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ObjectiveTo study the mechanism of Danggui Sinitang in mitigating gouty arthritis （GA） in rats by regulating autophagy via the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathway. MethodSixty male SD rats were randomly assigned into normal， model， colchicine （0.3 mg·kg-1）， and low-， medium-， and high-dose Danggui Sinitang （6.54， 13.08， and 26.16 g·kg-1） groups （n=10） and administrated with corresponding drugs by gavage. The rats in the normal group and model group were administrated with equal volume of normal saline by gavage for 7 days. One hour after administration on day 5， the GA model was established by injecting sodium urate suspension （50 g·L-1） into the right ankle joint of rats in other groups except the normal group， and the rats in the normal group were injected with sterile normal saline of the same volume. The swelling and pathological changes of the ankle joint were observed. The serum levels of tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， and IL-1β were determined. Western blot was employed to determine the protein levels of PI3K， phosphorylated PI3K （p-PI3K）， protein kinase B （Akt）， phosphorylated Akt （p-Akt）， mTOR， phosphorylated mTOR （p-mTOR）， microtubule-associated protein 1 light chain 3 Ⅱ/Ⅰ （LC3Ⅱ/Ⅰ）， autophagy effector Beclin-1， and ubiquitin-binding protein p62 in the synovial tissue. Real-time fluorescent quantitative PCR （Real-time PCR） was employed to determine the mRNA levels of PI3K， Akt， mTOR， LC3， Beclin-1 and p62. ResultCompared with the normal control， the model group showed increased joint swelling index （P<0.01）， elevated serum levels of TNF-α， IL-6， and IL-1β， inflammatory cell infiltration， and fibrous tissue hyperplasia. In addition， the model group showed up-regulated protein levels of PI3K， p-PI3K， Akt， p-Akt， mTOR， p-mTOR， and p62 and mRNA levels of PI3K， Akt， mTOR， and p62 in the synovial tissue， while it showed down-regulated protein levels of LC3Ⅱ/Ⅰ and Beclin-1 and mRNA levels of LC3 and Beclin-1 （P<0.01）. Compared with the model group， medium- and high-dose Danggui Sinitang alleviated the joint swelling （P<0.01）， lowered the serum levels of TNF-α， IL-6， and IL-1β （P<0.05）， and relieved the inflammatory cell infiltration in the synovial tissue of the ankle joint and the fibrous tissue hyperplasia. Moreover， they down-regulated the protein levels of PI3K， p-PI3K， Akt， p-Akt， mTOR， p-mTOR， and p62 and the mRNA levels of PI3K， Akt， mTOR， and p62 in the synovial tissue （P<0.05）， while they up-regulated the protein levels of LC3Ⅱ/Ⅰ and Beclin-1 and the mRNA levels of LC3 and Beclin-1 （P<0.05）. ConclusionDanggui Sinitang， especially at a high dose， can inhibit PI3K/Akt/mTOR signaling pathway to improve autophagy in the synovial tissue， thereby mitigating GA.
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Objective: To study the expressions of Renin, angiotensin converting enzyme (ACE), angiotensin receptor 1 (AT1R), and AT2R in synovial tissue of osteoarthritis (OA) at different stages. Methods: The patients who were treated with upper knee amputation because of trauma or total knee arthroplasty for OA between January 2018 and December 2018 were enrolled. Among them, 32 patients who met the selection criteria were included in the study. According to the Kellgren-Lawrence (K-L) X-ray classification, they were allocated to normal synovial group (group A, n=9), moderate OA synovial group (group B, n=11, K-L level 3), and advanced OA synovial group (group C, n=12, K-L level 4). The relative expressions of Renin, ACE, AT1R, and AT2R mRNAs and proteins were detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot. Results: The relative expressions of Renin, ACE, and AT1R mRNAs and proteins were significantly higher in group B and group C than in group A ( P<0.05). The relative expressions of ACE and AT1R mRNAs and proteins and Renin protein were significantly higher in group C than in group B ( P<0.05). However, the relative expressions of AT2R mRNA and protein were lower in group B and group C than in group A ( P<0.05), and in group C than in group B ( P<0.05). Conclusion: The expressions of Renin, ACE, and AT1R in synovial tissue of osteoarthritis significantly increase as the K-L level increased, and the expression of AT2R decreases. Renin, ACE, AT1R, and AT2R have a certain degree of correlation with the development of OA.
