Efficacy of Duhuo Jisheng Decoction in Treating Ankylosing Spondylitis: Clinical Evidence and Potential Mechanisms.

Background: Duhuo Jisheng Decoction (DHJSD) is an ancient compound widely used in the treatment of ankylosing spondylitis (AS). However, its efficacy is controversial, and its mechanism of action is not clear enough. Using meta-analysis and network pharmacology, our study evaluated the clinical efficacy of DHJSD in the treatment of AS and explored its mechanisms of action. Methods: We searched medical databases, including Embase, PubMed, the China National Knowledge Infrastructure databases, Wanfang, and the Chinese Scientific Journal Database, to identify studies that met the inclusion criteria. RevMan 5.3 software was used for the meta-analysis. The compounds and the potential protein targets of DHJSD were obtained from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and analysis platform. AS was treated as a search query in the NCBI, PharmGKB, TTD, DrugBank, and OMIM databases to obtain disease-related genes. The overlapping targets of DHJSD and AS were identified, and then Gene Ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed. Cytoscape was employed to construct a drug-compound-target network and a protein-protein interaction (PPI) network. CytoHubba was utilized to select the hub genes. Results: A total of 10 studies involving 860 participants were included in the meta-analysis. Compared with the control, DHJSD treatment significantly improved clinical symptoms; reduced the erythrocyte sedimentation rate (ESR), the C-reactive protein (CPR), and interleukin 6 (IL-6) levels; increased the degree of motion of the chest; reduced the visual analog scale (VAS) pain score; reduced Schober's test values; reduced the finger-to-floor distance; reduced the duration of morning stiffness. However, the differences were not statistically significant in the Bath Ankylosing Spondylitis Functional Index scores, the Bath Ankylosing Spondylitis Disease Activity Index scores, the bone Gla-containing protein (BGP) levels, or the bone alkaline phosphatase (BALP) levels. In terms of adverse events, DHJSD treatment of AS reduced the incidence of gastrointestinal events, the incidence of skin events, and the incidence of abnormal liver function; however, there was no statistically significant reduction in the incidence of adverse renal function events. Subgroup analysis showed that in the treatment of AS, the clinical effect of DHJSD for AS was better than that of the controls for both treatment durations, ≤2 months and >2 months. A total of 178 active compounds and 47 related potential targets were identified for DHJSD in the treatment of AS, including four hub genes (CXCL8, PTGS2, VEGFA, and STAT3). The core active ingredients of DHJSD in the treatment of AS were mainly quercetin, kaempferol, licochalcone A, and isorhamnetin. DHJSD treatment of AS-related pathways mainly involved the IL-17 signaling pathway, the TNF signaling pathway, and the rheumatoid arthritis signaling pathway. Conclusion: The above results suggest that DHJSD acts on AS through multiple targets, components, and pathways with significant clinical efficacy. Future studies may further explore the active components of DHJSD.

Nerve regeneration in rat peripheral nerve allografts: An assessment of the role of endogenous neurotrophic factors in nerve cryopreservation and regeneration.

Peripheral nerve injury is a common clinical problem that often leads to significant functional impairment or even complete paralysis. Allograft has been proposed as a potential repair strategy for peripheral nerve injuries. Furthermore, peripheral nerve cryopreservation may result in nearly unlimited supply of grafts. However, the concentration of neurotrophic factors secreted by Schwann cells (SCs) in the local micro-environment after transplantation may not be sufficient for the survival of neuronal soma and axonal regeneration. Here, we investigated the effect of endogenous neurotrophic factors (ENTFs) on nerve regeneration in rats after the allograft of a cryopreserved sciatic nerve. ENTFs were highly expressed in the sciatic nerves pretreated for 14 days. Although the number of surviving cells in the sciatic nerves and their immunogenicity were low in the 14-day group after 4 weeks of cryopreservation, they continued to express high levels of ENTFs in vitro. At 1 week postoperation, the 14-day Allo group showed low plasma levels of interleukin-2, interferon-γ and tumour necrosis factor-alpha and low cellular immune response. At 20 weeks postoperation, nerve regeneration and functional recovery in the 14-day Allo group was similar to that in the fresh isograft group but better than that in the cryopreserved-fresh allograft and fresh allograft groups. Thus, ENTFs were induced in vitro after pretreatment of the sciatic nerve. Following cryopreservation, the sciatic nerves with high levels of ENTFs continued to express high levels of ENTFs in vitro. The immune response after allograft was weak, which promoted recipient nerve regeneration.

