Effect of constant compressive stress induced by imitating Tuina stimulation with various durations on the cell cycle, cellular secretion, apoptosis, and expression of myogenic differentiation and myogenic factor 5 of rat skeletal muscle cells in vitro.

OBJECTIVE: To investigate the effect of constant compressive stress induced by imitating Tuina stimulation with various durations on the cell cycle, cellular secretion, apoptosis, and expression of myogenic regulatory factors (MRFs), myogenic factor 5(Myf5) and myogenic differentiation (MyoD) of rat skeletal muscle cells (RSkMCs) in vitro. METHODS: Third passage RSkMCs were subjected to constant compressive stresses with various durations at 2000 strain for 15, 30, 60, 90, and 120 min via a four-point bending system. The control group (CG) was cultured in the absence of mechanical loading. Alterations of the cell cycle and apoptosis rate were detected by flow cytometry (FCM). The concentrations of interleukin 6 (IL-6) / prostaglandin E2 (PGE2) and nitric oxide (NO) in supernatants were determined by enzyme-linked immunosorbent assays and the nitrate reductase method, respectively. Expression of Myf5 and MyoD was detected by immunohistochemistry. RESULTS: Compared with the CG, a significant alteration was observed in the synthesis phase fraction (SPF) (P < 0.01). The SPF and proliferation index (PI) were reduced from 15 to 90 min, but reached levels similar to those at 120 min. Apoptosis was increased significantly at 30 min (P < 0.05) and especially at 90 and 120 min (P < 0.01). Expression of MyoD and Myf5 was increased significantly at 15, 30, and 90 min (P < 0.01). Compared with 15 and 30 min, MyoD and Myf5 expression at 60 and 120 min was decreased significantly (P < 0.01). Compared with 60 min, MyoD expression at 90 min was increased significantly (P < 0.05), whereas MyoD and Myf5 expression at 120 min was significantly lower (P < 0.05). The IL-6 concentration was increased at 60 min compared with the CG and 15 min (P < 0.05), whereas the concentrations of PGE2 and NO were the highest at 15 and 30 min, respectively, compared with the CG and other time points (P < 0.05). CONCLUSION: The cell cycle, secretion, apoptosis, and Myf5 and MyoD expression of RSkMCs were regulated by compressive stress in a time-dependent manner. SPF and PI were inhibited at short durations (< 90 min), but NO and PGE2 secretion was the highest at shorter durations (< 30 min). With the prolongation of stimulation time, SPF, PI, and apoptosis were increased, but Myf5 and MyoD expression was decreased gradually at 15-30 min.

Moxibustion benignantly regulates circadian rhythm of REV-ERBα in RA rats.

The key clinical symptoms and previous findings of RA show a circadian variation, with more prominent joint swelling, stiffness, and pain occurring in the early morning. Moxibustion is able to relieve RA in various pass ways, however, there is no verifying study results for the pathological rhythm of RA. Therefore, we conducted this work to verify whether moxibustion could adjust RA circadian rhythm according to regulate core clock genes. Based on these previous findings that circadian timekeeping is disturbed in RA at molecular level, the aim of this study was to observe the influence of moxibustion on expression level and circadian rhythm of REV-ERBα at different tissues of RA rats. Furthermore, the expression level of core clock genes closely related to RA were evaluated by RT-PCR. 96 SD rats were randomly assigned as 1:1:1:1 ratio to 4 groups for normal control group, RA model group, 5-7 am moxibustion group, and 5-7 pm moxibustion group. RT-PCR was used to measure the relatively expression quantity of REV-ERBα, CLOCK, BMAL1, and PER2 in hypothalamus, hippocampus, and adrenal gland. In RA rats, the expression level of REV-ERBα mRNA were up-regulated in different tissues, and moxibustion potentially up-regulated them in different degrees. In untreated RA rats, the circadian rhythm of REV-ERBα mRNA in hippocampus and adrenal gland both disappeared (P>0.05) and moxibustion was able to recover them (P<0.05). The expression level of CLOCK and PER2 mRNA in hippocampus and adrenal gland were down-regulated significantly (P<0.05) in RA model rats, while moxibustion up-regulated both of them in hippocampus (P<0.05). These results suggested together that moxibustion can benign regulate circadian rhythm of REV-ERBα in different tissues of RA rats. It was revealed that moxibustion not only recovered the losing diurnal oscillation of REV-ERBα in hippocampus and adrenal gland, but also adjusted the circadian rhythm of REV-ERBα in hypothalamus, hippocampus, and adrenal gland to close the normal circadian pattern.

