A prospective review of the health-promoting potential of Jing Si Herbal Tea.

Traditional Chinese medicine (TCM) has gained considerable attention over the past few years for its multicomponent, multitarget, and multi-pathway approach to treating different diseases. Studies have shown that TCMs as adjuvant therapy along with conventional treatment may benefit in safely treating various disorders. However, investigations on finding effective herbal combinations are ongoing. A novel TCM formula, "Jing Si Herbal Tea (JSHT)," has been reported recently for their health-promoting effects in improving overall body and mental health. JSHT is a combination of eight herbs recognized in Chinese herbal pharmacopoeia for their anti-viral, anti-aging, and anti-cancer properties as well as protective effects against cardiovascular, metabolic, neural, digestive, and genitourinary diseases. Thus, to better understand the beneficial effects of the ingredients of JSHT on health, this review intends to summarize the preclinical and clinical studies of the ingredients of JSHT on human health and diseases, and possible therapeutic effects with the related mode of actions and future prospects for their application in complementary therapies.

RopB-regulated SpeB cysteine protease degrades extracellular vesicles-associated streptolysin O and bacterial proteins from group A Streptococcus.

Extracellular vesicles (EVs) can be released from gram-positive bacteria and would participate in the delivery of bacterial toxins. Streptococcus pyogenes (group A Streptococcus, GAS) is one of the most common pathogens of monomicrobial necrotizing fasciitis. Spontaneous inactivating mutation in the CovR/CovS two-component regulatory system is related to the increase of EVs production via an unknown mechanism. This study aimed to investigate whether the CovR/CovS-regulated RopB, the transcriptional regulator of GAS exoproteins, would participate in regulating EVs production. Results showed that the size, morphology, and number of EVs released from the wild-type strain and the ropB mutant were similar, suggesting RopB is not involved in controlling EVs production. Nonetheless, RopB-regulated SpeB protease degrades streptolysin O and bacterial proteins in EVs. Although SpeB has crucial roles in modulating protein composition in EVs, the SpeB-positive EVs failed to trigger HaCaT keratinocytes pyroptosis, suggesting that EVs did not deliver SpeB into keratinocytes or the amount of SpeB in EVs was not sufficient to trigger cell pyroptosis. Finally, we identified that EV-associated enolase was resistant to SpeB degradation, and therefore could be utilized as the internal control protein for verifying SLO degradation. This study revealed that RopB would participate in modulating protein composition in EVs via SpeB-dependent protein degradation and suggested that enolase is a potential internal marker for studying GAS EVs.

Protective effects of Scoparia dulcis L. extract on high glucose-induced injury in human retinal pigment epithelial cells.

Diabetic retinopathy (DR) is a major cause of vision loss in diabetic patients. Hyperglycemia-induced oxidative stress and the accumulation of inflammatory factors result in blood-retinal barrier dysfunction and the pathogenesis of DR. Scoparia dulcis L. extract (SDE), a traditional Chinese medicine, has been recently recognized for its various pharmacological effects, including anti-diabetic, anti-hyperlipidemia, anti-inflammatory, and anti-oxidative activities. However, there is no relevant research on the protective effect of SDE in DR. In this study, we treated high glucose (50 mM) in human retinal epithelial cells (ARPE-19) with different concentrations of SDE and analyzed cell viability, apoptosis, and ROS production. Moreover, we analyzed the expression of Akt, Nrf2, catalase, and HO-1, which showed that SDE dose-dependently reduced ROS production and attenuated ARPE-19 cell apoptosis in a high-glucose environment. Briefly, we demonstrated that SDE exhibited an anti-oxidative and anti-inflammatory ability in protecting retinal cells from high-glucose (HG) treatment. Moreover, we also investigated the involvement of the Akt/Nrf2/HO-1 pathway in SDE-mediated protective effects. The results suggest SDE as a nutritional supplement that could benefit patients with DR.

Recuperative herbal formula Jing Si maintains vasculature permeability balance, regulates inflammation and assuages concomitants of "Long-Covid".

