Modulation of T Cell Differentiation in Mice with COPD Combined with Lung Cancer Through Key Targets of PD-1 by Tao Hong Si Wu Tang.

The objective is to assess the anti-inflammatory effect of Tao Hong Si Wu Tang combined with anti-PD-1 in a mouse model of COPD combined with lung cancer, elucidating its mechanism through modulation of PD-1/PD-L binding, regulation of Th1/Th2 and Th17/Treg balance, inhibition of IL-4 and IL-17, and promotion of IFN-γ and TGF-ß levels in peripheral blood. One hundred male C57/BL6 mice were randomly allocated to five groups: A (blank control), B (model control), C (THSW), D (anti-PD-1), and E (THSW + anti-PD-1), with 20 mice in each group. The COPD model was induced using fumigation and LPS intra-airway drip, followed by the establishment of lung cancer by Lewis cell inoculation. Treatment groups received Tao Hong Si Wu Tang or/and PD-1 monoclonal antibody. Various indicators were assessed, including macroscopic observation, HE staining of lung tissue, ELISA for cytokines, flow cytometry for cell proportions, and immunohistochemistry/western blotting for protein expression. Lung tissue analysis revealed significant differences between groups, with marked tumor formation observed in groups B-E. Serum levels of IL-4, IFN-γ, IL-17, and TGF-ß were significantly altered, along with changes in CD4 + T/CD8 + T ratio and cytokine-producing cell populations. Expression levels of key proteins were also significantly affected across treatment groups. Tao Hong Si Wu Tang demonstrated anti-inflammatory effects comparable to anti-PD-1, potentially through modulation of PD-1/PD-L binding, correction of Th1/Th2 and Th17/Treg imbalance, and modulation of cytokine levels. These findings suggest a role for Tao Hong Si Wu Tang in ameliorating inflammation and immune dysregulation in COPD combined with lung cancer.

[Mechanism of electroacupuncture penetration needling for relieving synovial inflammation of knee osteoarthritis through TLR4/MyD88/NF-κB signal pathway].

OBJECTIVE: To observe the effects of electroacupuncture (EA) penetration needling on Toll-like receptors 4/myeloid differentiation factor 88/nuclear factor-kappa B (TLR4/MyD88/NF-κB) signaling pathway in rat synovium and the serum-related inflammatory factors, so as to explore the mechanism of EA penetration needling on synovial inflammation in rats with knee osteoarthritis (KOA). METHODS: SD male rats were randomly divided into sham-operation group, model group, EA+penetration needling group, and conventional EA group, with 16 rats in each group. The rats model was prepared by anterior cruciate ligment transection and these rats were forced to exercise for 8 weeks after operation. After successful modeling, in the EA+penetration needling group, the needles were inserted at "Dubi" (ST35) "Neixiyan" (EX-LE4), and at "Xuehai"(SP10) "Liangqiu"(ST34) on the right hind limb, towards each other, 5-8 mm in depth, respectively. In the conventional EA group, the needles were inserted at ST35 and EX-LE4 on the right hind limb, obliquely, at 30° angle to the skin, 3-5 mm in depth; and were inserted at SP10 and ST34 on the right hind limb perpendicularly, 3-5 mm in depth. In these two groups, electric stimulation was operated with dense-disperse wave, 2 Hz/10 Hz in frequency and 0.5-1.5 mA in intensity, retained for 20 min in each treatment. The treatment was given once daily, 10 days as 1 course of treatment, and 2 courses were required at the interval of 2 days. After the intervention, the knee joint effusion was observed by musculoskeletal ultrasound; the contents of IL-1ß, IL-6 and TNF-α in serum were determined by ELISA; the morphological changes in the synovium were observed after H.E. staining; the positive expression of NF-κB p65 in the synovial membrane was detected by immunohistochemical method; the expression levels of TLR4, MyD88, TRAF-6 and NF-κB p65 proteins in the synovial membrane were determined by Western blot. RESULTS: Compared with the sham-operation group, in the model group, the knee joint effusion was obviously increased, the synovial lining cells were distributed irregularly, the cells were disarranged, the pannus was formed largely, and a great number of the inflammatory cells were infiltrated; the contents of serum IL-1ß, IL-6 and TNF-α, the positive expression of NF-κB p65, the protein expression levels of TLR4, MyD88, TRAF-6 and NF-κB p65 in the synovial tissue were increased (P<0.05). Compared with the model group, the knee joint effusion was reduced, the synovial lining cells were proliferated, a small number of the inflammatory cells were infiltrated, and the pannus was formed lightly; the contents of serum IL-1ß, IL-6 and TNF-α, the positive expression of NF-κB p65, the protein expression levels of TLR4, MyD88, TRAF-6 and NF-κB p65 in the synovial tissue were lower (P<0.05) in the EA+penetration needling group and the conventional EA group. In the conventional EA group, the knee joint effusion was increased, the synovial lining cells were proliferated, the inflammatory cells were infiltrated largely, and the pannus was formed increasingly; the contents of serum IL-1ß, IL-6 and TNF-α, and the protein expression levels of TLR4, MyD88 and NF-κB p65 in the synovial tissue were increased when compared with the EA+penetration needling group (P<0.05). CONCLUSION: The EA+penetration needling can significantly relieve the synovial inflammatory reaction and the knee joint effusion in KOA rats. The mechanism is probably related to down-regulating the downstream inflammatory cascade through inhibiting the transduction of TLR4/MyD88/NF-κB signaling pathway.

