ObjectiveTo explore the potential mechanism underlying electroacupuncture therapy with the midnight-noon ebb-low method with hour-prescription for chronic obstructive pulmonary disease （COPD） with lung-qi deficiency syndrome from the perspective of brain and muscle basic helix-loop-helix ARNT-like 1 （BMAL1） as well as neutrophil extracellular traps （NETs）. MethodsThe experiment was conducted in two phases. For the therapeutic effectiveness observation， 24 rats were randomly allocated into control group 1， model group 1， midnight-noon electro-acupuncture group 1 and conventional electro-acupuncture group， with 6 rats per group. Rat models of COPD with lung-qi deficiency syndrome were established via cigarette smoke exposure combined with intratracheal instillation of lipopolysaccharide （LPS）. After successful modelling， rats in midnight-noon electro-acupuncture group 1 received electro-acupuncture at acupoints "Taiyuan （LU 9）"， "Feishu （BL 13）" and "Zusanli （ST 36）" during 05：00—07：00， 30 minutes per treatment once every other day for 14 consecutive days （7 sessions in total）. Rats in the conventional electro-acupuncture group received identical electroacupuncture manipulation at random daytime hours （08：00—18：00）. Pulmonary function parameters including forced expiratory volume in 0.3 second （FEV_0.3）， forced vital capacity （FVC） and FEV_0.3/FVC ratio were detected. Pulmonary histopathological changes were observed via hematoxylin-eosin （HE） staining. Plasma levels of pro-inflammatory factors interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α）， as well as pulmonary reactive oxygen species （ROS） content， BMAL1 protein expression and the levels of neutrophil extracellular traps （NETs） biomarkers such as myeloperoxidase （MPO）， neutrophil elastase （NE）， and citrullinated histone H3 （CitH3） in lung tissue were determined. For the mechanistic verification phase， 36 rats were divided into control group 2， model group 2， midnight-noon electro-acupuncture group 2， overexpression empty group， overexpression BMAL1 group and overexpression with midnight-noon electro-acupuncture group， with 6 rats in each group. At the 5th week of model construction， rats in the overexpression BMAL1 group and overexpression with midnight-noon electro-acupuncture group received intratracheal instillation of 100 μl adenoviral suspension carrying overexpressed Bmal1 gene. Rats in the overexpression empty group were injected with an equal titer and equal volume of blank adenovirus without the Bmal1 coding sequence into the lung at the identical time point. Midnight-noon electro-acupuncture group 2 and overexpression with midnight-noon electro-acupuncture group were consistent with the aforementioned protocol. Finally， pulmonary levels of MPO， NE and CitH3 were measured in all groups. ResultsCompared with the control group 1， the model group 1 exhibited decreased FEV_0.3， FVC and FEV_0.3/FVC ratio， elevated plasma IL-1β， TNF-α and pulmonary ROS levels， downregulated lung BMAL1 protein expression， and increased contents of MPO， NE and CitH3 （all P＜0.05）； typical NETs-like structures with sparse granular substances attached to fibrous networks were observed under scanning electron microscopy； HE staining revealed damaged alveolar architecture accompanied by inflammatory cell infiltration. Compared with model group 1， both the midnight-noon electro-acupuncture group 1 and conventional electro-acupuncture group achieved obvious improvements in all above indicators， with superior therapeutic effects in midnight-noon electro-acupuncture group 1 （P＜0.05）. Consistently， midnight-noon electro-acupuncture group 1 showed more prominent alleviation of NETs-like structure formation and lung pathological injury compared to the conventional electro-acupuncture group. Mechanistic validation results demonstrated that compared with model group 2， the average optical density of MPO as well as the protein levels of MPO， NE and CitH3 were markedly reduced in midnight-noon electro-acupuncture group 2， overexpression BMAL1 group and overexpression with midnight-noon electro-acupuncture group （P＜0.05）； furthermore， these parameters were significantly lower in the overexpression with midnight-noon electro-acupuncture group than in midnight-noon electro-acupuncture group 2 （P＜0.05）. ConclusionElectro-acupuncture based on the midnight-noon ebb-low method with hour-prescription may alleviate pulmonary inflammation in COPD by upregulating the expression of clock protein BMAL1 and inhibiting excessive accumulation of NETs in lung tissue.

