Mitochondria, functioning not only as the central hub of cellular energy metabolism but also as semi-autonomous organelles, orchestrate cellular fate decisions through their endogenous mitochondrial DNA (mtDNA), which encodes core components of the electron transport chain. Emerging research has identified microRNAs localized within mitochondria, termed mitochondria-located microRNAs (mitomiRs). Recent studies have revealed that mitomiRs are transcribed from nuclear DNA (nDNA), processed and matured in the cytoplasm, and subsequently transported into mitochondria. mitomiRs regulate mtDNA through diverse mechanisms, including modulation of mtDNA expression at the translational level and direct binding to mtDNA to influence transcription. Aberrant expression of mitomiRs leads to mitochondrial dysfunction and contributes to the pathogenesis of metabolic diseases. Restoring mitomiR expression to physiological levels using mitomiRs mimics or inhibitors has been shown to improve mitochondrial function and alleviate related diseases. Consequently, the regulatory mechanisms of mitomiRs have become a major focus in mitochondrial research. Given that mitomiRs are located in mitochondria, targeted delivery strategies designed for mtDNA can be adapted for the delivery of mitomiRs mimics or inhibitors. However, numerous intracellular and extracellular barriers remain, highlighting the need for more precise and efficient delivery systems in the future. The regulation of mtDNA expression mediated by mitomiRs not only expands our understanding of miRNA functions in post-transcriptional gene regulation but also provides promising molecular targets for the treatment of mitochondrial-related diseases. This review systematically summarizes recent research progress on mitomiRs in regulating mtDNA expression and discusses the underlying mechanisms of mitomiRs-mtDNA interactions. Additionally, it provides new perspectives on precision therapeutic strategies, with a particular emphasis on mitomiRs-based regulation of mitochondrial function in mitochondrial-related diseases.

Aim To discover the potential active compounds and possible mechanisms in rheumatoid arthritis (RA) treatment with Zhi-Huang-Zhi-Tong powder (ZHZTP) by using network pharmacology and in vitro study. Methods The active ingredient targets and disease targets of Zhihuang Zhitong Powder were searched and screened by database; they intersected to get a common target; and the "drug-component-target" relationship network diagram was constructed for GO and KEGG enrichment analysis of the overlapping genes; then the core components were docked with the core targets. Finally, based on the inflammation model of HUVECs in vitro, the efficacy and mechanism of Zhihuang Zhitong powder were verified by MTT method, plate scratch test and Western blot. Results Active compounds involved in RA treatment were screened in the present study, and the top two were ursolic acid and emodin, all playing crucial roles in RA treatment with ZHZTP. Additionally, the key target was AKTA, TNF and IL-6. GO and KEGG enrichment analysis revealed that ZHZTP regulated BP, MF and CC, and also focused on regulating AKTA, TNF and IL-6 signaling pathway. Molecular docking showed that interactions between key active compounds and key targets were stable. In vitro ZHZTP significantly inhibited cell viability and migration of TNF-a-stimulated HUVECs, and the involved mechanism may be associated with PI3K/AKT/m-TOR signaling. Conclusions The present study reveals that the potential active compounds of ZHZTP are ursolic acid and emodin, and moreover, the involved mechanisms of ZHZTP for RA treatment are associated with PI3 K/AKT/m-TOR signaling.

Aim To express and purify recombinant hCGH-CTP fusion protein in high-density suspension culture of Chinese hamster ovary cells (CHO-S), and to verify the lipid accumulation effect of rhCGH-CTP on 3T3-L1 mature adipocytes. Methods The recombinant protein expression vector (pcDNA3. 1-rhCGH-CTP) was constructed, achieved by fusing the human glycoprotein hormone beta 5/alpha 2 cDNA with CTP Linker. The expression plasmid was transiently transfected into the suspended CHO-S to express rhCGH-CTP protein and then purified, and the protein biological activity was verified. Intervention with 3T3-L1 mature adipocyte cells for 24 h was performed to detect the changes of intracellular triglyceride (TG) level. Results Western blot results showed that rhCGH-CTP protein was successfully expressed in CHO-S cells, and the yield was up to 715. 4 mg • L~ . The secreted protein was purified by AKTA pure system with higher purity that was up to 90% as identified by SDS-PAGE. In addition, the intracellular cAMP content of mature adipocytes with high expression of TSHR gene significantly increased after intervention with different concentrations of rhCGH-CTP protein by ELISA kit, indicating that rhCGH-CTP protein had biological activity. Oil red 0 staining showed that compared with the control group, the lipid content of mature adipocytes in the intervention groups with different concentrations of rhCGH-CTP protein significantly decreased (P < 0. 05) . Conclusions The rhCGH-CTP protein has been successfully expressed and purified with biological activity, and effectively reduce TG. This research provides an important theoretical basis for further revealing the physiological role of CGH protein and its potential application in clinical practice.

