Clinical significance of the expression levels of serum transforming growth factor-ß and CXC type chemokine ligand 13 in primary Sjogren's syndrome patients.

OBJECTIVE: The aim of this study was to investigate the expression levels of the serum transforming growth factor-ß1 (TGF-ß1) CXC type chemokine ligand 13 (CXCL13) in primary Sjogren's syndrome (pSS) patients and its correlation with disease severity. METHOD: Thirty patients with pSS admitted to Nanjing Traditional Chinese Medicine Affiliated Hospital of Nanjing University of Traditional Chinese Medicine from January 2021 to December 2022 were included as the pSS group, while 30 patients who underwent physical examination during the same period were included as the control group. The levels of TGF-ß1 and CXCL13 were detected. The diagnostic value of TGF-ß1 and CXCL13 for pSS was analyzed. Detection of serum TGF-ß1 and CXCL13 levels in pSS patients with different disease activities and lip gland pathological grading of pSS was done. We compared the correlation between TGF-ß1 and CXCL13 levels and disease activity and labial gland pathological grading in pSS patients. RESULT: The TGF-ß1 and CXCL13 levels in the pSS group were higher than those in the control group. The area under the receiver operating characteristic (ROC) curve (AUC) for TGF-ß1 and CXCL13 diagnosis of pSS was 0.790 (95% confidence interval (CI): 0.720~0.861) and 0.838 (95% CI: 0.778~0.898), respectively. The serum TGF-ß1 and CXCL13 levels of pSS patients significantly increase with the increase of disease activity and lip gland pathological grading. The TGF-ß1 and CXCL13 levels in pSS patients were positively correlated with disease activity and lip gland pathological grading. CONCLUSION: The levels of TGF-ß1 and CXCL13 in pSS patients were increased, and it was closely related to disease activity and lip gland pathological grading, which can be used as an effective indicator for the diagnosis of pSS. Key Points • The TGF-ß1 and CXCL13 levels in the pSS group were higher than those in the control group. • The TGF-ß1 and CXCL13 levels in pSS patients were positively correlated with disease activity and lip gland pathological grading. • TGF-ß1 and CXCL13 can be used as an effective indicator for the diagnosis of pSS.

Ruscogenin Ameliorated Sjögren's Syndrome by Inhibiting NLRP3 Inflammasome Activation.

This article investigated the role and the specific mechanism of Ruscogenin in Sjögren's syndrome (SS). NOD/ShiLtJ mice were treated with Ruscogenin, and acinar cells isolated from submandibular glands were treated with TNF-α, Ruscogenin and transfected with NLRP3 overexpression plasmid. Salivary flow rate (SFR) was measured at weeks 11, 13, 15, 17, and 20. Histological analysis of the submandibular glands was conducted by hematoxylin-eosin staining assay. IL-6, IL-17, TNF-α, and IL-1ß mRNA expression was detected through qRT-PCR. AQP 5, AQP 4, P2X7R, NLRP3, caspase 1, IL-1ß, Bax, and Bcl-2 protein levels were tested by western blot. Cell apoptosis was assessed through acridine orange and propidium iodide (AO/PI) staining assay and flow cytometry assay. Ruscogenin ameliorated the SFR and submandibular gland inflammation of NOD/ShiLtJ mice. Ruscogenin promoted the preservation of acinar cells and suppressed inflammation-related factors (P2X7R, NLRP3, caspase 1, and IL-1ß) in submandibular gland tissues of NOD/ShiLtJ mice. Ruscogenin inhibited acinar cell apoptosis in NOD/ShiLtJ mice and reversed TNF-α-induced apoptosis and inflammation of acinar cells. NLRP3 overexpression reversed the repressive effect of Ruscogenin on TNF-α-induced inflammation and apoptosis of acinar cells. Ruscogenin ameliorated SS by inhibiting NLRP3 inflammasome activation.

Influence of total glucosides of paeony on PD-1/PD-L1 expression in primary Sjögren's syndrome.