Effect of electroacupuncture at the ST36 and GB39 acupoints on apoptosis by regulating the p53 signaling pathway in adjuvant arthritis rats.

p53 and mouse double minute 2 homolog (MDM2) serve key regulatory roles in the apoptosis of synovial cells. The present study aimed to investigate the effects of electroacupuncture (EA) at the 'Zusanli' (ST36) and 'Xuanzhong' (GB39) acupoints on apoptosis in an adjuvant arthritis (AA) rat model. A total of 40 male Sprague­Dawley rats were randomly divided into Control, AA, AA + EA and AA + sham EA groups (n=10 rats in each group). Rats in all the groups, with the exception of the control group, were injected with Complete™ Freund's adjuvant into the bilateral hindlimb footpad to establish the AA model. Rats in the AA + EA group were treated with EA at the ST36 and GB39 acupoints. Rats in the AA + sham EA group were treated with percutaneous electrical stimulation at a position of 5 mm away from the ST36 and GB39 acupoints. The arthritis index scores and hindlimb paw volumes of the rats in each group were recorded. Subsequently, pathological changes in the synovial tissue were evaluated by hematoxylin and eosin (H&E) staining, and the apoptotic rate of the synovial cells was detected by TUNEL staining. In addition, the expression levels of the apoptosis­associated proteins, Bax, phorbol­12­myristate­13­acetate­induced protein 1 (Noxa) and p53 upregulated modulator of apoptosis (PUMA), were determined by western blot analysis. The expression of both the gene and protein of p53 and MDM2 in synovial tissue was detected by reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blot analysis, respectively. The results indicated that the arthritis index scores and hindlimb paw volumes upon EA stimulation were significantly decreased compared with those of the AA group (P<0.05). H&E staining revealed that the synovial inflammation of EA stimulation was significantly decreased compared with the AA group (P<0.05). The TUNEL assay results indicated that the apoptotic rate of synovial cells in the AA + EA group was significantly increased compared with that in the AA group (P<0.05). Furthermore, an increased expression of proapoptotic proteins was confirmed by the increased expression levels of Bax, Noxa and PUMA in the AA + EA group. The results of RT­qPCR and western blot analysis demonstrated that, compared with the AA group, EA stimulation led to a marked increase in p53 (P<0.05) and a significant decrease in MDM2 (P<0.05) gene and protein expression. Taken together, these results demonstrated that EA performed on the ST36 and GB39 acupoints led to a significant amelioration in AA injury of model rats, by regulating the p53 signaling pathway and inducing apoptosis.

Moxibustion of Zusanli (ST36) and Shenshu (BL23) Alleviates Cartilage Degradation through RANKL/OPG Signaling in a Rabbit Model of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a systemic and chronic autoimmune inflammatory disease characterized by severe synovial hyperplasia associated with progressive cartilage degradation. Due to the severe pain and disability caused by RA, effective therapeutic strategies that could simultaneously alleviate the inflammatory response and delay the disease progression are urgently needed. As a major alternative therapy in traditional Chinese medicine, moxibustion has been demonstrated that it could reduce the chronic inflammatory responses of a series of musculoskeletal diseases; however, whether moxibustion has protective effects on RA is still unclear. To investigate the effects of moxibustion on RA, moxibustion was applied to Zusanli (ST36) and Shenshu (BL23) acupoints in a RA rabbit model. HE staining of articular cartilage showed that moxibustion alleviated the cartilage degradation and bone destruction. In addition, moxibustion decreased the osteoclast number in RA rabbits. Real-time PCR revealed that moxibustion decreased the expression of RANKL mRNA while increased the expression of OPG mRNA, indicating a restoration of the balance between osteogenesis and osteoclastogenesis. Taken together, our results indicated that moxibustion had promising antiarthritic effects and could be an useful alternative method in RA therapeutics.