Umbilical cord mesenchymal stem cells (UC-MSCs) have been widely used in regenerative medicine, but there is limited research on the stability of UC-MSCs formulation during production. This study aims to assess the stability of the cell stock solution and intermediate product throughout the production process, as well as the final product following reconstitution, in order to offer guidance for the manufacturing process and serve as a reference for formulation reconstitution methods. Three batches of cell formulation were produced and stored under low temperature (2-8 ℃) and room temperature (20-26 ℃) during cell stock solution and intermediate product stages. The storage time intervals for cell stock solution were 0, 2, 4, and 6 h, while for intermediate products, the intervals were 0, 1, 2, and 3 h. The evaluation items included visual inspection, viable cell concentration, cell viability, cell surface markers, lymphocyte proliferation inhibition rate, and sterility. Additionally, dilution and culture stability studies were performed after reconstitution of the cell product. The reconstitution diluents included 0.9% sodium chloride injection, 0.9% sodium chloride injection + 1% human serum albumin, and 0.9% sodium chloride injection + 2% human serum albumin, with dilution ratios of 10-fold and 40-fold. The storage time intervals after dilution were 0, 1, 2, 3, and 4 h. The reconstitution culture media included DMEM medium, DMEM + 2% platelet lysate, 0.9% sodium chloride injection, and 0.9% sodium chloride injection + 1% human serum albumin, and the culture duration was 24 h. The evaluation items were viable cell concentration and cell viability. The results showed that the cell stock solution remained stable for up to 6 h under both low temperature (2-8 ℃) and room temperature (20-26 ℃) conditions, while the intermediate product remained stable for up to 3 h under the same conditions. After formulation reconstitution, using sodium chloride injection diluted with 1% or 2% human serum albumin maintained a viability of over 80% within 4 h. It was observed that different dilution factors had an impact on cell viability. After formulation reconstitution, cultivation in medium with 2% platelet lysate resulted in a cell viability of over 80% after 24 h. In conclusion, the stability of cell stock solution within 6 h and intermediate product within 3 h meets the requirements. The addition of 1% or 2% human serum albumin in the reconstitution diluent can better protect the post-reconstitution cell viability.

AIM To investigate the effects of different compatibility ratios and processing method on the content of rutin,isoquercetin,ferulic acid,quercetin,isotoosendanin,kaempferol,toosendanin,α-pinene,trans-anethole in the combination use of Toosendan Fructus and Foeniculi Fructus,and to explore the optimal compatibility ratio for its use.METHODS The analysis of HPLC-DAD was performed on a 30℃thermostatic ZORBAX SB C18 column(4.6 mm×250 mm,5 μm),with the mobile phase comprising of acetonitrile-0.1%phosphoric acid flowing at 1.0 mL/min in a gradient elution manner,and the use of DAD detector.SPSS 24.0 software was used to analyze the data differences.RESULTS Nine constituents showed good linear relationships within their own ranges(r≥0.999 1),whose average recoveries were 96.19%-103.13%with the RSDs of 1.86%-2.67%.Generally higher total content of nine constituents were detected in the combination use groups when Toosendan Fructus-Foeniculi Fructus were at ratios of 1 ∶ 1,1 ∶ 2,and 2 ∶ 1 than those single uses(P<0.05),and among which the 1 ∶ 1 ratio contributed the highest total content.After salt processing,decreased content of toosendanin and isotoosendanin,α-pinene and trans-anethole(P<0.05,P<0.01)),increased isoquercetin content(P<0.01),and no significant content changes of other ingredients were detected.CONCLUSION Through this method of high accuracy and good reproducibility,we learn that the combination use of Toosendan Fructus and Foeniculi Fructus promotes the dissolution of the nine constituents,and the maximum content is achieved at ratio of 1 ∶ 1.