The effects of triptolide on the cellular activity of cryopreserved rat sciatic nerves and nerve regeneration after allotransplantation.

Purpose: To investigate the effects of triptolide (T10) on the cellular activity of cryopreserved rat sciatic nerves and nerve regeneration after allotransplantation.Materials and methods: After the optimal T10 concentration was determine, sciatic nerve fragments from Sprague-Dawley (SD) rats were randomly divided into 5 groups: the fresh nerve group (group A), the Dulbecco's modified Eagle's medium (DMEM)-preservation group (group B), the T10-preservation group (group C), the T10-pretreatment, DMEM-preservation group (group D), and the T10-pretreatment, T10-preservation group (group E). The nerves in the preservation groups were preserved at 4 °C for 4 w. Then, either cryopreserved or fresh nerves were used to repair 10-mm sciatic nerve defects in Wistar rats (group A', group B', group C', group D', and group E', which correspond to the nerve groups described above); in addition, one fresh homologous transplantation group (group F') was established.Results: Nerve growth factor (NGF) was expressed at significantly higher levels in the groups treated with the T10 solution at 37 °C. After rat sciatic nerves were cryopreserved for 4 w, group E had increased numbers of live nerve cells and increased levels of biological activity (P < 0.001) and reduced levels of immunogenicity (P < 0.001) when compared with those for the other groups. Sixteen weeks after transplantation, recipient nerve regeneration in group E' was increased compared with that in groups A', B', C', and D' (P < 0.05).Conclusions: The application of T10 in vitro induced the expression of neurotrophic factors in rat sciatic nerves, increased the biological activity of cryopreserved nerves, reduced immunogenicity, and promoted recipient nerve regeneration after allotransplantation.

Nerve regeneration in rat peripheral nerve allografts: Evaluation of cold-inducible RNA-binding protein in nerve storage and regeneration.

The prevalence of peripheral nerve injury has attracted increased attention. Allografting has been proposed as a potential treatment strategy for peripheral nerve injury. Moreover, cryopreservation may provide almost unlimited graft material. We investigated whether cold-inducible RNA-binding protein (CIRP) could protect peripheral nerves during cryopreservation to promote regeneration postoperation. First, CIRP was highly expressed after pretreatment at 32°C. After 4 weeks of cryopreservation, the increased live cells, low Bax/Bcl-2 ratio and high nerve growth factor and glial cell-derived neurotrophic factor levels in the 32°C group demonstrated high nerve graft viability. At 4 weeks postoperation, 32°C-Allo group demonstrated low plasma levels of interleukin-6 and interferon-gamma and a diminished cellular immune response. At 20 weeks postoperation, nerve regeneration in the 32°C-Allo group was similar to that in the fresh isograft group and superior to that in the 4°C-Allo and 15°C-Allo groups. Moreover, the compound muscle action potential and the motor nerve conduction velocity of the 32°C-Allo group were equal to those of the fresh isograft group. In conclusion, CIRP induction increased Schwann cell biological activity, inhibited cell apoptosis, reduced immune rejection, and promoted recipient nerve regeneration. Thus, CIRP could exert protective effects during nerve storage and stimulate regeneration in peripheral nerve reconstruction.

[Effects of emodin on oxidative stress and inflammatory response in rats with acute spinal cord injury].

