High blood pressure (BP) is not restricted to adults; children and adolescents may also be affected, albeit less frequently. Aside from unfavorable environmental factors, such as obesity and sedentary life leading to early-onset essential hypertension (HT), several secondary causes must be investigated in the occasional hypertensive child/adolescent. Endocrine causes are relevant and multiple, related to the pituitary, thyroid, parathyroid, gonads, insulin, and others, but generally are associated with adrenal disease. This common scenario has several vital components, such as aldosterone, deoxycorticosterone (DOC), cortisol, or catecholamines, but there are also monogenic disorders involving the kidney tubule that cause inappropriate salt retention and HT that simulate adrenal disease. Finally, a blood vessel disease was recently described that may also participate in this vast spectrum of pediatric hypertensive disease. This review will shed some light on the diagnosis and management of conditions, focusing on the most prevalent adrenal  disturbances causing HT. Although HT is highly prevalent (34%) in adults, children and adolescents are not exceptions to this condition; the prevalence of HT in adolescents and young adults (12-19 years) is approximately 4% in the USA ( Hypertension (HT) in infancy is defined as blood pressure (BP) levels above the 95 adults 1-19 yearsrevalent 4% in aduAdrenocortical hormones, glucocorticoids (GC) and mineralocorticoids (MC), and adrenomedullary catecholamines exert essential effects on the components of BP: intravascular volume, peripheral retention of sodium and fluids, expansion of extracellular volume, hypokalemia, and suppression of plasma renin activity   . ActivaAdvances in molecular genetics (Next Generation Sequencing era) have allowed the diagnosis of several forms of adrenal-mediated HT. Thus, this review will emphasize monogenic and sporadic adrenal-linked diseases that cause pediatric HT to be didactically discussed. Although several endocrine diseases may be associated with HT in their clinical pictures, such as thyroid and ovarian dysfunctions and prolonged use of certain medications, this review will focus on the most prevalent adrenal  disturbances. Accordingly, the subject will be divided by the dominant pathophysiological players: 1) aldosterone; 2) DOC (deoxycorticosterone); 3) cortisol; all three syndromes of excess MC production or activity causing volume expansion and its consequences; 4) catecholamines ; 5) the kidney tubule ; and 6) the blood vessel (syndrome of muscle proliferation of smooth vessels and brachydactyly)  and bilateral adrenal hyperplasia (BAH), accounting for 90%-95% of cases. The remaining 5%-10% are familial forms with autosomal dominant inheritance, predominantly affecting young people.Over the last 10-15 years, knowledge of the genetic basis of PA has allowed the identification of Mendelian forms of PA, which are highly prevalent in children and adolescents   – 8 .In brief, the diagnostic management of PA comprises three steps:Screening : PA is biochemically suspected by an increased ratio of plasma aldosterone concentration (PAC) to plasma renin activity (PRA). A cutoff of 27 for this PAC:PRA ratio (ARR) ( o (ARR)    (with Po (ARR)   , 11 Confirmation : Lack of response to suppressive maneuvers confirms autonomous aldosterone secretion  with an adrenal protocol initially excluding aldosterone-producing carcinoma. As adrenal incidentaloma (AI) on CT increases with age, favoring false-positive diagnosis of APA or BAH, selective adrenal vein sampling (AVS) is considered the gold standard for differentiating unilateral from bilateral disease. However, in younger patients (<35 years), presenting with a typical unilateral adenoma (>1 cm), hypokalemia, and increased levels of PAC (30 ng/dL), AVS does not need to be performed  and controlled by ACTH, and the latter encodes aldosterone synthase in zona glomerulosa (ZG) under angiotensin and potassium regulation. The mutated chimeric gene comprises the regulatory sequences of CYP11B1 fused to the coding region of CYP11B2 , leading to abnormal expression of aldosterone synthase in the ZF, which is dependent on ACTH , on ACTH   .CYP11B1/CYP11B2 gene by extended polymerase chain reaction (PCR) can confirm the disease. Therapy with long-acting GC may reduce ACTH, but the lowest possible dose that normalizes BP and K should be used. Iatrogenic Cushing's syndrome and impaired linear growth are associated with overtreatment with GC. If BP remains uncontrolled, MC antagonists, spironolactone (SPL), or eplerenone, are necessary. The latter is preferred in children to avoid the common antiandrogenic effects of SPL.FH1 usually manifests before 20 years of age, and its prevalence is approximately 3% of pediatric HT   . AlthouCLCN2 gene, located on chromosome 3q27.1, has been associated with familial early-onset PA     . Other Kir3.4)   , 21 . PKir3.4)   , 11 . TKir3.4)   .CACNA1H (at chromosome 16p13) ( Germline mutations in  16p13)   , 23 , w 16p13)   , 22 . S 16p13)   .CACNA1D , which encodes the alpha-1 subunit of the voltage-dependent