Purpose::The reconstruction of Allen's type IV fingertip amputation is a clinical challenge. Our team designed bilateral unequal-sized hallux osteo-onychocutaneous free flaps for the long-term reconstruction of Allen's type IV fingertip amputation and conducted a retrospective study with a 5-year follow-up aims to evaluate the effects of this technique.Methods::A retrospective analysis with a 5-year follow-up including 13 patients with Allen's type IV fingertip amputation who were admitted to our hospital from January 2010 to January 2017 was conducted. The patients were treated with bilateral unequal-sized hallux osteo-onychocutaneous free flaps. The operation time, intraoperative blood loss, and complications were recorded, and the survival rate of the transplanted flaps was calculated. During the 5-year follow-up after operation, the nail growth time was recorded and the finger appearance was observed. At the last follow-up appointment, the length, width, and girth of the reconstructed fingertip and contralateral normal fingertip, range of motion of the reconstructed fingertip and contralateral normal fingertip, Semmes-Weinstein test (for the evaluation of tactile sensation), and two-point discrimination testing results were recorded. SPSS 22.0 software was used for the statistical analysis and the data are presented as mean ± SD.Results::The mean operation time was (5.62 ± 0.51) h, the mean intraoperative blood loss was (34.15 ± 3.13) mL, and the survival rate of the transplanted flaps was 100%. During the 5-year follow-up, the average nail growth time was (10.14 ± 1.98) months and the average bone union time was (3.78 ± 0.91) months. The length, width, and girth of the reconstructed fingertip were (31.52 ± 3.73) mm, (17.82 ± 1.74) mm, and (59.75 ± 3.04) mm, respectively, which did not differ from those of the contralateral normal fingertip. The range of motion of the reconstructed fingertip was (12.15 ± 2.79) degrees which is different from that of the contralateral normal fingertip. The average tactile sensation evaluated via the Semmes-Weinstein test and the average two-point discrimination test of the reconstructed fingertip were (0.39 ± 0.17) g and (7.46 ± 1.14) mm, respectively, which were not different from those of the contralateral normal fingertip. The average Maryland score of feet in the donor area was 87.66 ± 7.39, which was satisfactory.Conclusion::Bilateral unequal-sized hallux osteo-onychocutaneous free flaps are an effective method to reconstruct Allen's type IV fingertip amputations with a satisfactory appearance and good sensory function.

Objective To investigate the effects of oral use combined with percutaneous iontophoresis of Osteoking(mainly composed of Citri Reticulatae Pericarpium,Carthami Flos,Notoginseng Radix et Rhizoma,Eucommiae Cortex,Ginseng Radix et Rhizoma,Daturae Flos,Astragali Radix,Radixet Caulis Schizophragma Integrifolia,and Trionycis Carapax)on the pain,lumbar function and serological indicators of the patients with lumbar disc herniation(LDH).Methods Ninety-two patients with LDH were randomly divided into control group(31 cases),oral use group(31 cases)and combination group(30 cases).The control group was given oral administration of Celecoxib,the oral use group was given oral administration of Osteoking,and the combination group was given oral administration combined with percutaneous iontophoresis of Osteoking.The course of treatment for the 3 groups covered 6 weeks.The changes of pain visual analogue scale(VAS)score,lumbar function Japanese Orthopedic Association(JOA)score,serum tumor necrosis factor alpha(TNF-α)and matrix metalloproteinase 3(MMP-3)levels in the three groups were observed before and after treatment.Results(1)During the trial,there were 2 patients from both control group and oral use group lost to follow-up,but none of the combination group was lost to follow-up.Eventually,29 patients in the control group,29 patients in the oral group and 30 patients in the combination group were included in the statistical analysis.(2)After treatment,the pain VAS score and lumbar function JOA score in the three groups were significantly lower than those before treatment(P＜0.05).The intergroup comparison showed that the decrease of pain VAS score and lumbar function JOA score in the combination group was significantly superior to that in the control group and the oral use group(P＜0.05),while there was no significant difference between the control group and the oral use group(P＞0.05).(3)After treatment,the serum TNF-α level in the three groups was significantly lower than that before treatment(P＜0.05),while no significant difference of TNF-α level was presented among the three groups after treatment(P＞0.05).(4)After treatment,the serum MMP-3 level in the oral use group and combination group was significantly lower than that before treatment(P＜0.05),but no obvious change of MMP-3 level was shown in the control group(P＞0.05).The intergroup comparison showed that the decrease of serum MMP-3 level in the oral use group and combination group was significantly superior to that in the control group(P＜0.05),while no significant difference of TNF-αlevel was presented between the oral use group and combination group(P＞0.05).Conclusion Osteoking is effective on relieving the pain symptoms of LDH patients,improving the lumbar activity function,and decreasing the levels of serum TNF-α and MMP-3.The efficacy of Osteoking by oral use combined with percutaneous iontophoresis is stronger than that of Osteoking orally alone or Celecoxib.