This paper was aimed to investigate the effects of emodin on oxidative stress and inflammatory response in rats with acute spinal cord injury（SCI）, and to explore the protective mechanism of emodin on neurons after SCI. Rat SCI models were established using a modified Allen's method. One hundred and ninety five Sprague-Dawley rats were randomly divded into sham (group A), model (group B), emodin group of 20 mg·kg⁻¹(group C), emodin group of 40 mg·kg⁻¹(group D), emodin group of 80 mg·kg⁻¹(group E). Functional recovery was evaluated using the Basso-Beattie-Bresnahan (BBB) scale and inclined plate test on the 3rd, 7th, 14th and 28th day. On the 7th day after SCI, neuron nylon body was observed with toluidine blue staining. The changes of myelinated nerve fibers were observed by transmission electron microscope. Nrf2, HO-1, NQO1, GFAP, NF-κB protein were detected by Western blot. The content of TNF-α, IL-1ß, IL-6 were detected by ELISA. Immunofluorescence staining was performed to observe the expression of the GFAP, NG2 and ED-1. The BBB and inclined plate scores of group C, D and E were higher than group B on the 7th, 14th, 28th day,the difference was statistically significant (P<0.05). On the 7th day, Nylon Body of group B and C started to fuse,the fusion of group D and E were significantly alleviated than group B and C. Transmission electron microscope showed that the changes of demyelination were obvious in group B and C, group D and E were significantly improved than group B and C. Western blot showed that Nrf2, HO-1, GFAP, NF-κB protein expression,group C, D, E compared with group B, NQO1 protein expression, group D, E compared with group B, the difference was statistically significant (P<0.05). ELISA showed that the content of TNF-α, IL-6,group C, D, E compared with group B,the content of IL-1ß,group D, E compared with group B, the difference was statistically significant (P<0.05)；Immunofluorescence showed that the expressions of GFAP and NG2 in group C, D and E were higher than that in group B, and the group D and E were more obvious. The expression of ED-1 in group C, D, E were decreased significantly compared with group B. Emodin has protective effect on neurons after SCI. The mechanism may connect with activting Nrf2-ARE pathway, reducing the expression of NF-κB, ED-1, TNF-α, IL-1ß, IL-6, and promoting the expression of GFAP and NG2.

[Effects of tanshinone â ¡A sulfonate on nerve regeneration after cryopreserved sciatic nerve allograft in rats].

In order to investigate the protective effect of tanshinone ⅡA sulfonate on the sciatic nerve activty in rats after cryopreservation as well as the nerve regeneration and functional recovery after allograft and its possible mechanism, Sprague-Dawley (SD) rats were divded into four groups at different doses of tanshinone ⅡA sulfonate (A 0 mg·L⁻¹, B 80 mg·L⁻¹, C 160 mg·L⁻¹, D 480 mg·L⁻¹) cryopreserved at -80 °C for 24 weeks. Fresh control group nerve segments were harvested without cryopreservation. The ultrastructure and the viable cells of the nerve segments after cryopreservation were observed by electron microscopy, calcein-AM/propidium iodide staining, respectively. The expression of Bax and Bcl-2 was detected by Western blot. After cryopreservation, the nerve segments were cultured in vitro for one week, the mRNA and protein level of NGF and GDNF were detected by PCR and Western blot respectively. In addition, the above four cryopreserved groups transplanted to the Wistar rats by allografting (A', B', C', D'). At 16-week postoperation, muscle compound action potential latency and nerve conduction velocity were examined by electrophysiological. The number and the thickness of myelinated nerve fibers were analyzed by toluidine blue staining. The ultrastructure of the sciatic nerve by electron microscopy was observed. According to the results, after the cryopreserved for 24 weeks, compared with groups A and B, the nerve demyelination and vacuolation were weak, and the more viable cells, the decreased Bax and increased Bcl-2, the increased NGF and GDNF in group C and D. At 16-week poseoperation, the results demonstrated that the more larger and thickly regenerated myelinated axons, the shorter latency of muscle compound action potentials and higher nerve conduction velocity in groups C' and D' compared with groups A' and B'. According to these results, tanshinone ⅡA sulfonate exerted a significant protective effect on the viability of the nerves during cryopreservation at -80 °C and promoted nerve regeneration and functional recovery after transplantation especially in middle- and high-dose of tanshinone ⅡA sulfonate.