Coronavirus disease 2019 (COVID-19) is a worldwide health threat that has long-term effects on the patients and there is currently no efficient cure prescribed for the treatment and the prolonging effects. Traditional Chinese medicines (TCMs) have been reported to exert therapeutic effect against COVID-19. In this study, the therapeutic effects of Jing Si herbal tea (JSHT) against COVID-19 infection and associated long-term effects were evaluated in different in vitro and in vivo models. The anti-inflammatory effects of JSHT were studied in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and in Omicron pseudotyped virus-induced acute lung injury model. The effect of JSHT on cellular stress was determined in HK-2 proximal tubular cells and H9c2 cardiomyoblasts. The therapeutic benefits of JSHT on anhedonia and depression symptoms associated with long COVID were evaluated in mice models for unpredictable chronic mild stress (UCMS). JSHT inhibited the NF-ƙB activities, and significantly reduced LPS-induced expression of TNFα, COX-2, NLRP3 inflammasome, and HMGB1. JSHT was also found to significantly suppress the production of NO by reducing iNOS expression in LPS-stimulated RAW 264.7 cells. Further, the protective effects of JSHT on lung tissue were confirmed based on mitigation of lung injury, repression in TMRRSS2 and HMGB-1 expression and reduction of cytokine storm in the Omicron pseudotyped virus-induced acute lung injury model. JSHT treatment in UCMS models also relieved chronic stress and combated depression symptoms. The results therefore show that JSHT attenuates the cytokine storm by repressing NF-κB cascades and provides the protective functions against symptoms associated with long COVID-19 infection.

Neuroprotective Effects of Probiotic Lactobacillus reuteri GMNL-263 in the Hippocampus of Streptozotocin-Induced Diabetic Rats.

Diabetes-related brain complications have been reported in clinical patients and experimental models. The objective of the present study was to investigate the neuroprotective mechanisms of Lactobacillus reuteri GMNL-263 in streptozotocin (STZ)-induced diabetic rats. In this study, three different groups, namely control group, STZ-induced (55 mg/kg streptozotocin intraperitoneally) diabetic rats (DM), and DM rats treated with Lactobacillus reuteri GMNL-263 (1 × 109 CFU/rat/day), were utilized to study the protective effect of GMNL-263 in the hippocampus of STZ-induced diabetic rats. The results demonstrated that GMNL-263 attenuated diabetes-induced hippocampal damage by enhancing the cell survival pathways and repressing both inflammatory and apoptotic pathways. Histopathological analysis revealed that GMNL-263 prevented structural changes in the hippocampus in the DM group and decreased the level of inflammation and apoptosis in the hippocampus of DM rats. The IGF1R cell survival signaling pathway also improved after GMNL-263 treatment. These results indicate that probiotic GMNL-263 exerts beneficial effects in the brain of diabetic rats and has potential ability for clinical application.

Clinical course of patients with severe SARS-CoV-2 infection co-treatment with Jin Si Herbal Tea in Eastern Taiwan: A retrospective cohort study.

Introduction: Coronavirus disease-2019 (COVID-19) has affected more than 608 million people and has killed 6.5 million people in the world. A few studies showed traditional Chinese medicine can be beneficial for COVID-19 treatment. An herbal preparation Jin Si Herbal Tea (JS) was formulated with herbal extracts known for their potential to decrease spike protein and ACE2 interaction, 3CL, and TRPMSS2 protease activity, and thus aimed to evaluate the clinical course of JS co-treatment along with the usual treatment schedule given for severe COVID-19 patients. Methods: This retrospective cohort study included patients with severe COVID-19 admitted to Hualien Tzu Chi Hospital between June and July 2021. All the patients were co-treated with JS and the primary outcome was death. The secondary outcomes included laboratory exam, Ct value, clinical course, and hospital stays. There were 10 patients recruited in this study and divided into < 70 years and ≧ 70 years groups (n = 5 in each group). Results: Older patients (≧70 years) had a higher Charlson Comorbidity Index, VACO index, and lower hemoglobin levels than < 70 years patients. The trend of lymphocyte count, LDH, D-dimer, and Ct value of non-survivors was not consistent with previous studies. The death rate was 20% and the recovery rate to mild illness in 14 days was 40%. Conclusion: In conclusion, this is the first clinical study of JS co-treatment in severe COVID-19 patients. JS co-treatment might reduce death rate and recovery time. Further large-scale clinical trials would be expected.