In recent years,the development of acupuncture and moxibustion(shortened as acup-moxibustion)has flourished.With the verification of clinical efficacy of acup-moxibustion,its basic research has gradually drawn the attention of the practitioners accordingly.But how to scientifically perform the basic research of acup-moxibustion and to serve the clinic effectively has become a major problem for the contemporary Chinese medicine practitioners.By analyzing the characteristics of acup-moxibustion-related research projects funded by the National Natural Science Foundation of China,this paper outlined the current status of domestic research of acup-moxibustion,and proposed four suggestions after analyzing the problems and weaknesses of acup-moxibustion basic research in China:①the clinical evidence-based system in the current acup-moxibustion should be further constructed and the basic research should be focused on the area of advantages;② the key problems of acup-moxibustion basic research should be clarified,and the proportion of original researches should be increased;③ the integration of production,teaching and research of acup-moxibustion should be enhanced to adapt to the era of big science;④ the funding system and its polity and structure needed to be reformed.This study will help to increase the discipline ranking of acup-moxibustion,enhance its high-quality development,and promote its internationalization.

Objective To investigate the role and underlying mechanisms of miR-34a/SIRT1 in intensive care unit acquired weakness(ICU-AW).Methods(1)C2C12 mouse skeletal muscle cells were induced to differentiate into myotubes,and were divided into two groups:model group[ICU-AW group,treated with lipopolysaccharides(LPS)for 12 hours]and normal control group(treated with the same amount of sterile water for 12 hours).Western blotting was used to detect the protein expression level of Muscle ring finger 1(MuRF-1),atrophy gene 1(Atrogin-1)and Sirtuin-1(SIRT1).RT-qPCR was used to assess the mRNA expression level of microRNA-34a(miR-34a),MuRF-1,Atrogin-1 and SIRT1,and light microscope was used to observe the growth and differentiation of C2C12 skeletal muscle cells in each group.(2)ICU-AW cells were further subdivided into control group(treated with siRNA transfection agent intervention),Scra siRNA group(treated with transfection agent and non-specific siRNA),miR-34a siRNA group(treated with transfection agent and specific siRNA intervention),vehicle group(treated with agonist solvent dimethyl sulfoxide)and SRT1720 group(treated with SIRT1 agonist SRT1720).Western blotting was used to detect the protein expression level of SIRT1,Atrogin-1 and MuRF-1 in each group.RT-qPCR was used to detect the miR-34a and the mRNA expression level of SIRT1,Atrogin-1 and MuRF-1 in each group.(3)In addition,another group of ICU-AW cells were divided into control group(treated with siRNA transfection),miR-34a siRNA group(treated with transfection agent and specific siRNA intervention),miR-34a siRNA+vehicle group(treated with transfection agent,specific siRNA and Dimethyl sulfoxide intervention)and miR-34a siRNA+EX-527 group(treated with transfection agent,specific siRNA and SIRT1 inhibitor EX-527).Western blotting was used to detect the protein expression level of Atrogin-1 and MuRF-1.RT-qPCR was used to assess the mRNA expression level of Atrogin-1 and MuRF-1.Results Myotube differentiation was observed on the 4th day.Compared with control group,myotube atrophy was obvious in ICU-AW group.RT-qPCR and Western blotting results revealed that,compared with normal control group,in ICU-AW group,the mRNA