Zhen-Wu-Tang ameliorates lupus nephritis by diminishing renal tissue-resident memory CD8+ T cells via suppressing IL-15/STAT3 pathway.

Zhen-Wu-Tang (ZWT), a conventional herbal mixture, has been recommended for treating lupus nephritis (LN) in clinic. However, its mechanisms of action remain unknown. Here we aimed to define the immunological mechanisms underlying the effects of ZWT on LN and to determine whether it affects renal tissue-resident memory T (TRM) cells. Murine LN was induced by a single injection of pristane, while in vitro TRM cells differentiated with IL-15/TGF-ß. We found that ZWT or mycophenolate mofetil treatment significantly ameliorated kidney injury in LN mice by decreasing 24-h urine protein, Scr and anti-dsDNA Ab. ZWT also improved renal pathology and decreased IgG and C3 depositions. In addition, ZWT down-regulated renal Desmin expression. Moreover, it lowered the numbers of CD8+ TRM cells in kidney of mice with LN while decreasing their expression of TNF-α and IFN-γ. Consistent with in vivo results, ZWT-containing serum inhibited TRM cell differentiation induced by IL-15/TGF-ß in vitro. Mechanistically, it suppressed phosphorylation of STAT3 and CD122 （IL2/IL-15Rß）expression in CD8+ TRM cells. Importantly, ZWT reduced the number of total F4/80+CD11b+ and CD86+, but not CD206+, macrophages in the kidney of LN mice. Interestingly, ZWT suppressed IL-15 protein expression in macrophages in vivo and in vitro. Thus, we have provided the first evidence that ZWT decoction can be used to improve the outcome of LN by reducing CD8+ TRM cells via inhibition of IL-15/IL-15R /STAT3 signaling.

Association Between Serum Neurofilament Light Chain and Neurochemistry Deficits in Patients with Spinocerebellar Ataxia Type 3.

Extensive evidence supports the claim that the serum neurofilament light chain (sNfL) can be used as a biomarker to monitor disease severity in patients with spinocerebellar ataxia type 3 (SCA3). However, little is known about the associations between sNfL levels and neurochemical alterations in SCA3 patients. In this study, we performed a cross-sectional study to analyze the association between sNfL and brain metabolic changes in SCA3 patients. The severity of ataxia was assessed by using the Scale for the Assessment and Rating of Ataxia (SARA) and the International Cooperative Ataxia Rating Scale (ICARS). The sNfL levels and brain metabolic changes, represented by N-acetyl aspartate (NAA)/creatine (Cr) and choline complex (Cho)/Cr ratios, were measured by a single-molecule array and proton magnetic resonance spectroscopy, respectively. In this cohort, we observed consistently elevated sNfL levels and reduced brain metabolites in the cerebellar hemispheres, dentate nucleus, and cerebellar vermis. However, this correlation was further validated in the cerebellar cortex after analysis using pairwise comparisons and a Bonferroni correction. Taken together, our results further confirmed that sNfL levels were increased in SCA3 patients and were negatively correlated with metabolic changes in the cerebellar cortex. Our data also support the idea that sNfL levels are a promising potential complementary biomarker for patients with SCA3.

Methotrexate and electrostimulation cooperate to alleviate the relapse of psoriasiform skin inflammation by suppressing memory T cells.