Exosomes may be the carrier of acupuncture treatment for major depressive disorder.

The incidence of major depressive disorder (MDD) is increasing all over the world. There is a great need for complementary or alternative therapies with high safety, few side effects, and precise efficacy to care for MDD. In China, acupuncture has significant laboratory data and clinical trials to demonstrate its antidepressant efficacy. However, there is no clear answer as to how it works. Exosomes are membranous vesicles that rely on cellular multivesicular bodies (MVBs) fused to the cell membrane for release into the extracellular matrix. Almost all cell types are capable of producing and releasing exosomes. As a result, exosomes contain complex RNAs and proteins from their relatives (Cells that secretes exosomes). They can cross biological barriers and participate in biological activities, such as cell migration, angiogenesis, and immune regulation. These properties have made them a popular research topic. Some experts have suggested that exosomes may serve as delivery vehicles for acupuncture to work. This presents both an opportunity and a new challenge for improving the protocols of acupuncture as a treatment for MDD. To better define the relationship between MDD, exosomes, and acupuncture, we reviewed the literature from the last few years. Inclusion criteria included randomized controlled trials and basic trials evaluating acupuncture in the treatment or prevention of MDD, the role of exosomes in the development and progression of MDD, and the role of exosomes in acupuncture. We believe that acupuncture may affect the distribution of exosomes in vivo, and exosomes may be a new carrier for acupuncture treatment of MDD in the future.

[Eye acupuncture improves cerebral ischemia reperfusion injury by improving autophagy via ATF6 pathway in cerebral ischemia reperfusion injury rats].

OBJECTIVE: To explore the effect of eye acupuncture on autophagy and expressions of autophagy-related proteins Beclin1, LC3B, ATF6 and XBP1 in the infarction area of brain tissue in rats with acute cerebral ischemia-reperfusion injury (CIRI), so as to explore its mechanisms underlying improvement of CIRI. METHODS: Male SD rats were randomly divided into sham operation, model and eye acupuncture groups (n=16 in each group). The CIRI model was prepared by occlusion of the middle cerebral artery. Eye acupuncture stimulation was applied to bilateral "Gan"(Liver), "Shangjiao"(Upper-energizer), "Xiajiao"(Lower-energizer) and "Shen"(Kidney) regions at 0, 12 and 24 h after CIRI, 30 min each time. The neurological deficit score was given by referring to Longa's method, and TTC staining used to determine the success of model replication. After the treatment, the pathological changes of the cerebral infarction area were observed under light microscope, and the autophagosomes were observed by electron microscope. The protein expression levels of LC3B, Beclin1, ATF6 and XBP1 in the infarction area of brain tissue were detected by Western blot. The immunoactivity of Beclin1 and the immunofluorescence density of ATF6 and XBP1 in the infarction area of brain tissue were determined by immunohistochemistry. RESULTS: The Longa's score, and the protein expression levels of LC3B, Beclin1, ATF6 and XBP1 and immunoactivity or immunofluorescence density of Beclin1, ATF6 and XBP1 were significantly higher in the model group than those in the sham operation group (P<0.01), and considerably lower in the eye acupuncture group than those in the model group (P<0.01). Under light microscope, the model group had typical ethmoidal reticular cerebral infarction, while the eye acupuncture group had significantly smaller areas and clearer edges. Under electron microscope, there were more autophagosomes in the cytoplasm of neurons in the model group, and fewer autophagosomes in the eye acupuncture group (in contrast to the model group). CONCLUSION: Eye acupuncture can improve the neurological function and mitigate cerebral injury in CIRI rats which may be associated with its function in inhibiting autophagy in the brain tissue by regulating ATF6 pathway.

Huangqi Guizhi Wuwu Decoction can prevent and treat oxaliplatin-induced neuropathic pain by TNFα/IL-1ß/IL-6/MAPK/NF-kB pathway.

OBJECTIVE: This study explored the effects and mechanisms of Huangqi Guizhi Wuwu Decoction on chemotherapy-induced neuropathic pain (CINP). METHODS: Bodyweight and related behavioral testing of the rat model were utilized to investigate the effects of Huangqi Guizhi Wuwu Decoction on CINP. ELISA was used to measure the levels of TNF-α, IL-1ß, and IL-6, in the serum of chronic CINP rats. Immunohistochemistry and Western blot analysis were performed to detect the expression of MAPK pathway related-proteins namely ERK1/2, p38, and JNK, and the expression of downstream essential proteins such as c-Fos, CREB, and NF-κB. RESULTS: Body weight and related behavioral testing of the rat model suggests that Huangqi Guizhi Wuwu Decoction can improve the slow weight gain of oxaliplatin-induced chronic CINP model rats and effectively prevent and treat oxaliplatin-induced regular CIPN rat model of hyperalgesia. It can also oppress the mechanical pain threshold, cold pain threshold, and heat pain threshold decreased. Furthermore, by ELISA, immunohistochemistry, and western blot analysis, we found that Huangqi Guizhi Wuwu Decoction can down-regulate the levels of TNF-α, IL-1ß, and IL-6 in the serum of chronic CINP rats induced by oxaliplatin. It also suppresses the expression of MAPK pathway related-proteins ERK1/2, p38, and JNK. This results in a decrease in the expression of downstream essential proteins, c-Fos, CREB, and Nf-κB. CONCLUSIONS: In conclusion, we found that Huangqi Guizhi Wuwu Decoction can combat nerve cell injury, reduce pain sensitization, and prevent and repair the damage of nerve cells in the oxaliplatin CINP model rats via TNFα/IL-1ß/IL-6/MAPK/NF-kB pathway.