[Effect of tetramethylpyrazine added to vitrification solution on peripheral nerve allografts regeneration].

OBJECTIVE: To investigate the effect of tetramethylpyrazine (TMP) with a certain concentration added to vitrification solution on peripheral nerve allografts regeneration. METHODS: Forty-eight healthy clean SD male rats were selected as donors, and 96 healthy clean Wistar male rats as recipients, all rats being 3 months old and weighing 200-250 g. The sciatic nerves segments of 15 mm were removed from the donors, then randomly divided into 4 groups according to vitrification solution containing TMP. No TMP was used in group A as the control group; 100 mg/L, 200 mg/L and 400 mg/L TMP were used in group B, group C and group D, respectively. Then them were cryo-preserved at -196 degrees C for 3 weeks. Nerve defect of 10 mm in length was made in the sciatic nerves of recipients. After rewarming, the allografts were transplanted to the corresponding rats. The gross appearance, the morphological and electrophysiological changes, the image analysis of axons and motor end-plate were detected at 4, 8, 12 and 16 weeks. RESULTS: All rates survived to the end of the experiment. The adhesion and edema of allografts in group A and group B were obvious 4 weeks after operation; then adhesion and edema was obvious in group A and were improved in the other groups 8 weeks after operation. Adhesion was observed in groups A and B; no adhesion was observed in groups C and D at 12 weeks. The number of regeneration nerve, the latent, the amplitude, the nerve conduction velocity, the medullary sheath/microm2, the medullary sheath density/microm2 and the image analysis of axons and motor end-plate in groups A and B were significantly lower than those in groups C and D (P < 0.01); and there were no significant differences between groups C and D (P > 0.05). The observation of transmission electron microscope showed that medullated nerve fibers and myelin sheath of groups C and D were thicker than groups A and B, layers of groups C and D were clear. CONCLUSION: The vitrification solution with 200 mg/L tetramethylpyrazine has protective effect on regeneration of peripheral nerve allografts.

[Effects of tripterygium glycoside on apoptosis of the skeletal muscle after nerve allograft].

OBJECTIVE: To explore the effect of tripterygium glycoside (TG) on the skeletal muscle atrophy and apoptosis after nerve allograft. METHODS: Twenty Wistar male rats were adopted as donors, weighing 200-250 g, and the sciatic nerves were harvested. Fifty SD male rats were adopted as recipients, weighing 200-250 g. Fifty SD rats were made the models of 10 mm right sciatic nerve defect randomly divided into five groups (n=10): group A, group B, group C, group D and group E. groups A and B received fresh nerve allograft, groups C and D received sciatic nerve allograft pretreated with TG, and group E received autograft. The SD rats were given medicine for 5 weeks from the second day after the transplantation: groups A and E were given physiological saline, groups B and D TG 5 mg/(kg x d), and group C TG 2.5 mg/(kg x d). At 3 and 6 weeks, respectively, after nerve transplantation, general observation was performed; the structure of skeletal muscles was observed by HE staining; the diameter of skeletal muscles was analyzed with Image-Pro Plus v5.2; the ultrastructure of skeletal muscles was observed by TEM; the expressions of Bax and Bcl-2 were detected by immunohistochemical staining; and the apoptosis of skeletal muscles was detected by TUNEL. RESULTS: All rats survived to the end of the experiment. In general observation, the skeletal muscles of SD rates atrophied to different degrees 3 weeks after operation. The muscular atrophy in group A was more serious at 6 weeks, and that in the other groups improved. The wet weight, fiber diameter and expression of Bcl-2 in group A were significantly lower than those in groups B, C, D and E (P < 0.01); those in groups B, C and D were lower than those in group E (P < 0.05); and there were no significant differences among groups B, C and D (P > 0.05). The apoptosis index and expression of Bax in group A were significantly higher than those in groups B, C, D and E (P < 0.01); those in groups B, C and D were higher than in group E (P < 0.05); and there were no significant differences among groups B, C and D (P > 0.05). Three weeks after nerve allograft, under the light microscope, the muscle fibers became thin; under the TEM, the sarcoplasmic reticulum was expanded. Six weeks after nerve allograft, under the light microscope, the gap of the muscle fibers in group A was found to broaden and connective tissue hyperplasia occurred obviously; under the TEM, sarcomere damage, serious silk dissolution and fragmentary Z lines were seen in group A, but the myofibrils were arranged tidily in the other groups, and the light band, dark band and sarcomere were clear. CONCLUSION: TG can decrease the skeletal muscle atrophy and apoptosis after nerve allograft. The donor's nerve that is pretreated with TG can reduce the dosage of immunosuppressant for the recipient after allograft.