Methotrexate (MTX) is an immunosuppressant used to treat autoimmune diseases, including psoriasis. However, like other immunosuppressants, MTX alone does not prevent their recurrence. Electrostimulation (ES) has been utilized to treat some inflammatory disorders without any major side-effect. But it remains unknown if ES alone, or together with MTX, ameliorates autoimmune disease relapse: a sticky medical problem. In particular, the mechanisms underlying ES action remain unclear. The objective of this study was to determine an impact of ES and/or MTX on psoriasis relapse and their potential cooperation. We found that regional ES, but not MTX, ameliorated psoriasiform skin inflammation recurrence. Interestingly, treatment with both MTX and ES further prevented psoriasis recurrence compared to ES alone. Moreover, ES downregulated potassium channel Kv1.3 on T-cells and reduced CD4+/CD8+ effector memory (TEM) and CD8+ skin-resident memory T (TRM) cells, while ES plus MTX further decreased CD8+ TEM/TRM cells compared to ES alone. However, ES failed to further attenuate psoriasis recurrence or suppress T cell memory in Kv1.3-deficient mice, whereas lack of Kv1.3 itself ameliorated psoriasis relapse by shrinking T cell memory pool. Importantly, ES moderately inhibited T-cell proliferation in vitro. ES also reduced human CD8+ TRM cells and attenuated human skin lesions in humanized mice grafted with lesional skin from patients with recurrent psoriasis, with an enhanced efficacy in mice treated with both ES and MTX. Thus, ES and MTX cooperated to prevent psoriasis relapse by reducing T-cell memory via targeting potassium channel Kv1.3. Our studies may be implicated for treating human psoriasis.

Punicalagin alleviates the hyperproliferation of keratinocytes in psoriasis through inhibiting SKP2 expression.

Psoriasis is a chronic inflammatory skin disorder characterized by abnormal keratinocytes proliferation and multiple immune cells infiltration in the dermis and epidermis. Although most psoriasis-related researches have been concentrated on the interleukin-23 (IL-23)/interleukin-17 (IL-17) axis, new data suggest that keratinocytes also play a pivotal role in psoriasis. Previously, we found that punicalagin (PUN), a bioactive ellagitannin extracted from Pericarpium Granati (the pericarpium of Punica granatum L.), exerts a therapeutic effect on psoriasis. However, the underlying mechanism, especially its potential modulatory effect on keratinocytes, remains obscure. Our study aims to reveal the potential regulatory effect and its underlying cellular mechanism of PUN on the hyperproliferation of keratinocytes. We used tumor necrosis factor α (TNF-α), IL-17A and interleukin-6 (IL-6) to induce abnormal proliferation of HaCaT cells (Human Keratinocytes Cells) in vitro. Then, we evaluated the effects of PUN through MTT assay, EdU staining and cell cycle detection. Finally, we explored the underlying cellular mechanisms of PUN via RNA-sequencing, WB in vitro and in vivo. Here, we found that PUN can directly and dose-dependently decrease TNF-α, IL-17A and IL-6-induced abnormal proliferation of HaCaT cells in vitro. Mechanically, PUN suppresses the hyperproliferation of keratinocytes through repressing S-phase kinase-associated protein 2 (SKP2) expression in vitro and in vivo. Moreover, overexpression of SKP2 can partly abolish PUN-mediated inhibition of aberrantly proliferative keratinocytes. These results illustrate that PUN can reduce the severity of psoriasis through directly repressing SKP2-mediated abnormal proliferation of keratinocytes, which gives new insight into the therapeutic mechanism of PUN on psoriasis. Moreover, these findings imply that PUN might be a promising drug candidate for the treatment of psoriasis.

[Research progress in strigolactones and application prospect in medicinal plants].

Strigolactones(SLs) are a class of sesquiterpenoids derived from the carotenoid biosynthesis pathway with the core carbon skeleton consisting of tricyclic lactone(ABC tricyclic ring) and α,ß-unsaturated furan ring(D ring). SLs are widely distributed in higher plants and are symbiotic signals between plants and Arbuscular mycorrhiza(AM), which play key roles in the evolution of plant colonizing terrestrial habitats. As a new type of plant hormone, SLs possess such important biological functions as inhibiting shoot branching(tillers), regulating root architecture, promoting secondary growth, and improving plant stress resistance. Therefore, SLs have attracted wide attention. The biological functions of SLs are not only closely related to the formation of "excellent shape and quality" of Chinese medicinal materials but also have important practical significance for the production of high-quality medicinal materials. However, SLs have been currently widely studied in model plants and crops such as Oryza sativa and Arabidopsis thaliana, and few related studies have been reported on SLs in medicinal plants, which need to be strengthened. This review focused on the latest research progress in the isolation and identification, biological and artificial synthesis pathways, biosynthesis sites and transport modes, signal transduction pathways and mechanisms, and biological functions of SLs, and prospected the research on the regulation mechanism of SLs in the growth and development of medicinal plants and their related application on targeted regulation of Chinese herbal medicine production, which is expected to provide some references for the in-depth research on SLs in the field of Chinese medicinal resources.