Combined effect of traditional Chinese herbal-based formulations Jing Si herbal tea and Jing Si nasal drop inhibits adhesion and transmission of SARS-CoV2 in diabetic SKH-1 mice.

Multiple studies show increased severity of SARS-CoV2-infection in patients with comorbidities such as hypertension and diabetes. In this study, we have prepared two herbal-based formulations, a pleiotropic herbal drink (Jin Si Herbal Tea, JHT) and a nasal drop (Jin Si nasal drop, JND), to provide preventive care against SARS-CoV2 infection. The effect of JHT and JND was determined in SARS-CoV2-S-pseudotyped lentivirus-infected bronchial and colorectal cell lines and in SKH-1 mouse models. For preliminary studies, ACE2 receptor abundant bronchial (Calu-3) and colorectal cells (Caco-2) were used to determine the effect of JHT and JND on the host entry of various variants of SARS-CoV2-S-pseudotyped lentivirus. A series of experiments were performed to understand the infection rate in SKH-1 mice (6 weeks old, n = 9), find the effective dosage of JHT and JND, and determine the combination effect of JHT and JND on the entry and adhesion of various variant SARS-CoV2-S-pseudotyped lentiviruses, which included highly transmissible delta and gamma mutants. Furthermore, the effect of combined JHT and JND was determined on diabetes-induced SKH-1 mice against the comorbidity-associated intense viral entry and accumulation. In addition, the effect of combined JHT and JND administration on viral transmission from infected SKH-1 mice to uninfected cage mate mice was determined. The results showed that both JHT and JND were effective in alleviating the viral entry and accumulation in the thorax and the abdominal area. While JHT showed a dose-dependent decrease in the viral load, JND showed early inhibition of viral entry from day 1 of the infection. Combined administration of 48.66 mg of JHT and 20 µL of JND showed rapid reduction in the viral entry and reduced the viral load (97-99%) in the infected mice within 3 days of treatment. Moreover, 16.22 mg of JHT and 20 µL JND reduced the viral infection in STZ-induced diabetic SKH-1 mice. Interestingly, combined JHT and JND also inhibited viral transmission among cage mates. The results, therefore, showed that combined administration of JHT and JND is a novel and an efficient strategy to potentially prevent SARS-CoV2 infection.

Ameliorative Potential of Hot Compress on Sciatic Nerve Pain in Chronic Constriction Injury-Induced Rat Model.

Hot compress modalities are used to ameliorate pain despite prevalent confusion about which modality should be used and when. Most recommendations for hot compresses are based on empirical experience, with limited evidence to support its efficacy. To obtain insight into the nerve transmission mechanism of hot compresses and to identify the nerve injury marker proteins specifically associated with sciatic nerve pain, we established a rat model of chronic constriction injury (CCI) and performed mechanical allodynia, electrophysiology, and histopathological analysis. All CCI rats exhibited geometric representation of the affected hind paw, which indicated a hyper-impact on both mechanical gait and asymmetry of gait on day 28. The CCI model after 28 days of surgery significantly reduced compound muscle action potential (CMAP) amplitude, but also significantly reduced latency. Administration of hot compress for 3 weeks (heated at 40-42°C, cycle of 40 min, and rest for 20 min, three cycles each time, three times per week) significantly increased the paw withdrawal thresholds in response to stimulation by Von Frey fibers and reversed the CCI-induced reduced sciatic functional index (SFI) scores. Hot compress treatment in the CCI model improved CMAP amplitude and latency. The S100 protein expression level in the CCI+Hot compression group was 1.5-fold higher than in the CCI group; it dramatically reduced inflammation, such as tumor necrosis factor alpha and CD68 expression in nerve injury sites. Synaptophysin (Syn) expression in the CCI+Hot compression group was less than threefold in the CCI group at both nerve injury sites and brain (somatosensory cortex and hippocampus). This finding indicates that local nerve damage and inflammation cause significant alterations in the sensorimotor strip, and hot compress treatment could significantly ameliorate sciatic nerve pain by attenuating Syn and inflammatory factors from local pathological nerves to the brain. This study determines the potential efficacy and safety of hot compress, and may have important implications for its widespread use in sciatic nerve pain treatment.

