture,when combined with hand function training,demonstrates signifi-cantly greater efficacy than hand function rehabilitation training alone in improving post-cerebral infarction hand dysfunction.It enhances grip strength and fine motor coordination of the affected limb,alleviates hand muscle spasticity and joint stiffness,and promotes recovery of upper limb motor function,thereby improving patients'activities of daily living.This integrated therapeutic approach warrants further clinical validation and in-depth research for broader implementation.

Objective To evaluate the clinical efficacy of"Tongdu Tiaoshen"acupuncture combined with hand function rehabilitation training in patients with hand dysfunction following cerebral infarction.Methods A total of 104 patients were randomly assigned to either the observation group(receiving"Tongdu Tiaoshen"acupunc-ture combined with hand function rehabilitation training)or the control group(undergoing hand function rehabilita-tion training alone),with 52 patients in each group.Both groups underwent a 4-week treatment regimen.The following outcome measures were assessed pre-and post-treatment:Modified Ashworth Scale(MAS),Modified Lindmark Scale(MLS),Brunnstrom Motor Function Score(BMS)for upper limbs,Fugl-Meyer Assessment(FMA)of upper limb motor function,Modified Barthel Index(MBI),and grip strength(GS).Adverse events were docu-mented,and the clinical efficacy in each group was evaluated by calculating the effective rate.Results After excluding dropouts,a total of 94 cases were included in the final statistical analysis,with 48 cases assigned to the observation group and 46 to the control group.Both groups demonstrated significant improvements in MLS,MAS,BMS,FMA,MBI,and GS scores compared to pre-treatment levels,with all differences being statistically significant(P<0.05).Following treatment,intergroup comparisons also revealed statistically significant differences in all mea-sured indicators(P<0.05).The total effective rate was higher in the observation group(91.7%)than in the control group(82.6%),and the difference in efficacy grades between the two groups was statistically significant(P<0.05).Conclusions"Tongdu Tiaoshen"acupuncture,when combined with hand function training,demonstrates signifi-cantly greater efficacy than hand function rehabilitation training alone in improving post-cerebral infarction hand dysfunction.It enhances grip strength and fine motor coordination of the affected limb,alleviates hand muscle spasticity and joint stiffness,and promotes recovery of upper limb motor function,thereby improving patients'activities of daily living.This integrated therapeutic approach warrants further clinical validation and in-depth research for broader implementation.

AIM:To investigate the role of specific long noncoding RNA SLC25a6(lncSLC25a6)in homocys-teine(Hcy)-induced cuproptosis in cardiomyocytes.METHODS:Rat cardiomyocytes were cultured in vitro and divided into control group and Hcy group.After 48 h of intervention,the expression levels of cuproptosis-related proteins,ferre-doxin 1(FDX1)and heat shock protein 70(HSP70),were detected by Western blot and immunofluorescence staining.The oxidative stress state of cardiomyocytes was assessed using fluorescence staining,and the intracellular Cu2+levels were measured using a copper ion assay kit.Furthermore,the impact of Hcy on the expression of cuproptosis-related proteins in cardiomyocytes was analyzed following overexpression of lncSLC25a6.RESULTS:Compared with the control group,80 μmol/L Hcy significantly accelerated cardiomyocyte damage,with a notable underexpression of lncSLC25a6(P<0.05).Western blot results indicated that,compared with the control group,the expression level of FDX1 in the Hcy intervention group was significantly reduced(P<0.05),while the expression level of HSP70 was significantly elevated(P<0.05),and the expression level of copper ions in cardiomyocytes of the Hcy group was increased(P<0.05).Immunofluorescence staining showed a significant reduction in FDX1 fluorescence intensity and a significant increase in HSP70 fluorescence in-tensity in the Hcy group.Further overexpression of lncSLC25a6 significantly mitigated Hcy-induced cuproptosis in cardio-myocytes,resulting in elevated expression of FDX1 and reduced expression of HSP70(P<0.05).Pearson correlation analysis demonstrated that the expression level of lncSLC25a6 was negatively correlated with FDX1 protein expression(r=-0.676,P=0.046)and positively correlated with HSP70 expression(r=0.680,P=0.044).CONCLUSION:lnc-SLC25a6 significantly mitigates Hcy-induced cuproptosis in cardiomyocytes,positioning it as a potential therapeutic target for managing Hcy-induced cardiac injury.