[Cloning and gene function of dicarboxylate-tricarboxylate carrier protein in Gastrodia elata].

The gene GeDTC encoding the dicarboxylate-tricarboxylate carrier protein in Gastrodia elata was cloned by specific primers which were designed based on the transcriptome data of G. elata. Bioinformatics analysis on GeDTC gene was carried out by using ExPASY, ClustalW, MEGA, etc. Positive transgenic plants and potato minituber were obtained by virtue of the potato genetic transformation system. Agronomic characters, such as size, weight, organic acid content, and starch content, of potato minituber were tested and analyzed and GeDTC gene function was preliminarily investigated. The results showed that the open reading frame of GeDTC gene was 981 bp in length and 326 amino acid residues were encoded, with a relative molecular weight of 35.01 kDa. It was predicted that the theoretical isoelectric point of GeDTC protein was 9.83, the instability coefficient was 27.88, and the average index of hydrophilicity was 0.104, which was indicative of a stable hydrophilic protein. GeDTC protein had a transmembrane structure and no signal peptide and was located in the inner membrane of mitochondria. The phylogenetic tree showed that GeDTC was highly homologous with DTC proteins of other plant species, among which GeDTC had the highest homology with DcDTC(XP＿020675804.1) in Dendrobium candidum, reaching 85.89%. GeDTC overexpression vector pCambia1300-35Spro-GeDTC was constructed by double digests, and transgenic potato plants were obtained by Agrobacterium-mediated gene transformation. Compared with the wild-type plants, transgenic potato minituber harvested by transplanting had smaller size, lighter weight, lower organic acid content, and no significant difference in starch content. It is preliminarily induced that GeDTC is the efflux channel of tricarboxylate and related to the tuber development, which lays a foundation for further elucidating the molecular mechanism of G. elata tuber development.

[Application of strigolactone analogs in storage of Gastrodia elata].

This study explored the preservation effect of strigolactone analogs on Gastrodia elata tubers and screened out the suitable preservation measures of G. elata to provide a safer and more effective method for its storage and preservation. Fresh G. elata tubers were treated with 7FGR24, 2,4-D isooctyl ester, and maleic hydrazide, respectively. The growth of flower buds, the activities of CAT, and MDA, and the content of gastrodin and p-hydroxybenzyl alcohol were measured to compare the effects of different compounds on the storage and preservation of G. elata. The effects of different storage temperatures on the preservation of 7FGR24 were compared and analyzed. The gibberellin signal transduction receptor gene GeGID1 was cloned, and the effect of 7FGR24 on the expression level of GeGID1 was analyzed by quantitative polymerase chain reaction(qPCR). The toxicity of the G. elata preservative 7FGR24 was analyzed by intragastric administration in mice to evaluate its safety. The results showed that compared with 2,4-D isooctyl ester and maleic hydrazide, 7FGR24 treatment had a significant inhibitory effect on the growth of G. elata flower buds, and the CAT enzyme activity of G. elata was the highest, indicating that its preservation effect was stronger. Different storage temperatures had different effects on the preservation of G. elata, and the preservation effect was the strongest at 5 ℃. The open reading frame(ORF) of GeGID1 gene was 936 bp in length, and its expression level was significantly down-regulated after 7FGR24 treatment, indicating that 7FGR24 may inhibit the growth of flower buds by inhibiting the gibberellin signal of G. elata, thereby exerting a fresh-keeping effect. Feeding preservative 7FGR24 had no significant effect on the behavior and physiology of mice, indicating that it had no obvious toxicity. This study explored the application of the strigolactone analog 7FGR24 in the storage and preservation of G. elata and preliminarily established a method for the storage and preservation of G. elata, laying a foundation for the molecular mechanism of 7FGR24 in the storage and preservation of G. elata.