Therapeutic effect of Patrinia villosa on TNBS-induced ulcerative colitis via metabolism, vitamin D receptor and NF-κB signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Ulcerative colitis (UC) is a chronic inflammatory disease affecting the colon. Patrinia villosa Juss. (P.V) is an important traditional Chinese medicine widely used for more than 2000 years from ShenNongBenCaoJing, a famous ancient Chinese medicinal literary. P.V is often used in the treatment of UC, but the pathogenesis is not clear. AIM OF THE STUDY: This study was designed to analysis the metabolic pathways and relevant mechanisms of P.V on UC rats induced by TNBS. MATERIALS AND METHODS: The rat model of UC was established by 2,4,6-trinitrobenzene sulfonic acid (TNBS)/ethanol method. Three doses of P.V (21 g/kg, 43 g/kg, 64 g/kg) were administrated for 14 days. Disease activity index (DAI) scoring system and H&E staining were used to evaluate the efficacy. A method for simultaneous detection of 96 endogenous metabolic components was established by UPLC-MS. The method was used to detect the metabolites in serum and liver of rats with UC induced by TNBS. PLS-DA and Metaboanalyst were used to analyze the main metabolic pathways involved in the treatment of UC. The contents of IL-1ß, TNF-α and IL-6 in the colonic homogenate of rats were detected by enzyme-linked immunosorbent assay (ELISA). The expression levels of VDR, NF-κB, p-NF-κB, NLRP3 and caspase-1 in colon tissues of rats were detected by the method of Western blot. RESULTS: DAI scoring system and H&E staining indicated that P.V have the obvious therapeutic effect on UC induced by TNBS as a dosage-dependent manner. 36 potential biomarkers in serum and 26 potential biomarkers in liver were found in positive and negative ion mode of UPLC-MS, which significantly affected Glycine, serine and threonine metabolism, Glycerophospholipid metabolism, Purine metabolism, Histidine metabolism, Alanine, aspartate, and glutamate metabolism, Arginine and proline metabolism in serum, and significantly affected Purine metabolism, Lycine, serine and threonine metabolism, Glutathione metabolism, Glyoxylate and dicarboxylate metabolism in the liver. The contents of pro-inflammatory cytokines and receptors related to NF-κB signaling axis of model group were significantly higher than those of the control group, compared with the model group, their contents of the P.V group were significantly decreased (p < 0.01). Compared with the model group, the expression of NF-κB, p-NF-κB, NLRP3 and caspase-1 in colon tissues of the rats in P.V group were significantly decreased (p < 0.01). The expression of VDR in model group were significantly reduced compared to that in the control group, compared with the model group, the expression of VDR in P.V group were significantly increased (p < 0.01). CONCLUSION: P.V has an obvious therapeutic effect on UC induced by TNBS by regulating the energy metabolism, amino acid metabolism, purine metabolism, bile acid metabolism and lipid metabolism. P.V exerts anti-inflammatory effect by impacting bile acid levels, activating VDR, and inhibiting the overactivation of NF-κB signaling pathways.

Electroacupuncture Inhibits Atherosclerosis through Regulating Intestinal Flora and Host Metabolites in Rabbit.

METHODS: In this study, general rabbit conditions, vascular histology, metabolites, and intestinal flora structures were analyzed. Integrated analysis of metabolomics and 16S rRNA sequencing were performed. All the rabbits were randomly divided into four groups. The rabbit model of atherosclerosis was established. The histopathological change in the common carotid artery was assessed by HE staining and the structural change in the flora by 16S rRNA sequencing. HPLC-TOF-MS and Agilent MPP 12.1 were integrated to identify and screen out differential metabolites. Correlational analyses of every differential metabolite with intestinal flora were integrated on Omicshare platform. RESULTS: Atherosclerotic rabbits showed obvious changes in general conditions, significant fibrous cap and necrotic center on carotid artery, abnormal intestinal bacteria structure, and metabolites levels. Electroacupuncture improved the conditions, reduced lipid deposition on the carotid artery wall, diversified intestinal flora, and normalized host metabolism. Integrated analysis showed that 149 altered metabolites were related to 22 intestinal flora, among which eight intestinal floras and 21 metabolites have relationships with atherosclerosis. CONCLUSION: Electroacupuncture can effectively reverse atherosclerosis through manipulating the structural feature of intestinal flora to influence the host metabolites. The possible mechanisms involved activating signal pathways through host metabolites or affecting the activity of cardiovascular-related enzymes, or regulating host lipid metabolism directly.

Nucleotide-binding oligomerization domain protein 1 enhances oxygen-glucose deprivation and reperfusion injury in cortical neurons via activation of endoplasmic reticulum stress-mediated autophagy.