Objective To study the role of ubiquitin-conjugating enzyme 9(UBC9)in the pyroptosis of homocysteine-induced macrophages mediated by small ubiquitin-like modifier(SUMO)modification.Methods First,the effects of homocysteine at different concentrations(0 μmol/L,50 μ.mol/L,100 μmol/L,150 μmol/L and 200 μmol/L)on the viability and pyrodeath of mouse macrophages(RAW264.7)were detected by CCK-8 and Western blot.Western blot was used to detect the expression levels of UBC9,SUMO-1,and the inflammatory cytokine IL-1β in different groups of cells.qRT-PCR was used to detect the mRNA expression of UBC9 before and after RNA interference and the expression of UBC9,pyrogen-related protein,and SUMO-1 after RNA interference.Results After stimulation with 100 μmol/L homocysteine,the effect of macrophage activity was minimal,and NLRP3 and Caspase-1 were the proteins with the most obvious increase in expression(P＜0.05).Compared with the Control group,the Hcy group's expression of IL-1β and SUMO-1 was increased(P＜0.01).Compared with the Control group,the Hcy group's UBC9 protein and mRNA levels were increased(P＜0.05).The expression of NLRP3,Caspase-1,IL-1β,UBC9,and SUMO-1 was decreased in the si-UBC9+Hcy group compared with the si-NC+Hcy group(P＜0.01).Conclusions Homocysteine induces pyroptosis in macrophages,and its mechanism of action is related to the up-regulation of UBC9 to induce SUMO modification.

BACKGROUND:High-methionine diet can cause liver injury in Cbs+/-mice,and hyperhomocystinemia is related to the occurrence and progression of various liver-related diseases,such as hepatic steatosis,autoimmune hepatitis,and alcoholic fatty liver disease.MicroRNAs(miRNAs)are involved in various cellular processes including cell survival,differentiation and autophagy,which are of great significance. OBJECTIVE:To investigate the critical role of miR-144-3p on Cbs+/-mouse hepatocyte autophagy induced by high methionine die. METHODS:(1)Ten male cystathione-β-synthase normal(Cbs+/+)mice and another 10 male mice with single gene knockout(Cbs+/-)of similar body mass,4 weeks of age,were fed a high-methionine diet and executed after 12 weeks to take liver tissue.(2)Human hepatocytes(HL-7702)were cultured in vitro and divided into control[0 μmol/L homocysteine(Hcy)],Hcy(100 μmol/L Hcy),mimic-NC(transfected with mimic-NC),mimic-NC + Hcy(mimic-NC transfecton+100 μmol/L Hcy),miR-144-3p mimic(transfected with miR-144-3p mimic),and miR-144-3p mimic + Hcy(miR-144-3p mimic transfection+100 μ mol/L Hcy),inhibitor-NC(transfected with inhibitor-NC),inhibitor-NC + Hcy(inhibitor-NC transfection + 100 μmol/L Hcy),miR-144-3p inhibitor(transfected with miR-144-3p inhibitor),and miR-144-3p inhibitor + Hcy(miR-144-3p inhibitor transfection + 100 μmol/L Hcy).Quantitative real-time PCR was used to detect the expression of miR-144-3p in liver tissue and hepatocytes.After transfection of miR-144-3p mimic or inhibitor,quantitative real-time PCR and western blot were used to detect the transfection efficiency of miR-144-3p and its effect on the expression of autophagy-related proteins LC3B and p62.The levels of alanine transferase and aspartate aminotransferase in hepatocyte supernatants were determined by enzyme linked immunosorbent assay.The correlation between the expression of miR-144-3 in hepatocyte and the levels of alanine transferase and aspartate aminotransferase in hepatocyte supernatants were analyzed by Pearson correlation analysis. RESULTS AND CONCLUSION:Compared with the Cbs+/+ group and control group,the expression of miR-144-3p in the liver tissue of the Cbs+/-group and in hepatocytes of the Hcy group was decreased(P<0.01).The expression of LC3B-Ⅱ/Ⅰ was decreased in hepatocyte after transfection of miR-144-3p mimic,while the protein expression of p62 was increased(P<0.01).The opposite results were obtained after transfection of miR-144-3p inhibitor(P<0.01).Compared with the mimic-NC group,the levels of alanine transferase and aspartate aminotransferase were decreased in the miR-144-3p mimic group(P<0.01),while the opposite results were obtained in the inhibitor-NC group(P<0.01).The expression of miR-144-3p in hepatocytes was negatively correlated with the levels of alanine transferase(P<0.01,r=-0.887 6)and aspartate aminotransferase(P<0.01,r=-0.829 9)in the supernatant of hepatocytes.To conclude,Hcy promotes hepatocyte autophagy by inhibiting the expression of miR-144-3p,which subsequently aggravates liver injury.