Regenerative plantlets with improved agronomic characteristics caused by anther culture of tetraploid potato (Solanum tuberosum L.).

Objective: As the primary means of plant-induced haploid, anther culture is of great significance in quickly obtaining pure lines and significantly shortening the potato breeding cycle. Nevertheless, the methods of anther culture of tetraploid potato were still not well established. Methods: In this study, 16 potato cultivars (lines) were used for anther culture in vitro. The corresponding relation between the different development stages of microspores and the external morphology of buds was investigated. A highly-efficient anther culture system of tetraploid potatoes was established. Results: It was shown in the results that the combined use of 0.5 mg/L 1-Naphthylacetic acid (NAA), 1.0 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D), and 1.0 mg/L Kinetin (KT) was the ideal choice of hormone pairing for anther callus. Ten of the 16 potato cultivars examined could be induced callus with their respective anthers, and the induction rate ranged from 4.44% to 22.67% using this hormone combination. According to the outcome from the orthogonal design experiments of four kinds of appendages, we found that the medium with sucrose (40 g/L), AgNO3 (30 mg/L), activated carbon (3 g/L), potato extract (200 g/L) had a promotive induction effect on the anther callus. In contrast, adding 1 mg/L Zeatin (ZT) effectively facilitated callus differentiation. Conclusion: Finally, 201 anther culture plantlets were differentiated from 10 potato cultivars. Among these, Qingshu 168 and Ningshu 15 had higher efficiency than anther culture. After identification by flow cytometry and fluorescence in situ hybridization, 10 haploid plantlets (5%), 177 tetraploids (88%), and 14 octoploids (7%) were obtained. Some premium anther-cultured plantlets were further selected by morphological and agronomic comparison. Our findings provide important guidance for potato ploidy breeding.

Dihydroartemisinin ameliorates psoriatic skin inflammation and its relapse by diminishing CD8+ T-cell memory in wild-type and humanized mice.

Conventional immunosuppressants cause side effects and do not prevent the recurrence of autoimmune diseases. Moreover, they may not inhibit autoimmunity mediated by pathogenic memory T-cells. Dihydroartemisinin (DHA) has been shown to regulate autoimmunity. However, it remains unknown whether DHA impacts psoriasis and its recurrence. The objective of this study was to determine therapeutic effects of DHA on psoriasis and its relapse as well as its underlying mechanisms. Methods: We established animal models of imiquimod (IMQ)-induced psoriasis-like wild-type mice and humanized NSG mice receiving lesional human skin from patients with psoriasis. Many immunoassays, including immunohistochemistry, flow cytometry, quantitative RT-PCR and Western blotting, were performed. Results: We found that DHA not only ameliorated acute skin lesion of psoriatic mice, but also alleviated its recurrence by diminishing CD8+ central memory T (TCM) and CD8+ resident memory T (TRM) cells. It attenuated epidermal pathology and T-cell infiltration in the skin of IMQ-induced psoriatic mice while suppressing expression of IL-15, IL-17 and other proinflammatory cytokines in the skin. Surprisingly, DHA reduced the frequency and number of CD8+, but not CD4+, subset of CD44highCD62Lhigh TCM in psoriatic mice, whereas methotrexate (MTX) lowered CD4+, but not CD8+, TCM frequency and number. Indeed, DHA, but not MTX, downregulated eomesodermin (EOMES) and BCL-6 expression in CD8+ T-cells. Furthermore, DHA, but not MTX, reduced the presence of CD8+CLA+, CD8+CD69+ or CD8+CD103+ TRM cells in mouse skin. Interestingly, treatment with DHA, but not MTX, during the first onset of psoriasis largely prevented psoriasis relapse induced by low doses of IMQ two weeks later. Administration of recombinant IL-15 or CD8+, but not CD4+, TCM cells resulted in complete recurrence of psoriasis in mice previously treated with DHA. Finally, we demonstrated that DHA alleviated psoriatic human skin lesions in humanized NSG mice grafted with lesional skin from psoriatic patients while reducing human CD8+ TCM and CD103+ TRM cells in humanized mice. Conclusion: We have provided the first evidence that DHA is advantageous over MTX in preventing psoriasis relapse by reducing memory CD8+ T-cells.