Artemisia Leaf Extract protects against neuron toxicity by TRPML1 activation and promoting autophagy/mitophagy clearance in both in vitro and in vivo models of MPP+/MPTP-induced Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder involving the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Cellular clearance mechanisms, including the autophagy-lysosome pathway, are commonly affected in the pathogenesis of PD. The lysosomal Ca2+ channel mucolipin TRP channel 1 (TRPML1) is one of the most important proteins involved in the regulation of autophagy. Artemisia argyi Lev. et Vant., is a traditional Chinese herb, that has diverse therapeutic properties and is used to treat patients with skin diseases and oral ulcers. However, the neuroprotective effects of A. argyi are not explored yet. HYPOTHESIS: This study aims is to investigate the neuroprotective effects of A. argyi in promoting the TRPML1-mediated autophagy/mitophagy-enhancing effect METHODS: In this study, we used 1-methyl-4-phenyl-pyridinium (MPP+)-induced PD model established in an SH-SY5Y human neuroblastoma cell line as well as in a 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP)-induced PD model in C57BL/6 J mice. MTT assay was conducted to measure the cell viability and further MitoSoX and DCFDA assay were used to measure the ROS. Western blot analysis was used to access levels of TRPML1, p-DRP1 (ser616), p-AKT, PI3K, and ß-catenin, Additionally, IF and IHC analysis to investigate the expression of TRPML1, LC3B, ß-catenin, TH+, α-synuclein. Mitotracker stain was used to check mitophagy levels and a lysosomal intracellular activity kit was used to measure the lysosomal dysfunction. Behavioral studies were conducted by rotarod and grip strength experiments to check motor functions. RESULTS: In our in vitro study, A. argyi rescued the MPP+-induced loss of cell viability and reduced the accumulation of mitochondrial and total reactive oxygen species (ROS). Subsequently, it increased the expression of TRPML1 protein, thereby inducing autophagy, which facilitated the clearance of toxic accumulation of α-synuclein. Furthermore, A. argyi played a neuroprotective role by activating the PI3K/AKT/ß-catenin cell survival pathway. MPP+-mediated mitochondrial damage was overcome by upregulation of mitophagy and downregulation of the mitochondrial fission regulator p-DRP1 (ser616) in SH-SY5Y cells. In the in vivo study, A. argyi ameliorated impaired motor function and rescued TH+ neurons in the SNpc region. Similar to the results of the in vitro study, TRPML1, LC3B, and ß-catenin expression was enhanced in the SNpc region in the A. argyi-treated mice brain. CONCLUSION: Thus, our results first demonstrate that A. argyi can exert neuroprotective effects by stimulating TRPML1 and rescuing neuronal cells by boosting autophagy/mitophagy and upregulating a survival pathway, suggesting that A. argyi can further be exploited to slow the progression of PD.

Novel anti-aging herbal formulation Jing Si displays pleiotropic effects against aging associated disorders.