Ca2+ L-type Cav1.3 channel, were identified in two children with PA with no adrenal abnormalities . The steroid products androstenedione (andro) and dehydroepiandrosterone (DHEA) are immediate precursors of adrenal and gonadal androgens and estrogens. Impaired steroids produce a typical female phenotype (46XY DSD - a disorder of sex development) and absent Müllerian structures. Both XX and XY may present with hypergonadotropic hypogonadism (HH), lack of development of secondary sex characteristics (absence of pubic and axillary hair), primary amenorrhea, eunuchoid habitus, and bone mass impairment , accounting for 5%-7% of cases   . The pr11OHD is generally considered the second most common cause of CAH (5%-8%), except in Brazil and possibly China, where 17OHD is second   . SimilaNR3C1 gene located on chromosome 5q31  may be part of the steroid admixture produced by an adrenocortical carcinoma.The leading cause of pediatric CS is exogenous exposure to synthetic GC. Regarding the endogenous source, hypercortisolism can be divided into ACTH-dependent causes (ACTH-producing pituitary adenoma and ectopic ACTH secretion) and ACTH-independent causes    . Males TP 53, a tumor suppressor located on chromosome 17p13.1. Other phenotypic features of LFs may be sarcomas, osteosarcomas, breast carcinoma, brain tumors, leukemia/lymphoma, adenomas, and adrenocortical carcinomas ( CTNNB1 ), a prevalent cause of ACT in adults and related to poor prognosis , and the incidence in southwest Brazil is approximately 4.2 cases/million/year. It occurs at any age, with a bimodal distribution: a first peak occurring before 5 years of age and a second between the fourth and fifth decades   . This ucinomas   . Additiognosis   , are noognosis   . CS andCushing's disease (CD) is the most frequent cause of endogenous CS in childhood (after five years of age) and adolescence. The average age at presentation is 14.1 years   , 47 . ACarney complex (CNC) is a rare multiple neoplasia syndrome inherited in an autosomal-dominant manner caused by loss-of-function mutations of the PRKAR1A gene located at 17q22-24, which encodes the regulatory subunit type I alpha of protein kinase A (PKA)   . CNC isGNAS1 gene)    . Gsα pr1 gene)   .AMES results from the deficiency of 11-β-hydroxysteroid-dehydrogenase type 2 (11HSD2), an enzyme expressed predominantly at nephron distal tubules and collector MC receptor, colon, salivary glands, and placenta. This enzyme converts cortisol (F) into its inactive metabolite, cortisone (E), preventing the activation of the MC receptor. Both PAC and F can activate the MC receptor, but the latter has 1,000-fold higher concentrations than the former steroid   . Thus, HSD11B2 gene mutations (chromosome 16q22), which encode the 11HSD2 enzyme , and neurofibromatosis type 1 (NF1). Then, an extended genetic evaluation is mandatory in the pediatric population . However, when the diagnosis is delayed, mortality rates are high   . Initiaulation        , 60 . Ae 4q31)   . There e 4q31)   .SCNN1A , SCNN1B , and SCNN1G genes, which code the α, β, and γ subunits, respectively, from the epithelial sodium channel (ENaC), also called the amiloride-sensitive channel. These mutations increase the activity of ENaC and promote sodium retention at distal nephrons and collector tubules, regardless of the presence of aldosterone , and PHAII-C by mutations in the WNK1 gene (12p12.3.). Finally, germline variations in KLHL3 (5q31.2) and CUL3 (2q36) are related to PHAII-D and E, respectively , also known as Gordon syndrome (GS), is a rare autosomal dominant disease with low renin HT   . Five sctively   , 67 . Tctively   . Metaboctively   .WNK4 (whose function is reduced by WNK1 and other factors) is a negative regulator of the thiazide-sensitive Na-Cl cotransporter (NCCT) in the distal convoluted tubule (DCT). KLHL3-CUL3 E3 ubiquitin ligase regulates the levels of WNK1 and WNK4. Pathogenic variations of all four genes result in increased NCCT activity in the DCT, leading to the PHAII phenotype. Then, excess sodium and chloride reabsorption is associated with volume expansion, HT, and hyperchloremia. On the other hand, mutated PHAII genes may exacerbate the inhibition of aldosterone-sensitive renal outer medullary potassium channels , worsening the hyperkalemia of GS   . NCCT aPDE3A gene that lead to HT due to increased peripheral vascular resistance ( Bilginturan syndrome is a rare autosomal dominant disease with high penetrance, characterized by early-onset salt-independent HT, short stature, brachydactyly, and death before 50 years of age, possibly due to stroke   . Intereistance   . Thus, In summary, even though most diseases described above have been considered rare, they may lead to organ damage and increase early mortality risk if not diagnosed and managed correctly. Thus, it is essential to keep in mind and investigate adrenocortical and other monogenic secondary causes of HT, as therapeutic options may be distinct from primary HT. Additionally, knowing the genetic basis of adrenal and other monogenic causes of HT will permit better approaches and promote better patient quality of life.