Cerebral ischemia-reperfusion (CIR) can regulate multiple transcription factors to enhance or attenuate injury. Nucleotide-binding oligomerization domain protein 1 (NOD1) has been reported to be involved in autophagy and endoplasmic reticulum (ER) stress. Moreover, autophagy and ER stress play important roles in CIR injury. Hence, the function of NOD1 in CIR injury was explored in this study. Primary rat cortical neurons were treated with oxygen-glucose deprivation and reperfusion (OGD/R) in vitro. NOD1 level was measured using immunofluorescence, real-time quantitative PCR and western blotting and its ubiquitination using co-immunoprecipitation. Results showed that OGD/R up-regulated NOD1 level but inhibited NOD1 ubiquitination. Then the effect of NOD1 on OGD/R-induced changes in cell viability, apoptosis, autophagy and ER stress was evaluated by methyl thiazolyl tetrazolium assay, lactate dehydrogenase release, Hoechst staining, detection of autophagy and ER stress-related proteins using western blotting and infection with GFP-LC3 lentiviruses. OGD/R decreased cell viability and increased cell apoptosis. NOD1 up-regulation promoted these changes, but NOD1 down-regulation reversed these changes. Moreover, OGD/R triggered autophagy and ER stress and NOD1 silencing reversed OGD/R-induced changes in autophagy and ER stress. To validate the role of autophagy in OGD/R injury, autophagy inducer rapamycin was used. Rapamycin promoted OGD/R-induced decrease in cell viability and counteracted NOD1 silencing-induced increase in cell viability. In addition, ER stress inducer tunicamycin was used to investigate the relationship between ER stress and autophagy. Tunicamycin promoted OGD/R-induced decrease in cell viability and reversed NOD1 silencing-induced increase in cell viability. Tunicamycin also enhanced OGD/R-induced autophagy and reversed NOD1 silencing-induced inhibition in autophagy. The results indicated that NOD1 promoted OGD/R injury in cortical neurons through activating ER stress-mediated autophagy. This study provides new insights for the target of CIR injury treatment.

[Effect of electroacupuncture on urodynamics and expression of Wnt-1, ß-catenin, and Ngn1 in the spinal cord in rats with bladder detrusor hyperreflexia due to supersacral spinal cord transection].

OBJECTIVE: To investigate the effect of electroacupuncture (EA) at "Dazhui" (GV14) and "Ciliao" (BL32) on rats with bladder detrusor hyperreflexia (DH) after supersacral spinal cord transection, as well as the mechanism of EA in improving the urinary function by regulating the expression of Wnt-1, ß-catenin and Neurogenin 1(Ngn1). METHODS: A total of 48 female Sprague-Dawley rats were randomly divided into sham-operation group, model control group, EA group, and EA control group, with 12 rats in each group. T10 spinal cord transection (SCT) was performed by surgery. The Basso, Beattie and Bresnahan (BBB) score was used to evaluate the motor function of SCT rat, and the Crede technique was used to assist urination. After the urine volume became stable, the urodynamic test was used to determine whether a rat model of DH was successfully established. The rats in the EA group were given EA at GV14 and BL32, and those in the EA control group were given EA (10 Hz/50 Hz, 20 min) at the acupuncture points at 1 cm next to GV14 and BL32 at both sides alternatively. EA was performed once a day for one week. Urodynamic parameters were used to evaluate urinary function. Western blot and immunohistochemistry were used to measure the expression of Wnt-1 and ß-catenin in the spinal cord, and immunofluorescence assay was used to measure the expression of Ngn1 in the spinal cord. RESULTS: The BBB score of the model control group significantly decreased compared with that of the sham-operation group(P<0.01), and the EA group was significantly higher than the model control group and the EA control group. Compared with the sham-operation group, the model control group had significant increases in bladder base pressure, maximum pressure, and leak point pressure (P<0.01) and significant reductions in maximum bladder capacity and compliance (P<0.01). Compared with the model control group, the EA group had significant reductions in bladder base pressure, maximum pressure, and leak point pressure (P<0.01) and significant increases in maximum bladder capacity and compliance (P<0.01, P<0.05). Compared with the EA group, the EA control group had significant increases in bladder base pressure, maximum pressure, and leak point pressure (P<0.01) and significant reductions in maximum bladder capacity and compliance (P<0.01, P<0.05). Compared with the sham-operation group, the model control group had significant increases in the protein expression of Wnt-1 and ß-catenin (P<0.05, P<0.01) and a signi-ficant reduction in the protein expression of Ngn1 in the spinal cord (P<0.01). Compared with the model control group, the EA group had significant increases in the protein expression of Wnt-1, ß-catenin and Ngn1 in the spinal cord (P<0.01). Compared with the EA group, the EA control group had significant reductions in the protein expression of Wnt-1, ß-catenin, and Ngn1 in the spinal cord (P<0.01). CONCLUSION: EA at GV14 and BL32 can significantly improve urinary function in rats with bladder DH due to SCT, partially by activating the Wnt/ß-catenin signaling pathway and promoting the protein expression of Wnt-1, ß-catenin and Ngn1.

[Effect of deep and shallow electroacupuncture stimulation at "Huantiao"(GB30) on expression of phosphorylated-p38 and phosphorylated-p53 proteins and apoptosis in dorsal root ganglia in sciatic nerve injury rats].