Aim To investigate the role of miR-125 b and its DNA methylation in homocysteine ( Hcy )-in-duced vascular smooth muscle cells( VSMCs) prolifera-tion. Methods VSMCs were stimulated with 0,50, 100, 200, 500 μmol · L-1 Hcy respectively. Then qRT-PCR was used to detect the mRNA levels of miR-125b,and nested-touchdown methylation-specific PCR ( ntMS-PCR) was used to detect the methylation levels of miR-125b. VSMCs were transfected with miR-125b precursor or the inhibitor of miR-125b ,then 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyl tetrazolium bromide ( MTT ) assay was used to reflect the proliferation of VSMCs. The distribution of CpG islands of miR-125b promoter region was analyzed by bioinformatics meth-ods. VSMCs were stimulated with 100 μmol·L-1 Hcy and transfected with or without DNA methylation inhib-itors 5-nitrogen impurity cytidine ( AZC) , then the ex-pression of miR-125b was detected by qRT-PCR. Re-sults The mRNA levels of miR-125 b were decreased in 100,200,500 μmol·L-1 Hcy group compared with 0 μmol·L-1 Hcy group. The precursor of miR-125b could inhibit the proliferation activity and the inhibitor of miR-125 b could increase the proliferation activity of VSMCs cells. Bioinformatics analysis indicated that MiR-125 b promoter region had a CpG island whose length was 792 bp ( 1881-2672 ) . The miR-125 b pro-moter region methylation levels increased after Hcy in-tervention ( P <0. 01 ) . The expression level of miR-125 b increased after AZC intervention ( P <0. 05 ) . Conclusions ① Hcy promotes vascular smooth mus-cle cell proliferation maybe by down-regulating the ex-pression of miR-125b. ② Hcy down-regulates the ex-pression of miR-125 maybe by up-regulating the methy-lation levels of miR-125b promoter region.

Aim To investigate the possible mecha-nisms of the levels of NO decrease induced apoptosis in human placental trophoblast cells. Methods Human placental trophoblast cells ( HTR-8 ) were cultured in 5 ml DMEM-F12 culture medium with 37℃ 5% CO2 . Then, the old culture medium was discarded and re-placed with 10,100,500,1 000 μmol·L-1 L-NAME, and the group without L-NAME was set as the control group, cultured for 48h. The effects of L-NAME on the survival of cells were detected by methylthiazolyldiphe-nyl tetrazolium bromide ( MTT); the content of NO in cells was tested by nitrate reductive enzymatic;trans-mission electron microscopy, flow cytometry analysis and Annexin-V FITC dyeing were used to test the effects of L-NAME on apoptosis in HTR-8 cells;restore Fe3+ colorimetric assay was applied for detection of to-tal antioxidant capacity ( T-AOC ) , xanthine oxidase for detection of superoxide dismutase ( SOD) activity, and thiobarbituric acid colorimetry for determination of content of MDA. Results Compared with the control group, the survival rate of HTR-8 cells and the levels of NO in 100,500,1 000 μmol·L-1 L-NAME group were significantly reduced(P<0.05,P<0.01). Flow analysis and Annexin-V FITC staining showed that L-NAME could induce cell apoptosis in a dose-dependent manner. The number of cell apoptosis was negatively correlated with the content of NO ( r = -0.5210 ) in HTR-8 cells. Transmission electron microscopy results showed that compared with the control group, the ex-perimental group's cell nucleus shape was irregular, nuclear pyknosis in irregular shape, the chromatin ag-glutination or side the collection, mitochondrial swell-ing or enrichment, crest fracture or dissolved, even vanished, forming the vacuole, especially in 100 μmol ·L-1 L-NAME group, the apoptotic bodies obviously appeared. At the same time, T-AOC, SOD levels in HTR-8 cells decreased ( P <0.05 ) , and the MDA content increased ( P<0.05 ) . The number of cell ap-optosis was negatively correlated with the level of T-AOC ( r= -0.3212 ) , SOD ( r= -0.2779 ) in HTR-8 cells , while positively correlated with the content of MDA(r=0.2807). Conclusion Oxidative stress may play an important role in the levels of NO decrease in-duced apoptosis in human placental trophoblast cells.

Aim To explore the role of ERO1 αand its DNA methylation in homocysteine (Hcy)-induced in-hibition of hepatocytes proliferation.Methods The hepatocytes stimulated with 0 μmol·L -1 Hcy were set as the normal group (NC group)and the hepatocytes stimulated with 1 00 μmol·L -1 Hcy as the experimen-tal group (Hcy group).Methyl thiazolyl tetrazolium (MTT)reduction assay was used to reflect the prolifer-ation of the hepatocytes;qRT-PCR and Western blot were used to detect the mRNA and protein levels of ERO1 α;the expression of green fluorescence protein was observed in hepatocytes after the recombinant plas-mid of ERO1 α was constructed,which was used to confirm if the recombinant plasmid into hepatocytes was successful,then the mRNA and protein levels of ERO1 αwere assayed and the proliferation of the hepa-tocytes was also detected;ntMSP was used to detect the change of ERO1 αDNA methylation.Results The mRNA and protein levels of ERO1 αwere decreased in Hcy group compared with NC group,and the prolifera-tion activity of hepatocytes in Hcy group was de-creased.Sequencing result showed that the recombi-nant plasmid of ERO1 αwas constructed successfully. QRT-PCR and Western blot revealed that ERO1 αwas overexpressed. The result of MTT suggested that ERO1 αoverexpression restored hepatocyte proliferation inhibited by Hcy.Hcy caused ERO1 αDNA hyperm-ethylation.Conclusions Hcy inhibits hepatocyte pro-liferation by downregulating the expression of ERO1 α, and methylation of ERO1 αpromoter may play a role in this process.