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OBJECTIVE: To observe the effect of moxibustion on Toll-like receptor 4/nuclear factor-κB (TLR 4/NF-κB) signaling in the synovial tissue of the ankle joint in rats with rheumatoid arthritis (RA), so as to analyze its biological mechanism underlying improvement of RA. METHODS: Fifty male SD rats were randomly divided into normal control, model, moxibustion, moxibustion ＋ TLR 4 agonist, and moxibustion ＋ TLR 4 antagonist groups (n＝10 rats in each). The RA model was established by subcutaneous injection of Freund's complete adjuvant (FCA, 0.1 mL/rat) at the right hind-paw and by being raised in a wind (air fan blowing), cold (about 10 ℃) and wet (purling) environment for 20 days. After 3 days of modeling, mild moxibustion was applied to bilateral "Shenshu" (BL 23) and "Zusanli" (ST 36) for 20 minutes, once daily for successive 10 days. The TLR 4 agonist (lipopolysaccharide) or TLR 4 antagonist (TAK-242) (1 mg/mL) was separately administered via the tail vein 30 min before performing moxibustion every time in the agonist group and the antagonist group. The expression of NF-κB inhibitory factor ɑ (IκBɑ), IκB kinase complex β(IκKβ), myeloid differentiation factor 88 (MyD 88), TLR 4, and NF-κB p 65 proteins in the synovial tissue of the ankle joint was detected by using Western blot. RESULTS: Following modeling, the girth of the swollen ankle joint was obviously bigger (P0.05), suggesting that activation of TLR 4 reduced the anti-inflammatory effect of moxibustion intervention. CONCLUSION: Moxibustion can reduce the ankle joint swelling in RA rats, which may be closely associated with its effect in down-regulating the expression of IκBɑ， IκKβ， TLR 4, MyD 88, and NF-κB p 65 proteins and in inhibiting TLR 4/NF-κB signaling in the synovial tissue of the ankle joint.
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OBJECTIVE: To explore the effect of electroacupuncture (EA) on the expression of synovial AMP-activated protein kinase (AMPK) protein α， arginase-1 mRNA, nitric oxide synthase 2 (NOS 2) mRNA, NOD-like receptor protein 3 (NLRP 3) mRNA, and interleukin-1 β (IL-1 β) mRNA in acute gouty arthritis (AGA) rats, so as to explore its mechanisms underlying improvement of AGA via M 1/M 2 macrophage polarization. METHODS: Male Wistar rats were randomly divided into normal control, model, medication (colchicine) and EA groups (n＝15 rats in each group). The AGA model was established by injection of sodium urate crystal (MSU) suspension (0.2 mL) into the articular cavity of the left knee. The rats of the normal control group received articular injection of normal saline (0.2 mL) of the left knee, and those of the medication group were treated by gavage of the colchicine (0.3 mg•kg－1•d－1) once daily for 7 days. EA (2 Hz/10 Hz, 1.0 mA) was applied to "Zusanli"(ST 36) and "Sanyinjiao" (SP 6) of the left hind limb for 10 min, once daily for 7 days. The inflammatory conditions of the synovial membrane tissue of the left knee joint were observed by H．E. staining. The expression levels of phosphorylated AMPKα (p-AMPKα) protein, and arginase-1 (a maker of M 2 macrophages) mRNA, NOS 2 (a maker of M 1 macrophages) mRNA, NLRP 3 mRNA, and IL-1 β mRNA in the knee joint synovial tissue were detected by Western blot and quantitative real-time PCR, respectively. RESULTS: Compared with the normal group, the inflammatory cell infiltration of the synovial tissue was more severe, the expression of p-AMPKα protein was significantly decreased (P0.05), except higher up-regulation of arginase-1 mRNA in the medication group (P<0.05)．. CONCLUSION: EA intervention can up-regulate the expression of arginase-1 mRNA and p-AMPKα protein, and down-regulate the expression of NOS 2, IL-1 β and NLRP 3 mRNAs in synovial tissues in AGA rats, which may contribute to its anti-inflammatory effect by promoting conversion of macrophages from M 1 pro-inflammatory phenotype to M 2 anti-inflammatory phenotype.