OBJECTIVE: To observe the effect of deep electroacupuncture (EA) stimulation at "Huantiao"(GB30) on hindlimb motor function and expression of p38 mitogen-activated protein kinase (p38 MAPK ) and p53 proteins in dorsal root ganglia (DRG) in rats with chronic constrictive injury (CCI) of sciatic nerve. METHODS: Forty-eight SD rats (half male and half female) were randomly divided into control, model, shallow EA (SEA) stimulation and deep EA (DEA) stimulation groups (n=12 in each group). The CCI model was constructed by implanting a silicone tube close to the sciatic nerve of the left hind limb. For DEA group and SEA group, filiform acupuncture needles were inserted into GB30 about 12-14 mm deep and 5-8 mm deep (monitored by using a high-frequency ultrasound device), respectively, followed by electrical stimulation (2 Hz/100 Hz, 1 mA) using an EA stimulator. The intervention was conducted for 15 min every time, once daily for 14 days. The sciatic nerve function index (SFI) calculated to assess the motor function status. Histopathological changes of the sciatic nerve were displayed by H.E. staining. The expression levels of phosphorylated-p38 MAPK (p-p38) and phosphorylated-tumor protein p53 (p-p53) in DRGs of L4-L5 on the affected side were observed by immunohistochemical staining. RESULTS: Following modeling, the SFI were significantly decreased (P<0.01), and the expression levels of p-p38 and p-p53 proteins of L4-L5 DRGs were considerably increased in the model group (P<0.05). After the intervention, the SFI were obviously increased, and the expression levels of p-p38 and p-p53 proteins notably down-regulated in both DEA and SEA groups relevant to the model group (P<0.01, P<0.05). The therapeutic effect of DEA was significantly superior to that of SEA in raising SFI and down-regulating expression le-vels of p-p38 and p-p53 proteins (P<0.01, P<0.05). H.E. staining showed disordered arrangement of the sciatic nerve fibers and myelin, disaggregation of the myelin and axons with deformity and vacuolation in some of them and with an increase of Schwann cells in the model group, which was relatively milder in both DEA and SEA groups. CONCLUSION: Both DEA and SEA at GB30 can obviously improve the motor function in CCI rats, which may be associated with its function in down-regulating the expression of p-p38 and p-p53 proteins in L4-L5 DRGs, restraining p38 MAPK signaling. The therapeutic effect of DEA is evidently better than that of SEA.

Metabolomics analysis of baicalin on ovalbumin-sensitized allergic rhinitis rats.

Allergic rhinitis (AR) is a global health problem that appears in all age groups and affects approximately 15-30% of people. Baicalin has been used for the treatment of various allergic diseases, including AR. However, the metabolic mechanisms of AR and baicalin against AR have not been systematically studied. Here, ovalbumin-sensitized AR rats were used as a model, and animal behaviour, histological analysis, enzyme-linked immunosorbent assay (ELISA) and metabolomics were used to elucidate the mechanism of baicalin for AR. The results indicated that baicalin has a protective effect on AR rats by inhibiting the release of immunoglobulin E (IgE), histamine, interleukin-1 beta (IL-1ß), interleukin-4 (IL-4), interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF-α). In addition, ovalbumin-induced AR included modulation of arachidonic acid, leukotriene A4 (LTA4), leukotriene B4 (LTB4), α-ketoglutaric acid, phosphatidylcholine PC (20 : 4/0 : 0), PC (16 : 0/0 : 0), citric acid, fumarate, malate, 3-methylhistidine, histamine and other amino acids that are involved in arachidonic acid, histidine metabolism, the TCA cycle and amino acid metabolism. Thus, AR could be alleviated or reversed by baicalin.

[Anti-apoptotic Mechanism of Deep Electroacupuncture at "Huantiao" (GB 30) in Repair of the Injured Sciatic Nerve in Rats].

OBJECTIVE: To observe the effect of deep electroacupuncture (EA) at "Huantiao" (GB 30) on functional and pathological changes of the damaged sciatic nerve and apoptosis-related factors in lumbar dorsal root ganglia (DRGs) in rats, so as to study its mechanisms underlying relieving sciatica. METHODS: Forty-eight SD rats were randomly divided into normal, mo-del, deep EA (DEA) and shallow EA (SEA) groups (n＝12 in each group)．The sciatic nerve injury model was established by silicone tube extrusion of the sciatic nerve stem. For DEA group, acupuncture needles were inserted into GB 30 about 8 mm deep to induce nerve impulse under the guidance of high-frequency ultrasound, and the needles were inserted into GB 30 about 3-5 mm in the SEA group. The EA treatment was applied to bilateral GB 30 for 20 min beginning from the 15th day on after modeling, once daily for 14 days. The sciatic nerve function index (SFI) and motor nerve conduction velocity (MNCV) were recorded and calculated. Pathological changes of the sciatic nerve were displayed by H. E. staining. The cell apoptosis and expression of PI 3 K and AKT proteins in lumbar 4-5 DRGs were detected by TUNEL staining, and immunohistochemistry, respectively. RESULTS: In comparison with the normal group, the SFI and MNCV were significantly decreased (P<0.01，P<0.05), the number of neuronal apoptosis and the expression of PI 3 K and AKT in L 4-L 5 DRGs were significantly increased in the model group (P<0.05). Following the treatment, the SFI, MNCV, and the number of neuronal apoptosis were reversed compared with the model group (P<0.01, P<0.05), both PI 3 K and AKT expression levels were significantly up-regulated in both DEA and SEA groups (P<0.05). The therapeutic effects were significantly better in the DEA group than in the SEA group in down-regulating cell apoptosis number and in up-regulating SFI, MNCV, and PI 3 K and AKT protein expression on day 28 of modeling (P<0.01，P<0.05). CONCLUSION: Deep EA at GB 30 can activate PI 3 K-AKT signaling pathway, inhibit the apoptosis of nerve cells in DRGs after sciatic nerve injury in rats, which may contribute to its effect in improving neurological impairment.