[Effects of moxibustion at different time points on foot swelling, serum level of TNF-α and circadian rhythm in rats with rheumatoid arthritis].

OBJECTIVE: To study the effects of moxibustion at different time points on serum level of tumor necrosis factor-α (TNF-α) in rats with rheumatoid arthritis (RA), and to explore its regulation mechanism on circadian rhythm. METHODS: A total of 96 Sprague-Dawley (SD) adult rats were randomly assigned into a blank group, a model group, a moxibustion at 5-7 AM group and a moxibustion at 5-7 PM group, 24 rats in each group, half male and half female. Each group was further divided into a 0 AM group, a 6 AM group, a 12 N group and a 6 PM group, 6 rats in each group. All rats were treated with the 12 h/12 h light-dark cycle in the whole process of experiment. Except for the blank group, all rats were treated with intracutaneous injection of freund's complete adjuvant (FCA) at right foot pad to establish the RA model. The rats at the two moxibustion groups were treated with grain-sized moxibustion at "Shenshu" (BL 23) and "Zusanli" (ST 36) at 5-7 AM and 5-7 PM, respectively, one side per treatment, once a day; six treatments were taken as one course and 3 courses were given with an interval of one day between courses. The rats in the remaining groups were treated with identical fixation but without moxibustion intervention. The right foot volume was measured before model establishment, after model establishment and after treatment. The blood samples were collected after treatment and the serum level of TNF-α was measured by ELISA. The SPSS 21.0 software and Halberg Cosinor were adopted to analyze the experiment data. RESULTS: After treatment, compared with the blank group, the foot swelling severity was significantly increased in the model group, moxibustion at 5-7 AM group and moxibustion at 5-7 PM group (all P<0.01); compared with the model group, the foot swelling severity was significantly reduced in the moxibustion at 5-7 AM group and moxibustion at 5-7 PM group (both P<0.01). Compared with the blank group, the serum level of TNF-α was increased significantly in the model group and moxibustion at 5-7 AM group (both P<0.05); compared with the model group, the serum level of TNF-α was reduced significantly in the moxibustion at 5-7 AM group and moxibustion at 5-7 PM group (both P<0.05). The serum level of TNF-α showed circadian rhythm in all the groups (all P<0.05), and the peak appeared at night phase; compared with the blank group, the median value of TNF-α was increased significantly in the model group (P<0.05), the peak phase was delayed and the amplitude was increased (P<0.05); compared with the model group, the median value of TNF-α was significantly reduced in the moxibustion at 5-7 AM group and moxibustion at 5-7 PM group (P<0.01), the peak phase was advanced and the amplitude was reduced (P<0.05). CONCLUSION: Moxibustion could effectively reduce the serum level of TNF-α to relieve the foot swelling severity in RA rats. Moxibustion could regulate the circadian rhythm of TNF-α to play its effects on the inhibition of the synthesis of TNF-α. No efficacy is observed between the treatment at 5-7 AM and 5-7 PM.

[Anterior-posterior acupoint combination for low back pain].

The meridian diseases, abnormal psoas and abdominal muscles and abdominal pressure are key causes of low back pain. We treat low back pain mainly with lumbar back area, neglecting abdomen. The anterior-posterior acupoint combination, regulating yin to treat yang, is effective. Based on the Chinese meridian-collateral and contemporary anatomy related to low back pain, the combination method, tuina and its mechanism are introduced, so as to develop the thought and method for low back pain. A typical case is added.

[Current situation of research and problems in diagnosis and treatment of senile depression with integrative traditional Chinese and Western medicine].

This paper summarized the researches on diagnosis and treatment of senile depression with integrative Chinese and Western medicine in the recent 5 years, and some problems existed at present were also submitted. It is pointed out that it's necessary to expand the study with senior electrocerebral neurotransmitter analysis technique (SET) for the diagnosis and efficacy evaluation of senile depression by either traditional Chinese or Western medicine. By which it would also be able to offer convincing evidence for diagnosis of depression, TCM syndrome differentiation dependant therapy and the efficacy evaluation.