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Objective To measure the endogenous metabolites in synovial tissues of rheumatoid arthritis (RA) rabbits with cold syndrome;To investigate the specificity mechanisms of heat-reinforcing needling for RA. Methods A total of forty healthy purple blue rabbits were randomly allocated to normal group, model group, reinforcing-reducing needling group (RRN), twirling-reinforcing needling group (TRN) and heat-reinforcing needling group (HRN) 8 rabbits in each group. Arthritis rabbits cold model was made with ovalbumin and freezing. Except for normal groupand model group, RRN was given acupuncture of reinforcing-reducing needling at Zusanli (ST36), TRN was given acupuncture of twirling-reinforcing needling at Zusanli (ST36), HRN was administrated acupuncture of heat-reinforcing needling at Zusanli (ST36), once a day and retaining 30 min, a total of seven days. Fresh synovial tissues of rabbits knee joints were extracted after the intervention, then GC-Q/TOF-MS technology were employed to evaluate metabolic profiles. Results The synovial tissues metabolites of TCA cycle, carbohydrate and fatty acid metabolism in model group mainly increased compared with normal group (P<0.05); The synovial tissues metabolites of various kinds above-mentioned decreased compared with model group in all intervention of acupuncture group (P<0.05), and the metabolites of TCA cycle and carbohydrate metabolism in HRN were obviously less than that of RRN and TRN (P<0.05). Conclusion The specificity of heat-reinforcing needling for RA manifests in regulation of TCA cycle and carbohydrate metabolism.
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Background: Shoulder joint is a multiaxial, diarthrodial joint of ball and socket variety. The various components of shoulder joint i.e. capsule, various ligaments, joint cavity and synovial tissue start developing in early embryonic and foetal life roughly in between 6th to 12th week of foetal development. The present study aims to analyze the sequences of development of various structures of shoulder joint in human embryo and compare the findings with other observers to gain some insight regarding its development and an attempt is made to correlate these observations clinically to analyze cause and management of recurrent shoulder dislocations. Methods: Shoulder joints of 32 foetuses collected from areas in and around Jammu were dissected properly and decalcified in Gooding and Stewart’s solution. Sections were cut after obtaining blocks by paraffin wax embedding method. Slides were stained using Haematoxylin and Eosin, Masson’s trichrome and orcein staining and important findings were documented. Results: The bony structures - head of humerus and glenoid fossa and joint cavity appear by 10 weeks which prolongs into bicipital sulcus by 12-1/2 weeks. Synovial tissue appears by 10 weeks and synovial villi appear by 14 weeks. Glenoid labrum, capsular ligament, coracohumeral ligament and superior glenohumeral ligament are seen by 10 weeks. Middle glenohumeral ligament is seen at 12-1/2 weeks while inferior glenohumeral ligament is seen at 14 weeks. Tendon of biceps is seen at 10 weeks. Conclusions: By 10 weeks of gestational age various structures of shoulder joint develop in situ, resembling in form and arrangement as those of adults. From these early stages, development proceeds rapidly to achieve adult characteristics. There are no intermediate stages in between where structures similar to those of lower forms i.e. syn/amphi artroses appear temporarily.
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Objective: To investigate the anti-inflammatory, especially the inhibitory effects of TZT-5, an active component from the leaves of Triterygium wilfordii, on the rheumatoid arthritis of rats and to explore its pharmacodynamic effect using an arthritic model of rats. Methods: The ear swelling model induced by croton oil in mice, paw edema model induced by carrageenin in rats, chicken collagen II (CCII)-induced arthritic model, and adjuvant-induced arthritic (AIA) model in rats were established, respectively. The in vivo anti-inflammatory and inhibitory effects of TZT-5 on the rheumatoid arthritis of rats were observed. Results: TZT-5 could alleviate the symptoms of the above four inflammation animal models, ameliorate the infiltration of inflammatory cells in synovium, and inhibit the proliferation of synovial cells in both CCII-induced arthritic models and AIA models of rats. Conclusion: TZT-5 has the ideal anti-inflammatory effects on CCII-induced arthritic model and AIA model of rats and it is a candidate as a new anti-rheumatoid arthritis drug.