Bleomycin inhibits proliferation and induces apoptosis in TPC-1 cells through reversing M2-macrophages polarization.

Papillary thyroid carcinoma (PTC) is one of the most common types of thyroid malignancy. Previous studies have demonstrated that the density of tumor-associated macrophages (TAMs) within the tumor microenvironment affects the progression of PTC due to the imbalance in M1/M2 macrophage subtypes. M2 macrophages induce anti-inflammatory effects and promote tumor progression, whereas M1 macrophages destroy tumor cells. Therefore, reversing TAM polarization to M1 may be a novel strategy for the treatment of cancer. Although bleomycin (BLM) is a commonly used anti-cancer drug, which regulates the secretion of relevant cytokines, high dose and long-term treatment with BLM may lead to pulmonary fibrosis. In the present study, flow cytometry data revealed that low dose treatment with BLM (5 or 10 mU/ml) facilitated the expression of the M1 phenotype markers cluster of differentiation (CD)80 and C-C chemokine receptor 7, and concurrently suppressed the M2 marker CD206 on M2-macrophages. Reverse transcription-quantitative polymerase chain reaction data revealed that the expression levels of tumor necrosis factor-α and interleukin-1ß markedly increased, whereas the expression of IL-10 decreased in M2 macrophages treated with BLM. A fluorescein isothiocyanate-dextran uptake experiment revealed that BLM increased the phagocytic capacity of M2, however not M1 or M0 macrophages. In addition, to verify the effect of BLM-treated M2 macrophages on thyroid carcinoma cells, a co-culture system of macrophages and the human PTC cell line TPC-1, was established. BLM-treated M2 macrophages increased the number of cells in early and late apoptosis and inhibited the migration, proliferation and invasion of TPC-1 cells. These results suggest that a low dose and indirect effect of BLM may induce suppressive effects on PTC by selectively reversing M2 macrophage polarization to M1, which may provide a novel strategy for cancer treatment.

Scutellarin Prevents Nonalcoholic Fatty Liver Disease (NAFLD) and Hyperlipidemia via PI3K/AKT-Dependent Activation of Nuclear Factor (Erythroid-Derived 2)-Like 2 (Nrf2) in Rats.

BACKGROUND Nonalcoholic fatty liver disease (NAFLD) is a condition characterized by excessive fat accumulation in the form of triglycerides. The incidence of NAFLD and hyperlipidemia, with their associated risks of end-stage liver and cardiovascular diseases, is increasing rapidly. This study aimed to investigate the effects of scutellarin on the experimental NAFLD in high-fat diet fed and chronic stress rats, and its possible mechanism. MATERIAL AND METHODS Sprague-Dawley rats were fed with high-fat diet and subjected to chronic stress for 12 weeks, and administered orally with scutellarin for 4 weeks (n=8), and then blood and livers were harvested for analyzing. Enzyme activity assay, immunofluorescence, Western blot, and quantitative RT-PCR were performed to analyze the factors of the oxidant/antioxidant system and pathway. RESULTS After the high-fat diet and chronic stress administration for 12 weeks, serum and liver lipid metabolism of treatment groups with the different doses of SCU effectively improved and the degree of oxidative damage reduced. Using Western blot assay and immunofluorescence (IF) staining assay, Nrf2, HO-1, and PI3K, and AKT proteins significantly increased after SCU treatment for 4 weeks (P<0.01). The hepatic mRNA expression of HO-1, NQO1, and Nrf2 in SCU treatment groups was upregulated significantly through quantitative RT-PCR assay (P<0.05). However, compared to the positive control group, no difference was detected in the SCU (100 or 300 mg/kg) groups (P>0.05). These results indicate that SCU protects against NAFLD in rats via attenuation of oxidative stress. CONCLUSIONS The antioxidant effects of SCU on NAFLD are possibly dependent on PI3K/AKT activation with subsequent Nrf2 nuclear translocation, which increases expression of HO-1 and NQO1. We therefore suggest that breviscapine may be a potentially useful therapeutic strategy for NAFLD and hyperlipidemia.

Effects of total flavonoids of sea buckthorn ( Hippophae rhamnoides L.) on cytotoxicity of NK92-MI cells.