Common characteristics of aging include reduced somatic stem cell number, susceptibility to cardiac injuries, metabolic imbalances and increased risk for oncogenesis. In this study, Pleiotropic anti-aging effects of a decoction Jing Si herbal drink (JS) containing eight Traditional Chinese Medicine based herbs, with known effects against aging related disorders was evaluated. Adipose derived mesenchymal stem cells (ADMSCs) from 16 week old adult and 24 month old aging WKY rats were evaluated for the age-related changes in stem cell homeostasis. Effects of JS on self-renewal, klotho and Telomerase Reverse Transcriptase expression DNA damage response were determined by immunofluorescence staining. The effects were confirmed in senescence induced human ADMSCs and in addition, the potential of JS to maintain telomere length was evaluated by qPCR analysis in ADMSCs challenged for long term with doxorubicin. Further, the effects of JS on doxorubicin-induced hypertrophic effect and DNA damage in H9c2 cardiac cells; MPP+-induced damages in SH-SY5Y neuron cells were investigated. In addition, effects of JS in maintaining metabolic regulation, in terms of blood glucose regulation in type-II diabetes mice model, and their potential to suppress malignancy in different cancer cells were ascertained. The results show that JS maintains stem cell homeostasis and provides cytoprotection. In addition JS regulates blood glucose metabolism, enhances autophagic clearances in neurons and suppresses cancer growth and migration. The results show that JS acts on multiple targets and provides a cumulative protective effect against various age-associated disorders and therefore it is a candidate pleiotropic agent for healthy aging.

Exercise renovates H2S and Nrf2-related antioxidant pathways to suppress apoptosis in the natural ageing process of male rat cortex.

Ageing is a complex biological process that increases the probability of disease and death, which affects the organs of all species. The accumulation of oxidative damage in the brain contributes to a progressive loss of cognitive functions or even declined the energy metabolism. In this study, we tested the effects of exercise training on the apoptosis, survival, and antioxidant signaling pathways in the cerebral cortex of three age groups of male rats; 3, 12, and 18 months. We observed that H2S and the expression of Nrf2-related antioxidant pathways declined with age and increased after exercise training. IGF1R survival pathway was less increased in middle-aged rats; however, significantly increased after exercise training. The expression of mitochondrial-dependent apoptotic pathway components, such as Bak, cytochrome C, and caspase 3 in the ageing control group, were much higher than those of the exercise training groups. This study demonstrated that exercise training could reduce the apoptosis and oxidative stress that accrues throughout ageing, which causes brain damage.

AXL is crucial for E1A-enhanced therapeutic efficiency of EGFR tyrosine kinase inhibitors through NFI in breast cancer.

AXL which is a chemosensitizer protein for breast cancer cells in response to epidermal growth factor receptor-tyrosine kinase inhibitor and suppresses tumor growth. The clinical information show nuclear factor I (NFI)-C and NFI-X expression correlate with AXL expression in breast cancer patients. Following, we establish serial deletions of AXL promoter to identify regions required for Adenovirus-5 early region 1A (E1A)-mediated AXL suppression. All of the NFI family members were extensively studied for their expression and functions in regulating AXL. Moreover, E1A post-transcriptionally downregulates AXL expression through NFI. NFI-C and NFI-X, not NFI-A and NFI-B, resulting in cell death in response to EGFR-TKI. Our finding suggests that NFI-C and NFI-X are crucial regulators for AXL and significantly correlated with poor survival of breast cancer patients.

Intrathecal Injection of Dual Zipper Kinase shRNA Alleviating the Neuropathic Pain in a Chronic Constrictive Nerve Injury Model.

Dual leucine zipper kinase (DLK) is a member of mitogen-activated protein kinase kinase kinase (MAP3K) family mainly involved in neuronal degeneration. However, the role of DLK signaling in the neuropathic pain has not yet been fully determined. Chronic constrictive injury (CCI) was conducted by four 3-0 chromic gut ligatures loosely ligated around the sciatic nerve. Escalated DLK expression over the dorsal root ganglion was observed from one to four rings of CCI. Remarkable expression of DLK was observed in primary dorsal root ganglion cells culture subjected to electrical stimulation and attenuated by DLK short hairpin RNA (shRNA) treatment. Intrathecal injection of DLK shRNA attenuates the expression of DLK over the dorsal root ganglion and hippocampus neurons and increased the threshold of mechanical allodynia and decreased thermal hyperalgesia. In CatWalk gait analysis, significant decreases of print area, maximum contact maximum intensity, stand phase, single stance, and regular index by CCI were alleviated by the DLK shRNA administration. In conclusion, the expression of DLK was up-regulated in chronic constrictive injury and attenuated by the administration of DLK shRNA, which paralleled the improvement of neurobehavior of neuropathic pain. The modulation of DLK expression is a potential clinic treatment option for neuropathic pain.