Objective To observe the effects of Huoxue Tongjiang Decoction on the expressions of gelsolin(GSN),prosaposin(PSAP)and nuclear factor(NF)-κB in reflux esophagitis(RE)model rats;To explore its mechanism of protecting esophageal mucosa.Methods Totally 75 Wistar rats were randomly divided into control group,model group,Western medicine group,TCM low-and high-dosage groups,with 15 rats in each group.The control group was given celiac switch operation,the other groups were given modified partial cardiac myotomy and external pyloric partial ligation to prepare RE rat model.Seven days after operation,each group was given normal saline,Western medicine or corresponding dosage of Huoxue Tongjiang Decoction for gavage,twice a day for 14 days.HE staining was used to observe the morphology of esophageal mucosa and pathological score was evaluated,immunohistochemical staining was used to detect the positive expressions of GSN and PSAP in esophageal mucosa,and Western blot was used to detect the protein expressions of NF-κBp65 and p-NF-κBp65 in esophageal tissue,ELISA was used to detect the contents of serum tumor necrosis factor(TNF)-α and interleukin(IL)-6.Results Compared with the control group,the esophageal mucosa of the model group was obviously damaged,the pathological score increased,the expression of GSN in esophageal mucosa decreased,the expressions of PSAP,NF-κBp65 and p-NF-κBp65 protein increased,and the contents of TNF-α and IL-6 in serum increased,with statistical significance(P<0.01).Compared with the model group,the esophageal mucosal injury improved obviously in the Western medicine group and TCM low-and high-dosage groups,the pathological score decreased,the expression of GSN in esophageal mucosa increased,the expressions of PSAP,NF-κBp65 and p-NF-κBp65 protein decreased,and the contents of TNF-α and IL-6 in serum decreased,with statistical significance(P<0.05,P<0.01).Conclusion Huoxue Tongjiang Decoction can effectively improve the immune inflammation of esophageal mucosa in RE model rats,its mechanism may be related to regulating GSN,PSAP and NF-κB pathway and reducing the release of inflammatory factors TNF-α and IL-6.

Objective: To explore the role of N6-methyladenosine(m6A) modification of RNA and Methyltransferase like Protein 14 methyltransferase(METTL14) in the pathogenesis of early-onset preeclampsia(ePE). Methods: Placental tissues of 15 pregnant women with early-onset preeclampsia and 15 normal pregnant women were collected. The level of m6A was determined by colorimetry, and the expression of METTL14 was determined by RT-qPCR, Western blot and immunohistochemistry(IHC) experiments. By transfecting siRNA and plasmid, METTL14 levels of trophoblast cells were knocked down and overexpressed, and cell phenotype experiments were carried out in vitro. The effects of METTL14 on the proliferation, migration and invasion of trophoblast cells were investigated by CCK-8, scratch assay, Transwell assay and invasion assay. Results: The level of m6A in placental tissue of ePE was lower than that of normal pregnancy. METTL14 was mainly expressed in the nuclei of trophoblast cells. Compared with normal pregnancy, the expression of METTL14 in placental tissue of ePE decreased, and thelevel of METTL14 mRNA was positively correlated with the level of m6A in placental tissue. The results of the CCK-8 experiment showed that compared with the control group, knockdown of METTL14 expression in trophoblast cells significantly reduced the cell proliferation rate, while the proliferation ability of trophoblast cells with overexpressed METTL14 was enhanced. The results of the scratch test showed that compared with the control group, the relative healing rate of scratches was significantly reduced after METTL14 knockdown, while it increased after the overexpression of METTL14. The results of the Transwell assay and invasion assay showed that compared with the control group, after knockdown of METTL14, the number of trophoblast cells passing through the chamber was significantly reduced, while the number of trophoblast cells with overexpressed METTL14 passing through the chamber increased. Conclusion The total RNA m6A modification level in placental tissue of ePE is lower than that in the normal pregnancy group. The down-regulation of methyltransferase METTL14 is involved in the regulation of the total RNA m6A modification level. The overexpression of METTL14 can enhance the proliferation, migration and invasion abilities of trophoblast cells. It provides a new perspective for exploring the pathogenesis of early-onset preeclampsia.