Sea buckthorn ( Hippophae rhamnoides L.) has multifarious medicinal properties including immunoregulatory effect. The total flavonoids of Hippophae rhamnoides L. (TFH) are the main active components isolated from berries of sea buckthorn. The aim of this study was to evaluate the effects of TFH on the cytotoxicity of NK92-MI cells and its possible mechanisms. NK92-MI cells were treated with TFH (2.5 or 5.0 mg/L) or phosphate-buffered saline (PBS) for 24 h, the cytotoxicity against K562 was detected by measuring the release of lactate dehydrogenase (LDH), expression levels of NCRs (NKp30, NKp44, NKp46) and NKG2D were detected by flow cytometry, and expression levels of perforin and granzyme B were detected by western blot. Cytokine Antibody Arrays with 80 cytokine proteins were used to profile the effect of TFH on cytokines. Western blot was adopted to detect the effects of TFH on STAT1, STAT4, and STAT5 signal pathway. Compared with the normal control group, TFH could significantly enhance NK92-MI cell cytotoxicity against K562 cells, upregulate expressions of NKp44, NKp46, perforin, and granzyme B. TFH could upregulate expressions of IL-1α, IL-2, IL-7, IL-15, CSF-2, CSF-3, MCP-1, MIG, IFN-γ, TNF-α, and TNF-ß and downregulate expressions of IL-16, MIP-1ß, CX3CL-1, and MIF. TFH could increase expressions of phospho-STAT1 and phospho-STAT5. The results suggest that TFH stimulated NK92-MI cells to activate and enhance cytotoxicity of NK92-MI cells.

Moxibustion regulates inflammatory mediators and colonic mucosal barrier in ulcerative colitis rats.

AIM: To observe the efficacy and mechanism of grain-sized moxibustion at different acupoints in a rat model of ulcerative colitis (UC). METHODS: Sprague-Dawley rats were randomly divided into control, UC model, grain-sized moxibustion at a single acupoint (CV 12), grain-sized moxibustion at two acupoints (CV 12 and CV 4), grain-sized moxibustion at three acupoints (CV 12, CV 4, and ST 36), and medication groups (n = 8/group). The UC model was established by enema of trinitrobenzene sulfonic acid. Direct moxibustion was used once a day for 7 d. Disease activity index (DAI) was evaluated before and after the treatment. Morphologic changes of intestinal tissue were observed under an optical microscope. The expression of tumor necrosis factor (TNF)-α and p38 mitogen-activated protein kinase (p38MAPK) in colonic tissue was detected using Western blot, and the levels of occludin and zonula occludens-1 (ZO-1) mRNAs were detected using reverse transcription PCR. RESULTS: Compared with the control group, the intestinal mucosae were incomplete in the model group, glandular structures were irregular, and submucosae were edematous, hyperemic, and infiltrated with inflammatory cells. The DAI scores and expression of TNF-α and p38MAPK were increased significantly in the model group compared to controls (Ps < 0.01), while the mRNA levels of occludin and ZO-1 were reduced significantly (Ps < 0.01). Compared with the model group, colonic mucosa and the arrangement of glands were complete and regular in the treatment groups. DAI scores and the expression of TNF-α and p38MAPK were reduced significantly in moxibustion groups compared to controls (Ps < 0.01), while the mRNA levels of occludin and ZO-1 were increased significantly (Ps < 0.01). The improvements in the above indices in the three acupoints group and the medication group were superior to those in the single and two acupoints groups (all P < 0.05). CONCLUSION: Reduction of TNF-α and p38MAPK and increased expression of occludin and ZO-1 in colonic tissue represent a potential mechanism for improved intestinal mucosal tissue repair with grain-sized moxibustion.

Proteomics Study on Nonallergic Hypersensitivity Induced by Compound 4880 and Ovalbumin.

Nonallergic hypersensitivity reaction (NHR) accounts for more than 77% of all immune-mediated immediate hypersensitivity reactions and has become a serious threat to public health. Here, proteomics was used to study the NHR mechanism of two typical substances, the compound 4880 and ovalbumin. Twelve different proteins were suggested as potential biomarkers for examining the NHR mechanism, and our results revealed that the mechanism mainly encompassed 2 processes, i.e., generation and effect processes. The generation process could be classified as direct stimulation, complement (classical and alternative), coagulation, kallikrein-kinin, and integrated pathways. Thus glutathione peroxidase 1, terminal complement complex (complement factor 4d and Bb), coagulation 13, kininogen-1, and IgE could be used as candidate biomarkers for the indication of the corresponding pathways respectively, the proteins were further confirmed by ELISA. And the effect process was mainly composed of histamine as well as proteins such as DCD and MYLPF, which could be used as important indices for the symptoms of NHR. Our study differs from previous studies in that C4880 was found to not only be involved in the direct stimulation pathway, but also in the activated complement and kallikrein-kinin pathways through the coagulation pathway. We also report for the first time that ovalbumin-induced NHR could be a combination of the coagulation, classical complement, and integrated pathways.

Effect of ginseng polysaccharides on NK cell cytotoxicity in immunosuppressed mice.