Late administration of high-frequency electrical stimulation increases nerve regeneration without aggravating neuropathic pain in a nerve crush injury.

BACKGROUND: High-frequency transcutaneous neuromuscular electrical nerve stimulation (TENS) is currently used for the administration of electrical current in denervated muscle to alleviate muscle atrophy and enhance motor function; however, the time window (i.e. either immediate or delayed) for achieving benefit is still undetermined. In this study, we conducted an intervention of sciatic nerve crush injury using high-frequency TENS at different time points to assess the effect of motor and sensory functional recovery. RESULTS: Animals with left sciatic nerve crush injury received TENS treatment starting immediately after injury or 1 week later at a high frequency(100 Hz) or at a low frequency (2 Hz) as a control. In SFI gait analysis, either immediate or late admission of high-frequency electrical stimulation exerted significant improvement compared to either immediate or late administration of low-frequency electrical stimulation. In an assessment of allodynia, immediate high frequency electrical stimulation caused a significantly decreased pain threshold compared to late high-frequency or low-frequency stimulation at immediate or late time points. Immunohistochemistry staining and western blot analysis of S-100 and NF-200 demonstrated that both immediate and late high frequency electrical stimulation showed a similar effect; however the effect was superior to that achieved with low frequency stimulation. Immediate high frequency electrical stimulation resulted in significant expression of TNF-α and synaptophysin in the dorsal root ganglion, somatosensory cortex, and hippocampus compared to late electrical stimulation, and this trend paralleled the observed effect on somatosensory evoked potential. The CatWalk gait analysis also showed that immediate electrical stimulation led to a significantly high regularity index. In primary dorsal root ganglion cells culture, high-frequency electrical stimulation also exerted a significant increase in expression of TNF-α, synaptophysin, and NGF in accordance with the in vivo results. CONCLUSION: Immediate or late transcutaneous high-frequency electrical stimulation exhibited the potential to stimulate the motor nerve regeneration. However, immediate electrical stimulation had a predilection to develop neuropathic pain. A delay in TENS initiation appears to be a reasonable approach for nerve repair and provides the appropriate time profile for its clinical application.

Prevention of Axonal Degeneration by Perineurium Injection of Mitochondria in a Sciatic Nerve Crush Injury Model.

Background: Axon degeneration leads to cytoskeletal disassembly, metabolism imbalance, and mitochondrial dysfunction during neurodegeneration or nerve injury. Objective: In this study, we assess the possibility of mitigating axon degeneration by local injection of mitochondria in a crushed sciatic nerve. Methods: Sciatic nerve explants cocultured with mitochondria were assessed for the optimal dosage in local injection and nerve regeneration potential. The left sciatic nerve was crushed in Sprague-Dawley rats and then local injection of mitochondria into the distal end of the injured nerve was conducted for further assessment. Results: Mitochondrial coculture attenuated cytoskeletal loss and oxidative stress in isolated nerve explants. In Vivo analyses also showed that mitochondrial transplantation improved animal neurobehaviors, electrophysiology of nerve conduction, and muscle activities. Mitochondria injection significantly attenuated the oxidative stress and increased the expression of neurotrophic factors both in injured nerves and denervated muscles, as well as restored muscular integrity, and increased the pool of muscular progenitor cells and total muscle weight. Conclusion: Mitochondria injection can protect injured nerves from axonal degeneration both in Vitro and in Vivo. This improvement was accompanied with the expression of neurotrophic factors as well as the reduction of oxidative stress, which may account for the functional recovery of both injured nerves and denervated muscles.

Feasibility of Human Amniotic Fluid Derived Stem Cells in Alleviation of Neuropathic Pain in Chronic Constrictive Injury Nerve Model.