[Experimental study on demyelination and immunological rejection of rats' sciatic nerve allograft with tripterygium wilfordii's pretreatment].

OBJECTIVE: To investigate the appropriate concentration of tripterygium wilfordii and immunological rejection of rats' sciatic nerve allograft with the tripterygium wilfordii's pretreatment so as to explore tripterygium wilfordii's suppression. METHODS: Sixty SD rats (male, weighing 270-290 g), as sciatic nerve allograft acceptor were randomized into 5 groups (groups A, B, C, D and E, n = 12). To repair the sciatic nerve defect of SD rats, the Wistar rats' sciatic nerve allografts about 15 mm long were used with 24 hours' soak of different concentrations of tripterygium wilfordii (group A: 200 mg/L, group B: 400 mg/L, group C: 800 mg/L). The control groups (group D: the fresh sciatic nerve allograft from donors; group E: the fresh sciatic nerve allograft from themselves) were established. At different time points after operation, the morphological examinations (the observation of histology, light microscope, electron microscope), the detection of myelin basic protein's (MBP) content and the analyses of CD4+ and CD8+ T cells on the allografts in the acute phase were performed. RESULTS: There was no significant difference in morphology among groups A, B and C, the adhesions between allografts and connective tissue were milder than that of group D, and the allografts' amorphous and the inflammatory cell infiltration were better than that of group D. The degeneration of myelin sheath was observed at different levels and there was no significant difference between group B and group E (P > 0.05). There was a significant difference in immunological rejection between groups A, B, C and group D (P < 0.05). CONCLUSION: Tripterygium wilfordii can effectively suppress the acute immunological rejection in the early stage after operation, and protect the myelin sheath to a certain extent.

[Effects of electroacupuncture on synovia IL-1beta and TNF-alpha contents in the rabbit with knee osteoarthritis].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on synovia IL-1beta and TNF-alpha contents in rabbits with knee osteoarthritis (KOA). METHODS: A total of 40 New Zealand rabbits were randomly divided into control, model, massage and EA groups. KOA model was established by gypsum fixing method. EA (2 Hz, 1-3 mA) was applied to left "Xiyan" (EX-LE 5), "Yanglingquan" (GB 34), "Xuehai" (SP 10), "Zusanli" (ST 36) and "Liangqiu" (ST 34) for 30 min, once daily and continuously for 21 days. For massage group, the affected knee joint was pressed, kneaded, stretched and rotated repeatedly for 15 min every time, followed by forced running about 100 m. The intra-joint synovia was collected (0.4-0.6 mL) for detecting the contents of IL-1beta and TNF-alpha with radioimmunoassay. RESULTS: No IL-1beta and TNF-alpha were detected in the synovia in control group, while in the other 3 groups, synovia IL-1beta and TNF-alpha levels increased significantly. Before treatment, no significant differences were found among model, EA and massage groups in the levels of IL-1beta and TNF-alpha (P > 0.05), while after the treatment the two indexes both decreased considerably (P < 0.05). Compared with model group, the two indexes were remarkably lower (P < 0.05), but no significant differences were found between EA and massage groups in synovia IL-1beta and TNF-alpha contents (P > 0.05). CONCLUSION: Both EA and massage can effectively suppress the release of synovia IL-1beta and TNF-alpha in KOA of rabbits, which may contribute to the effect of acupuncture in the treatment of osteoarthritis.