The aim of the present study was to investigate the effects of Ginseng polysaccharides (GPS) on natural killer (NK) cell cytotoxicity in immunosuppressed mice. Cyclophosphamide (Cy) was used to construct an immunosuppressed mouse model. The mice in each group were submitted to gavages with 200 or 400 mg/kg GPS every day for 10 days. Magnetic-activated cell sorting was used to isolate spleen NK cells, and the NK cell cytotoxicity, blood distribution, expression levels of perforin and granzyme, and the mRNA expression levels of interferon (IFN)-γ were detected. Compared with the normal control group, the cytotoxicity and proportion of NK cells in the blood, and the expression levels of perforin, granzyme and IFN-γ mRNA in the Cy model group were significantly reduced (P<0.05). In addition, compared with the Cy model group, the cytotoxicity and proportion of NK cells in the whole blood, and the expression levels of perforin and granzyme in the NK cells in the Cy + low-dose GPS and Cy + high-dose GPS groups were significantly increased (P<0.05). However, the mRNA expression levels of IFN-γ in the NK cells did not significantly change (P>0.05). Compared with the normal control group, the cytotoxicity and proportion of NK cells in the whole blood, and the expression levels of perforin in the Cy + low-dose GPS and the Cy + high-dose GPS groups were significantly lower (P<0.05). However, the expression levels of granzyme in the NK cells was not significantly different, as compared with the normal control group (P>0.05). These results suggested that GPS promotes NK cell cytotoxicity in immunosuppressed mice by increasing the number of NK cells in the whole blood and upregulating the expression of perforin and granzyme. Thus, the present study investigated the molecular mechanism underlying NK cell activation by GPS, the research showed that GPS have a wide application prospects in the treatment of cancer and immunodeficiency diseases.

[Effect of Electroacupuncture Stimulation of Different Tissues at "Huantiao" (GB 30) Acupoint on Expression of Phosphorylated JNK and c-jun in Spinal Cord of Rats with Sciatic Nerve Injury].

OBJECTIVE: To observe the effects of electroacupuncture (EA) stimulation of different tissues (nerve stem, muscular layer) at "Huantiao" (GB 30) acupoint on expression of hosphorylated c-jun N-terminal kinase (p-JNK) and c-jun (p-c-jun) proteins in the lumbar spinal cord in rats with sciatic nerve injury, so as to explore its mechanism underlying improvement of peripheral neuropathic damage. METHODS: Forty-eight SD rats were randomly divided into normal, model (the left sciatic nerve severed), GB 30 deep needling (the acupuncture needle tip was inserted to the sciatic nerve trunk to elicit an instantaneous jerk of the hind limb) and GB 30 shallow needling (the needle tip was inserted to the muscle layer to evoke a local muscular contraction) groups (n = 12 rats in each group). EA stimuli were delivered at 2 Hz/100 Hz, 1 mA, 20 min in duration per treatment for 10 consecutive days. Histopathological changes were observed by Hematoxylin-eosin (HE) staining and immunohistochemical assay was carried out to examine the pathological change of spinal segments (L4-L5) and the expression of p-JNK and p-c-jun proteins, respectively. RESULTS: For rats with the sciatic nerve severed, the spinal neurons became swelling, degeneration or even apoptosis. Acupuncture intervention reduced the number of apoptosic neurons and improved the pathological change, which was relatively better in the.deep needling group than in the shallow needling group. Likewise, the elevated spinal p-JNK and p-c-jun expression levels of the model group were significantly reduced by EA intervention (deep needling vs shallow needling, P < 0.01. CONCLUSION: Acupuncture can improve the spinal pathological changes in rats with sciatic nerve injury, which is probably achieved by decreasing the p-JNK and p-c-jun expression and inhibiting the JNK signaling pathway, and thereby, reducing the apoptosis of the spinal neurons. Deep needling results in greater benefits than shallow needling.

Effects of acupuncturing Pishu combined with Ginsenoside Rg3 on the immune function of rats with chronic fatigue.

OBJECTIVE: This study was designed to investigate the effects of acupuncturing Pishu combined with Ginsenoside Rg3 on the immune function of rats with chronic fatigue. METHODS: Forty male SD rats were equally randomized into control group, chronic fatigue system group (CFS), Ginsenoside Rg3 (Rg3) group, acupuncture group and acupuncture combined with Ginsenoside Rg3 (A+Rg3) group. Rats with chronic fatigue were established by bounding and forced swimming in cold water once daily for 21 days except control group, then the rats in the acupuncture and A+Rg3 group were treated by manual acupuncture stimulation of bilateral "Pishu" once daily for 7 days. Ginsenoside Rg3 was administered by intravenous to the rats of the A+Rg3 and Rg3 group for 7 days in dosages of 2 mg/kg body weight, and two markers of physical fatigue were evaluated: body weight and blood lactic acid (LA). The percentages of CD3(+) lymphocytes, CD4(+) lymphocytes, and CD8(+) lymphocytes in the spleens of the rats were evaluated using flow cytometric analysis. Serum IFN-gamma (IFN-γ) and IL-4 contents were detected by ELISA. RESULTS: Increased body weight and reduced blood LA concentrations were found in the rat of Rg3 group and A+Rg3 group than that in CFS group. The rat of Rg3 group and A+Rg3 group also showed a significant increase in the percentage of CD4(+) lymphocytes and a significant decrease in the percentage of CD8(+) lymphocytes and correct CD4(+)/CD8(+) ratio. Compared with the CFS group, the level of IFN-γ in the Rg3, acupuncture and A+Rg3 groups was reduced and IL-4 was increased. CONCLUSIONS: Acupuncture and Rg3 can improve the immune system activity of CFS rats and acupuncturing Pishu combined with Rg3 was significantly superior compared with Rg3 and acupuncture, respectively.