Objective To explore the effect of moxibustion on bone metabolism and the mechanism of bone protection,the effect of moxibustion,a traditional Chinese treatment in patients with rheumatoid arthritis(RA),especially the levels of two biomarkers,interleukin 35(IL-35)and β-catenin in the serum of rheumatoid arthritis patients were investigated.Methods A total of 72 RA patients were randomly assigned equally to the treatment group(36 cases)and 36 patients each of the control group(36 cases).The control group was intervened with standardized Western medicine treatment.For the treatment group,moxibustion intervened on Zusanli,Shenshu,and Ashi acupoints three times a week for 5-week treatment,with the remaining treatments being the same as the control group.The clinical symptom-related scores,routine clinical test indicators,and serum IL-35,β-catenin,BMP-2,IL-1β contents of RA patients before and after treatment were compared between the two groups.Results After treatment,VAS score,DAS28 score,morning stiffness score and BMP-2,β-catenin and IL-1β in serum in 2 groups significantly decreased(P<0.01),while IL-35 content in treatment group significantly increased(P<0.01).VAS score,DAS28 score,morning stiffness score and BMP-2,β-catenin and IL-1β in serum showed the obvious advantage of the treatment group over the control group(P<0.05),but the two groups(P>0.05)have no significant discrepancy in serum IL-35 content.For the treatment group,the improvement values of morning stiffness score and joint swelling number were positively correlated with the improvement values of BMP-2 and β-catenin in serum(P<0.01,P<0.05,r>0).The improvement of serum IL-35 was negatively correlated with the improvement of DAS28 score in the treatment group(P<0.05,r<0).Conclusion On the basis of conventional western medicine treatment,moxibustion may inhibit the levels of IL-35 and IL-1β in serum of RA patients,regulate the Wnt/β-catenin signaling pathway,regulate the levels of β-catenin and BMP-2,reduce the inflammatory response of RA,regulate bone metabolism,and achieve the purpose of bone protection.Thus,bone and joint teratogenesis or disability in RA patients was prevented and delayed.

Objective To observe the effects of Huoxue Tongjiang Decoction on the expressions of gelsolin(GSN),prosaposin(PSAP)and nuclear factor(NF)-κB in reflux esophagitis(RE)model rats;To explore its mechanism of protecting esophageal mucosa.Methods Totally 75 Wistar rats were randomly divided into control group,model group,Western medicine group,TCM low-and high-dosage groups,with 15 rats in each group.The control group was given celiac switch operation,the other groups were given modified partial cardiac myotomy and external pyloric partial ligation to prepare RE rat model.Seven days after operation,each group was given normal saline,Western medicine or corresponding dosage of Huoxue Tongjiang Decoction for gavage,twice a day for 14 days.HE staining was used to observe the morphology of esophageal mucosa and pathological score was evaluated,immunohistochemical staining was used to detect the positive expressions of GSN and PSAP in esophageal mucosa,and Western blot was used to detect the protein expressions of NF-κBp65 and p-NF-κBp65 in esophageal tissue,ELISA was used to detect the contents of serum tumor necrosis factor(TNF)-α and interleukin(IL)-6.Results Compared with the control group,the esophageal mucosa of the model group was obviously damaged,the pathological score increased,the expression of GSN in esophageal mucosa decreased,the expressions of PSAP,NF-κBp65 and p-NF-κBp65 protein increased,and the contents of TNF-α and IL-6 in serum increased,with statistical significance(P<0.01).Compared with the model group,the esophageal mucosal injury improved obviously in the Western medicine group and TCM low-and high-dosage groups,the pathological score decreased,the expression of GSN in esophageal mucosa increased,the expressions of PSAP,NF-κBp65 and p-NF-κBp65 protein decreased,and the contents of TNF-α and IL-6 in serum decreased,with statistical significance(P<0.05,P<0.01).Conclusion Huoxue Tongjiang Decoction can effectively improve the immune inflammation of esophageal mucosa in RE model rats,its mechanism may be related to regulating GSN,PSAP and NF-κB pathway and reducing the release of inflammatory factors TNF-α and IL-6.

Objective To explore the effect of moxibustion on bone metabolism and the mechanism of bone protection,the effect of moxibustion,a traditional Chinese treatment in patients with rheumatoid arthritis(RA),especially the levels of two biomarkers,interleukin 35(IL-35)and β-catenin in the serum of rheumatoid arthritis patients were investigated.Methods A total of 72 RA patients were randomly assigned equally to the treatment group(36 cases)and 36 patients each of the control group(36 cases).The control group was intervened with standardized Western medicine treatment.For the treatment group,moxibustion intervened on Zusanli,Shenshu,and Ashi acupoints three times a week for 5-week treatment,with the remaining treatments being the same as the control group.The clinical symptom-related scores,routine clinical test indicators,and serum IL-35,β-catenin,BMP-2,IL-1β contents of RA patients before and after treatment were compared between the two groups.Results After treatment,VAS score,DAS28 score,morning stiffness score and BMP-2,β-catenin and IL-1β in serum in 2 groups significantly decreased(P<0.01),while IL-35 content in treatment group significantly increased(P<0.01).VAS score,DAS28 score,morning stiffness score and BMP-2,β-catenin and IL-1β in serum showed the obvious advantage of the treatment group over the control group(P<0.05),but the two groups(P>0.05)have no significant discrepancy in serum IL-35 content.For the treatment group,the improvement values of morning stiffness score and joint swelling number were positively correlated with the improvement values of BMP-2 and β-catenin in serum(P<0.01,P<0.05,r>0).The improvement of serum IL-35 was negatively correlated with the improvement of DAS28 score in the treatment group(P<0.05,r<0).Conclusion On the basis of conventional western medicine treatment,moxibustion may inhibit the levels of IL-35 and IL-1β in serum of RA patients,regulate the Wnt/β-catenin signaling pathway,regulate the levels of β-catenin and BMP-2,reduce the inflammatory response of RA,regulate bone metabolism,and achieve the purpose of bone protection.Thus,bone and joint teratogenesis or disability in RA patients was prevented and delayed.