Antitumor effects of immunity-enhancing traditional Chinese medicine.

Traditional Chinese Medicine (TCM) has been traditionally used to treat patients with cancers in China. It not only alleviates the symptoms of tumor patients and improves their quality of life, but also controls the size of tumors and prolongs the survival of tumor patients. While some herbs of TCM may exert therapeutic effects by directly targeting cancer cells or reducing side effects caused by antitumor drugs, others can control tumor growth and metastasis via enhancing antitumor immunity. In particular, TCM can exert antitumor effects by upregulating immune responses even in immunosuppressive tumor microenvironment. For instance, it reduces the number of M2-type macrophages and Treg cells in the tumor tissue. Although extensive reviews on directly killing cancer cells by TCM have been conducted, a review of anticancer activity of TCM solely based on its immunity-enhancing capacity is unusual. This review will summarize research progress of antitumor TCM that regulates the immune system, including both innate immunity, such as macrophages, dendritic cells, natural killer cells and MDSCs, and adaptive immunity, including CD4+/CD8+ T lymphocytes, regulatory T cells (Tregs) and B cells. As cancer immunotherapy has recently achieved certain success, it is expected that the clinical applications of immunity-enhancing TCM or traditional medicine for treating various cancer patients will be expanded. Further studies on the mechanisms by which TCM regulates immunity will provide new insights into how TCM controls tumor growth and metastasis, and may help improve its therapeutic effects on various cancers in clinic.

Herbal Components of a Novel Formula PSORI-CM02 Interdependently Suppress Allograft Rejection and Induce CD8+CD122+PD-1+ Regulatory T Cells.

A recipient usually rejects a transplanted organ and thus needs immunosuppressive treatments to prevent rejection. Achieving long-term allograft survival without continuous global immunosuppression is highly desirable in transplantation as long-term immunosuppression causes various side effects. Therefore, it is necessary to search for medicine with potentially less side effects. Traditional Chinese medicine PSORI-CM01 (Yin Xie Ling), a formula with seven natural herbs, has been used to treat patients with psoriasis. Here, we investigated a "sharpened" formula, PSORI-CM02 consisting of only five herbs from PSORI-CM01: Curcumae rhizoma, Radix paeoniae rubra, Rhizoma smilacis glabrae, Mume fructus, and Sarcandrae herba. We examined whether or not PSORI-CM02 would suppress alloimmunity and found that PSORI-CM02 significantly inhibited murine skin allograft rejection and reduced graft-infiltration of CD3+ T cells. Interestingly, omitting any single herbal component rendered the whole formula ineffective in suppression, indicating that these herbal components exert their effects cooperatively as a whole. Moreover, PSORI-CM02 increased CD8+CD122+PD-1+ Treg frequency with CD4+FoxP3+ Tregs remaining unchanged in recipient mice, whereas CsA reduced CD4+FoxP3+ Treg frequency. PSORI-CM02 also hindered CD11c+ DC maturation posttransplantation. Importantly, PSORI-CM02-induced CD8+CD122+PD-1+ Tregs were more potent in suppression of allograft rejection in Rag-/- mice than control Tregs. On the other hand, PSORI-CM02 suppressed T cell proliferation in vitro and reduced their phosphorylation of P70S6K and P50/P65, suggesting that it inhibits both mTOR and NFκB signaling pathways. It also increased IL-10 production while reducing IFNγ level in the supernatant of activated T cells co-cultured with CD8+CD122+PD-1+ Tregs. Furthermore, HPLC fingerprinting ruled out that PSORI-CM02 contained CsA or rapamycin. PSORI-CM02 also did not cause any illness and toxic injury in recipient mice. Thus, we demonstrate that PSORI-CM02 formula suppresses allograft rejection without toxicity.