AIM: To study the influence of total glucosides of paeony (TGP) on the expression of peripheral blood programmed cell death protein 1 (PD-1) and its ligand (PD-L1) in patients with primary Sjögren's syndrome (pSS). METHOD: Ten patients with new-onset pSS were selected as the experimental group and were treated with 1.8 g of TGP (the main ingredient is Radix Paeoniae Alba) daily for 3 months; furthermore, 10 physically healthy individuals were selected as the control group. Peripheral blood mononuclear cells were isolated, and flow cytometry was used to detect PD-1 expression on the surface of CD4+ T and CD8+ T lymphocytes and PD-L1 expression on the surface of CD14+ monocytes and CD19+ B cells before and after treatment in the experimental and control groups. Furthermore, plasma levels of soluble PD-1 (sPD-1), interleukin (IL)-10, and IL-17A were also determined using enzyme-linked immunosorbent assay. RESULTS: The PD-1 expression on the surface of CD4+ T and CD8+ T lymphocytes in the peripheral blood of patients with pSS were significantly higher than in the control group (P < 0.001). However, PD-L1 expression on the surface of CD14+ monocytes declined but not significantly (P > 0.05), and PD-L1 expression on the surface of CD19+ B cells increased significantly (P < 0.001). Moreover, sPD-1 and IL-17A levels in the plasma of the experimental group were significantly higher than in the control group (P < 0.001), but the IL-10 level was significantly lower than in the control group (P < 0.001). After TGP treatment, PD-1 expression on the surface of CD4+ T and CD8+ lymphocytes in the peripheral blood of patients with pSS had decreased significantly (P < 0.001); the PD-L1 expression on the surface of CD19+ cells had decreased significantly (P < 0.001); and the PD-L1 expression on the surface of CD14+ monocytes did not differ significantly (P > 0.05). Furthermore, the levels of sPD-1 and IL-17A in plasma had decreased (P < 0.01) and IL-10 levels had increased after TGP treatment (P < 0.01). CONCLUSION: PD-1/PD-L1 molecules expressed on the surface of T cells, B cells, and monokaryon participated in the pathogenesis and development of SS through interactions. Therefore, TGP, which may increase the expression of PD-1 and its relevant ligand PD-L1 in the peripheral blood mononuclear cells, may play a role in the pathogenesis and development of SS through the PD-1/PD-L1 pathway by regulating regulatory T cells/T helper cell 17.

The genome of the medicinal plant Andrographis paniculata provides insight into the biosynthesis of the bioactive diterpenoid neoandrographolide.

Andrographis paniculata is a herbaceous dicot plant widely used for its anti-inflammatory and anti-viral properties across its distribution in China, India and other Southeast Asian countries. A. paniculata was used as a crucial therapeutic treatment during the influenza epidemic of 1919 in India, and is still used for the treatment of infectious disease in China. A. paniculata produces large quantities of the anti-inflammatory diterpenoid lactones andrographolide and neoandrographolide, and their analogs, which are touted to be the next generation of natural anti-inflammatory medicines for lung diseases, hepatitis, neurodegenerative disorders, autoimmune disorders and inflammatory skin diseases. Here, we report a chromosome-scale A. paniculata genome sequence of 269 Mb that was assembled by Illumina short reads, PacBio long reads and high-confidence (Hi-C) data. Gene annotation predicted 25 428 protein-coding genes. In order to decipher the genetic underpinning of diterpenoid biosynthesis, transcriptome data from seedlings elicited with methyl jasmonate were also obtained, which enabled the identification of genes encoding diterpenoid synthases, cytochrome P450 monooxygenases, 2-oxoglutarate-dependent dioxygenases and UDP-dependent glycosyltransferases potentially involved in diterpenoid lactone biosynthesis. We further carried out functional characterization of pairs of class-I and -II diterpene synthases, revealing the ability to produce diversified labdane-related diterpene scaffolds. In addition, a glycosyltransferase able to catalyze O-linked glucosylation of andrograpanin, yielding the major active product neoandrographolide, was also identified. Thus, our results demonstrate the utility of the combined genomic and transcriptomic data set generated here for the investigation of the production of the bioactive diterpenoid lactone constituents of the important medicinal herb A. paniculata.

Identification and functional characterization of diterpene synthases for triptolide biosynthesis from Tripterygium wilfordii.

Tripterygium wilfordii, which has long been used as a medicinal plant, exhibits impressive and effective anti-inflammatory, immunosuppressive and anti-tumor activities. The main active ingredients are diterpenoids and triterpenoids, such as triptolide and celastrol, respectively. A major challenge to harnessing these natural products is that they are found in very low amounts in planta. Access has been further limited by the lack of knowledge regarding their underlying biosynthetic pathways, particularly for the abeo-abietane tri-epoxide lactone triptolide. Here suspension cell cultures of T. wilfordii were found to produce triptolide in an inducible fashion, with feeding studies indicating that miltiradiene is the relevant abietane olefin precursor. Subsequently, transcriptome data were used to identify eight putative (di)terpene synthases that were then characterized for their potential involvement in triptolide biosynthesis. This included not only biochemical studies which revealed the expected presence of class II diterpene cyclases that produce the intermediate copalyl diphosphate (CPP), along with the more surprising finding of an atypical class I (di)terpene synthase that acts on CPP to produce the abietane olefin miltiradiene, but also their subcellular localization and, critically, genetic analysis. In particular, RNA interference targeting either both of the CPP synthases, TwTPS7v2 and TwTPS9v2, or the subsequently acting miltiradiene synthase, TwTPS27v2, led to decreased production of triptolide. Importantly, these results then both confirm that miltiradiene is the relevant precursor and the relevance of the identified diterpene synthases, enabling future studies of the biosynthesis of this important bioactive natural product.

[Efficacy and Safety Evaluation of Liujin Runzao Concentrated Decoction in Treating Primary Sjögren's Syndrome].