Cervical cancer is the leading cause of cancer death in women in the developing countries.The treatment based on surgery,radiotherapy and chemotherapy is often accompanied by intolerable complications.Clinical practice has proved that TCM therapy has a positive effect on the complications related to the treatment of cervical cancer,but there is still a lack of scientific and standardized application reference opinions.Based on Delphi method,our research group constructed and formulated an expert consensus study on the complications related to the treatment of cervical cancer with TCM therapies,so as to provide a reference for clinical treatment of such diseases.

Objective:To explore the locations of the lumbosacral nerve roots by use of the magnetic stimulation.Methods:Thirty healthy subjects were studied. The projections of the right L 2 to S 1 intervertebral foramina on their body surfaces were determined manually with ultrasound assistance. Magnetic stimulation was applied to different nerve root segments to induce compound muscle action potentials (CMAP) in the vastus medialis, tibialis anterior, and gastrocnemius muscles of the lower limbs. The changes in latency, amplitude, and motor threshold were observed. Results:Magnetic stimulation on the L 2-L 3 segment resulted in a significant direct excitation of the vastus medialis. That on the L 5-S 1 segment evoked a significant direct excitatory effect on the tibialis anterior and gastrocnemius, with a motor threshold below 40%, an amplitude exceeding 1mV, and many effective responses. However, during the magnetic stimulation on the L 4 segment, the amplitude of the vastus medialis was above 1mV, with no significant differences in the number of effective responses among the muscle groups. Moreover, there was a stepwise change in the latency of effective muscle responses to magnetic stimulation at different segments. The CMAP latencies of 12+ ms for the tibialis anterior and 13+ ms for the gastrocnemius indicated activation of the L 5 and L 4 nerve roots, respectively, while those of 6+ ms, 7+ ms, and 8+ ms for the vastus medialis suggested activation of the L 4, L 3, and L 2 nerve roots, respectively. Conclusions:Based on the responses (CMAP latency, amplitude and motor threshold) of the vastus medialis, tibialis anterior and gastrocnemius to magnetic stimulation at different L 2 to S 1 segments, the spinal nerve root segments responsible for innervation can be inferred.

To examine the molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae (CRKP) and investigate the horizontal transmission of blaKPC and blaNDM genes for the prevention and treatment of CRKP. A total of 49 clinically isolated CRKP strains were retrospectively analyzed from January to December 2022 at The First Hospital of Hunan University of Chinese Medicine. Phenotypic screening was performed using modified carbapenem inactivation assay (mCIM) and EDTA-carbapenem inactivation assay (eCIM). Polymerase chain reaction (PCR) was utilized to identify carbapenem resistance genes, β-lactamase resistance genes, and virulence genes, while multi-locus sequence analysis (MLST) was employed to assess the homology of CRKP strains. Conjugation experiments were conducted to infer the horizontal transmission mechanism of blaKPC and blaNDM genes. The results showed that the study included 49 CRKP strains, with 44 carrying blaKPC and 8 carrying blaNDM, Three strains were identified as blaKPC+ blaNDM-CRKP. In this study, 28 out of 49 CRKP strains (57.2%) were found to carry virulence genes. Additionally, one CRKP strain tested positive in the string test and was found to carry both Aerobactin and rmpA virulence genes. MLST results revealed a total of 5 ST types, with ST11 being predominant (41/49, 83.7%). Successful conjugation was observed in all 3 blaKPC-CRKP strains, while only 1 out of 3 blaNDM-CRKP strains showed successful conjugation. The transconjugant exhibited significantly reduced susceptibility to imipenem and cephalosporin antibiotics. In conclusion, the resistance mechanism of CRKP in this study is primarily attributed to the production of KPC enzymes, along with the presence of multiple β-lactamase resistance genes. Additionally, there is a local prevalence of hv-CRKP and blaKPC+ blaNDM-CRKP. blaKPC and blaNDM can be horizontally transmitted through plasmids, with varying efficiency among different strains.