PURPOSE: The neurobehavior of neuropathic pain by chronic constriction injury (CCI) of sciatic nerve is very similar to that in humans, and it is accompanied by a profound local inflammation response. In this study, we assess the potentiality of human amniotic fluid derived mesenchymal stem cells (hAFMSCs) for alleviating the neuropathic pain in a chronic constriction nerve injury model. METHODS AND METHODS: This neuropathic pain animal model was conducted by four 3-0 chromic gut ligatures loosely ligated around the left sciatic nerve in Sprague-Dawley rats. The intravenous administration of hAFMSCs with 5x105 cells was conducted for three consecutive days. RESULTS: The expression IL-1ß, TNF-α and synaptophysin in dorsal root ganglion cell culture was remarkably attenuated when co-cultured with hAFMSCs. The significant decrease of PGP 9.5 in the skin after CCI was restored by administration of hAFMSCs. Remarkably increased expression of CD 68 and TNF-α and decreased S-100 and neurofilament expression in injured nerve were rescued by hAFMSCs administration. Increases in synaptophysin and TNF-α over the dorsal root ganglion were attenuated by hAFMSCs. Significant expression of TNF-α and OX-42 over the dorsal spinal cord was substantially attenuated by hAFMSCs. The increased amplitude of sensory evoked potential as well as expression of synaptophysin and TNF-α expression was alleviated by hAFMSCs. Human AFMSCs significantly improved the threshold of mechanical allodynia and thermal hyperalgesia as well as various parameters of CatWalk XT gait analysis. CONCLUSION: Human AFMSCs administration could alleviate the neuropathic pain demonstrated in histomorphological alteration and neurobehavior possibly through the modulation of the inflammatory response.

Improved neurological outcome by intramuscular injection of human amniotic fluid derived stem cells in a muscle denervation model.

PURPOSE: The skeletal muscle develops various degrees of atrophy and metabolic dysfunction following nerve injury. Neurotrophic factors are essential for muscle regeneration. Human amniotic fluid derived stem cells (AFS) have the potential to secrete various neurotrophic factors necessary for nerve regeneration. In the present study, we assess the outcome of neurological function by intramuscular injection of AFS in a muscle denervation and nerve anastomosis model. MATERIALS AND METHODS: Seventy two Sprague-Dawley rats weighing 200-250 gm were enrolled in this study. Muscle denervation model was conducted by transverse resection of a sciatic nerve with the proximal end sutured into the gluteal muscle. The nerve anastomosis model was performed by transverse resection of the sciatic nerve followed by four stitches reconnection. These animals were allocated to three groups: control, electrical muscle stimulation, and AFS groups. RESULTS: NT-3 (Neurotrophin 3), BDNF (Brain derived neurotrophic factor), CNTF (Ciliary neurotrophic factor), and GDNF (Glia cell line derived neurotrophic factor) were highly expressed in AFS cells and supernatant of culture medium. Intra-muscular injection of AFS exerted significant expression of several neurotrophic factors over the distal end of nerve and denervated muscle. AFS caused high expression of Bcl-2 in denervated muscle with a reciprocal decrease of Bad and Bax. AFS preserved the muscle morphology with high expression of desmin and acetylcholine receptors. Up to two months, AFS produced significant improvement in electrophysiological study and neurological functions such as SFI (sciatic nerve function index) and Catwalk gait analysis. There was also significant preservation of the number of anterior horn cells and increased nerve myelination as well as muscle morphology. CONCLUSION: Intramuscular injection of AFS can protect muscle apoptosis and likely does so through the secretion of various neurotrophic factors. This protection furthermore improves the nerve regeneration in a long term nerve anastomosis model.

Comprehensive analysis of neurobehavior associated with histomorphological alterations in a chronic constrictive nerve injury model through use of the CatWalk XT system.