and protein expression levels of Atrogin-1 and MuRF-1 significantly increased(P＜0.05),and the expression level of miR-34a significantly increased(P＜0.05),while the mRNA and protein expression levels of SIRT1 significantly decreased(P＜0.05).RT-qPCR results showed that,compared with control group(treated with siRNA transfection agent intervention)and Scra siRNA group,the expression of miR-34a and mRNA expression of Atrogin-1 and MuRF-1 in miR-34a siRNA group significantly decreased(P＜0.05),while the mRNA expression of SIRT1 significantly increased(P＜0.05),meanwhile the protein expression of Atrogin-1 and MuRF-1 decreased significantly(P＜0.01),and the protein expression of SIRT1 significantly increased(P＜0.05).RT-qPCR results also showed that,compared with vehicle group,the mRNA expression of Atrogin-1 and MuRF-1 in SRT1720 group decreased significantly(P＜0.05),while SIRT1 increased significantly(P＜0.05).Western blotting results demonstrated that,compared with control group and Scra siRNA group,the protein expression of Atrogin-1 and MuRF-1 in miR-34a siRNA group decreased significantly(P＜0.05),while SIRT1 increased significantly(P＜0.05).RT-qPCR and Western blotting results indicated that,compared with miR-34a siRNA+vehicle group,the mRNA and protein expression of Atrogin-1 and MuRF-1 in miR-34a siRNA+EX-527 group increased significantly(P＜0.05).Conclusion Overactivation of miR-34a in ICU-AW contributes to skeletal muscle atrophy by inhibiting the expression of SIRT1,which may play an important role in the pathogenesis of ICU-AW.

Objective:To evaluate the efficacy and safety of hypomethylating agents (HMA) in patients with myelodysplastic syndromes (MDS) .Methods:A total of 409 MDS patients from 45 hospitals in Zhejiang province who received at least four consecutive cycles of HMA monotherapy as initial therapy were enrolled to evaluate the efficacy and safety of HMA. Mann-Whitney U or Chi-square tests were used to compare the differences in the clinical data. Logistic regression and Cox regression were used to analyze the factors affecting efficacy and survival. Kaplan-Meier was used for survival analysis. Results:Patients received HMA treatment for a median of 6 cycles (range, 4-25 cycles) . The complete remission (CR) rate was 33.98% and the overall response rate (ORR) was 77.02%. Multivariate analysis revealed that complex karyotype ( P=0.02, OR=0.39, 95% CI 0.18-0.84) was an independent favorable factor for CR rate. TP53 mutation ( P=0.02, OR=0.22, 95% CI 0.06-0.77) was a predictive factor for a higher ORR. The median OS for the HMA-treated patients was 25.67 (95% CI 21.14-30.19) months. HMA response ( P=0.036, HR=0.47, 95% CI 0.23-0.95) was an independent favorable prognostic factor, whereas complex karyotype ( P=0.024, HR=2.14, 95% CI 1.10-4.15) , leukemia transformation ( P<0.001, HR=2.839, 95% CI 1.64-4.92) , and TP53 mutation ( P=0.012, HR=2.19, 95% CI 1.19-4.07) were independent adverse prognostic factors. There was no significant difference in efficacy and survival between the reduced and standard doses of HMA. The CR rate and ORR of MDS patients treated with decitabine and azacitidine were not significantly different. The median OS of patients treated with decitabine was longer compared with that of patients treated with azacitidine (29.53 months vs 20.17 months, P=0.007) . The incidence of bone marrow suppression and pneumonia in the decitabine group was higher compared with that in the azacitidine group. Conclusion:Continuous and regular use of appropriate doses of hypomethylating agents may benefit MDS patients to the greatest extent if it is tolerated.