Objective To evaluate the efficacy and safety of Liujin Runzao Concentrated Decoction (LRCD) for the treatment of primary Sjögren's syndrome (pSS). Methods Forty pSS patients with fluid depletion and distribution obstacles syndrome (FDDOS) were randomly assigned to the experimen- tal group and the control group according to 1:1 proportion. All patients received standard therapy: Radix Paeoniae alba total glycosides 600 mg, twice per day. Patients in the experimental group additionally took LRCD, 30 mL each time, twice per day. The therapeutic course for all was 4 weeks, and two courses for all. The improvement of dry mouth and dry eyes were comprehensively evaluated. Each outcome of composite index constitutions (integrals of dry eyes and dry mouth, salivary flow rate, Schirmer test) was respectively reported. Schirmer test and salivary flow rate were determined as well. Score of TCM syndrome, blood sedimentation,'immunoglobulin, and adverse drug reactions were observed. Results The effective rate of comprehensive effect for dry eyes and dry mouth improvement at the end of 8 weeks was 80% in the experimental group and 35% in the control group, with statistical difference (X² =8. 286, P <0. 05). As for the composition of comprehensive effect for dry eyes and dry mouth improvement: The score for dry eyes and dry mouth decreased in the two groups more after treatment than before treatment. The difference in pre-post treatment score for dry eyes and dry mouth at week 8 was higher in the experimental group than in the control group. The difference in pre-post treatment score at week 8 was 1. 71 (95% Cl: -0. 37 -3. 78) between the two groups (P <0. 05). The difference in pre-post treatment Schirmer test and salivary flow rate at week 8 was higher in the experimental group than in the control group, but with on statistical difference (P >0. 05). The difference in pre-post treatment Schirmer test and salivary flow rate at week 8 was 2. 74 mL/15 min (95% Cl: 0. 49 -4.98) and 0. 13 mm/5 min (95% Cl: 0. 92 -1. 23) between the two groups (P <0. 05). The score of TCM syndrome decreased more in the two groups, as compared with before treatment. The difference in pre-post treatment score of TCM syn- drome at week 8 was 1. 71 (95% CI: -1. 40 -4. 81) between the two groups (P >0. 05). One case of uri- nary tract infections occurred in the control group, while no obvious adverse event occurred in the exper- imental group. Conclusion Standard treatment combined LRCD showed better comprehensive effect for dry eyes and dry mouth in pSS patients with FDDOS, and was more safe.

Identification of syn-pimara-7,15-diene synthase reveals functional clustering of terpene synthases involved in rice phytoalexin/allelochemical biosynthesis.

Rice (Oryza sativa) produces momilactone diterpenoids as both phytoalexins and allelochemicals. Accordingly, the committed step in biosynthesis of these natural products is catalyzed by the class I terpene synthase that converts syn-copalyl diphosphate to the corresponding polycyclic hydrocarbon intermediate syn-pimara-7,15-diene. Here, a functional genomics approach was utilized to identify a syn-copalyl diphosphate specific 9beta-pimara-7,15-diene synthase (OsDTS2). To our knowledge, this is the first identified terpene synthase with this particular substrate stereoselectivity and, by comparison with the previously described and closely related ent-copalyl diphosphate specific cassa-12,15-diene synthase (OsDTC1), provides a model system for investigating the enzymatic determinants underlying the observed difference in substrate specificity. Further, OsDTS2 mRNA in leaves is up-regulated by conditions that stimulate phytoalexin biosynthesis but is constitutively expressed in roots, where momilactones are constantly synthesized as allelochemicals. Therefore, transcription of OsDTS2 seems to be an important regulatory point for controlling production of these defensive compounds. Finally, the gene identified here as OsDTS2 has previously been mapped at 14.3 cM on chromosome 4. The class II terpene synthase producing syn-copalyl diphosphate from the universal diterpenoid precursor geranylgeranyl diphosphate was also mapped to this same region. These genes catalyze sequential cyclization steps in momilactone biosynthesis and seem to have been evolutionarily coupled by physical linkage and resulting cosegregation. Further, the observed correlation between physical proximity and common metabolic function indicates that other such class I and class II terpene synthase gene clusters may similarly catalyze consecutive reactions in shared biosynthetic pathways.

Functional identification of rice syn-copalyl diphosphate synthase and its role in initiating biosynthesis of diterpenoid phytoalexin/allelopathic natural products.

Rice produces a number of phytoalexins, and at least one allelopathic agent, from syn-copalyl diphosphate (CPP), representing the only known metabolic fate for this compound. Thus, the class II terpene synthase that converts the universal diterpenoid precursor geranylgeranyl diphosphate to syn-CPP catalyzes the committed step in biosynthesis of these natural products. Here the extensive sequence information available for rice was coupled to recombinant expression and functional analysis to identify syn-copalyl diphosphate synthase (OsCPSsyn). In addition, OsCPSsyn mRNA was found to be specifically induced in leaves by conditions that stimulate phytoalexin biosynthesis. Therefore, transcription of OsCPSsyn seems to be an important regulatory point for controlling the production of these defensive compounds. Finally, alignments carried out with OsCPSsyn revealed that class II terpene synthases exhibit a sequence conservation pattern substantially different from that of the prototypical class I enzymes. One particularly notable feature is the specific conservation of the functionally cryptic 'insertional' sequence element in class II terpene synthases, indicating that this region is important for the corresponding cyclization reaction.