OBJECT: Neuropathic pain is debilitating, and when chronic, it significantly affects the patient physically, psychologically, and socially. The neurobehavior of animals used as a model for chronic constriction injury seems analogous to the neurobehavior of humans with neuropathic pain. However, no data depicting the severity of histomorphological alterations of the nervous system associated with graded changes in neurobehavior are available. To determine the severity of histomorphological alteration related to neurobehavior, the authors created a model of chronic constrictive injury of varying intensity in rats and used the CatWalk XT system to evaluate neurobehavior. METHODS: A total of 60 Sprague-Dawley rats, weighing 250-300 g each, were randomly assigned to 1 of 5 groups that would receive sham surgery or 1, 2, 3, or 4 ligatures of 3-0 chromic gut loosely ligated around the left sciatic nerve. Neurobehavior was assessed by CatWalk XT, thermal hyperalgesia, and mechanic allodynia before injury and periodically after injury. The nerve tissue from skin to dorsal spinal cord was obtained for histomorphological analysis 1 week after injury, and brain evoked potentials were analyzed 4 weeks after injury. RESULTS: Significant differences in expression of nerve growth factor existed in skin, and the differences were associated with the intensity of nerve injury. After injury, expression of cluster of differentiation 68 and tumor necrosis factor-α was increased, and expression of S100 protein in the middle of the injured nerve was decreased. Increased expression of synaptophysin in the dorsal root ganglion and dorsal spinal cord correlated with the intensity of injury. The amplitude of sensory evoked potential increased with greater severity of nerve damage. Mechanical allodynia and thermal hyperalgesia did not differ significantly among treatment groups at various time points. CatWalk XT gait analysis indicated significant differences for print areas, maximum contact maximum intensity, stand phase, swing phase, single stance, and regular index, with sham and/or intragroup comparisons. CONCLUSIONS: Histomorphological and electrophysiological alterations were associated with severity of nerve damage. Subtle neurobehavioral differences were detected by the CatWalk XT system but not by mechanical allodynia or thermal hyperalgesia. Thus, the CatWalk XT system should be a useful tool for monitoring changes in neuropathic pain, especially subtle alterations.

NADPH oxidase subunit 4-mediated reactive oxygen species contribute to cycling hypoxia-promoted tumor progression in glioblastoma multiforme.

BACKGROUND: Cycling and chronic tumor hypoxia are involved in tumor development and growth. However, the impact of cycling hypoxia and its molecular mechanism on glioblastoma multiforme (GBM) progression remain unclear. METHODOLOGY: Glioblastoma cell lines, GBM8401 and U87, and their xenografts were exposed to cycling hypoxic stress in vitro and in vivo. Reactive oxygen species (ROS) production in glioblastoma cells and xenografts was assayed by in vitro ROS analysis and in vivo molecular imaging studies. NADPH oxidase subunit 4 (Nox4) RNAi-knockdown technology was utilized to study the role of Nox4 in cycling hypoxia-mediated ROS production and tumor progression. Furthermore, glioblastoma cells were stably transfected with a retroviral vector bearing a dual reporter gene cassette that allowed for dynamic monitoring of HIF-1 signal transduction and tumor cell growth in vitro and in vivo, using optical and nuclear imaging. Tempol, an antioxidant compound, was used to investigate the impact of ROS on cycling hypoxia-mediated HIF-1 activation and tumor progression. PRINCIPAL FINDINGS: Glioblastoma cells and xenografts were compared under cycling hypoxic and normoxic conditions; upregulation of NOX4 expression and ROS levels were observed under cycling hypoxia in glioblastoma cells and xenografts, concomitant with increased tumor cell growth in vitro and in vivo. However, knockdown of Nox4 inhibited these effects. Moreover, in vivo molecular imaging studies demonstrated that Tempol is a good antioxidant compound for inhibiting cycling hypoxia-mediated ROS production, HIF-1 activation, and tumor growth. Immunofluorescence imaging and flow cytometric analysis for NOX4, HIF-1 activation, and Hoechst 3342 in glioblastoma also revealed high localized NOX4 expression predominantly in potentially cycling hypoxic areas with HIF-1 activation and blood perfusion within the endogenous solid tumor microenvironment. CONCLUSIONS: Cycling hypoxia-induced ROS via Nox4 is a critical aspect of cancer biology to consider for therapeutic targeting of cycling hypoxia-promoted HIF-1 activation and tumor progression in GBM.