Clinical features and outcome in patients with osseomuscular type of Wilson's disease.

BACKGROUND: Wilson's disease with osseomuscular type is a rare condition, which often lacks typical hepatic and neurological symptoms and causes misdiagnoses easily. During the past 10 years, eight Chinese patients of osseomuscular type of Wilson's disease were identified in our clinic. METHODS: Clinical information was gathered from medical records and follow-ups. The genetic testing was performed in each patient. Serum ceruloplasmin, Kayser-Fleischer rings, liver function, brain magnetic resonance imaging and abdominal ultrasonography were also evaluated. RESULTS: The median age of onset is 12 years of age. The patients had their initial musculoskeletal conditions with arthralgia or joint deformity, while the hepatic or neurologic signs were minimal. Most patients (6/8) eventually developed clinical neurological symptoms afterwards with a median interval of 36 months. All of them had normal liver function and low serum ceruloplasmin (<0.1 g/L). Most patients (6/8) present with Kayser-Fleischer rings and abnormal hepatic ultrasonography. The arthralgia was resolved with copper chelation therapy. CONCLUSIONS: Wilson's disease with osseomuscular type occurs without typical hepatic or neurological symptoms, which makes the clinical diagnosis challenging. Serum ceruloplasmin, abdominal ultrasonography, ophthalmic examination and genetic testing help to establish the diagnosis. Early diagnosis can initiate an effective treatment and prevent the further damage.

Transcriptome Profiling of the Potato (Solanum tuberosum L.) Plant under Drought Stress and Water-Stimulus Conditions.

Drought stress can seriously affect tuberization, yield and quality of potato plant. However, the precise molecular mechanisms governing potato stolon's response to drought stress and water supply are not very well understood. In this work, a potato (Solanum tuberosum L.) variant, Ningshu 4, was subjected to severe drought stress treatment (DT) and re-watering treatment (RWT) at tuber bulking stage. Strand-specific cDNA libraries of stolon materials were constructed for paired-end transcriptome sequencing analyses and differentially expressed gene (DEG) examination. In comparison to untreated-control (CT) plants, 3189 and 1797 DEGs were identified in DT and RWT plants and 4154 solely expressed DEGs were screened out from these two comparison groups. Interestingly, 263 genes showed opposite expression patterns in DT and RWT plants. Among them, genes homologous to Protein Phosphatase 2C (PP2C), Aspartic protease in guard cell 1 (ASPG1), auxin-responsive protein, Arabidopsis pseudo response regualtor 2 (APRR2), GA stimulated transcripts in Arabidopsis 6 (GASA6), Calmodulin-like protein 19 (CML19), abscisic acid 8'-hydroxylases and calcium-transporting ATPase, et al. were related with drought-stress and water stimulus response. Sixteen DEGs involved in starch synthesis, accumulation and tuber formation exhibited significantly different expression upon re-watering. In addition, 1630, 1527 and 1596 transcription factor encoding genes were detected in CT, DT and RWT. DEGs of ERF, bHLH, MYB, NAC, WRKY, C2H2, bZIP and HD-ZIP families accounted for 50% in three comparison groups, respectively. Furthermore, characteristics of 565 gene ontology (GO) and 108 Kyoto Encyclopedia of Genes and Genomes pathways (KEGG) were analyzed with the 4154 DEGs. All these results suggest that the drought- and water-stimulus response could be implemented by the regulated expression of metabolic pathway DEGs, and these genes were involved in the endogenous hormone biosynthesis and signal transduction pathways. Our data provide more direct information for future study on the interaction between key genes involved in various metabolic pathways under drought stress in potato.