Tff2 and other selected genes in the stomach, pancreas, and the proximal, medial, and distal duodenum . The absence of the Tff2/Muc6 complex in the pancreas is due to a lack of Muc6. Based on its known motogenic, anti-apoptotic, and anti-inflammatory effects, we propose a protective receptor-mediated function of monomeric Tff2 for the pancreatic ductal epithelium. This view is supported by a report that a loss of Tff2 promotes the formation of pancreatic intraductal mucinous neoplasms.The lectin TFF2 belongs to the trefoil factor family (TFF). This polypeptide is typically co-secreted with the mucin MUC6 from gastric mucous neck cells, antral gland cells, and duodenal Brunner glands. Here, TFF2 fulfills a protective function by forming a high-molecular-mass complex with the MUC6, physically stabilizing the mucus barrier. In pigs and mice, and slightly in humans, TFF2 is also synthesized in the pancreas. Here, we investigated the murine stomach, pancreas, and duodenum by fast protein liquid chromatography (FPLC) and proteomics and identified different forms of Tff2. In both the stomach and duodenum, the predominant form is a high-molecular-mass complex with Muc6, whereas, in the pancreas, only low-molecular-mass monomeric Tff2 was detectable. We also investigated the expression of  TFF2 (formerly: (pancreatic) spasmolytic polypeptide ,2) is a In humans, TFF2 was reported as not being detectable in a normal pancreas ,18 or inIn addition to its exocrine secretion, there is also endocrine secretion of TFF2 (review: ), in parN,N’-diacetyllactosediamine (LacdiNAc) oligosaccharide [Xenopus laevis, which contains four TFF domains arranged in tandem [X. laevis skin, also consisting of two TFF domains [Mammalian TFF2 contains two cysteine-rich TFF domains, and the N- and C-terminal regions are connected with an additional disulfide bridge between Cys-6 and Cys-104, generating a circular structure , which shows the same highly restricted expression pattern as MUC6 and TFF2, i.e., gastric mucous neck cells, antral gland cells, and duodenal Brunner glands (review: [Griffonia simplicifolia [The molecular function of TFF2 in the stomach (and the duodenum) has been established as a lectin, which binds specifically to the terminal carbohydrate moiety of the mucin MUC6, i.e., the GlcNAcα1→4Galβ1→R epitope (review: ). This g of MUC6 . The key(review: ). The αGicifolia . Thus, Gicifolia ,39.The physical interaction of TFF2 and MUC6 probably creates a crosslinked mucous network, which affects the viscoelastic properties of mucous gels, as shown in vitro , and eve−8–10−6 M or above only, excluding TFF2 as a classical high-affinity peptide ligand  and inhibition of apoptosis . Foreview: ,28,52). ,52. −8–1. For. For−8–1 in a cancer cell line [Tff2KO mice would be highly interesting as changes might also explain the protection of Tff2KO mice from high-fat-induced obesity [There are also indications of the existence of different Tff2 receptors in embryonic pancreatic cells, with biological responses at a Tff2 concentration of 3 × 10−7 M . Tff2 wa−7 M . At that−7 M ). A combectively . These rell line . The effell line . Thus, a obesity , e.g., dTff2KO) mice do not show a severe phenotype -induced colitis is delayed  mice were bred with Tff2KO mice, the KC/Tff2KO offspring developed a greater number and higher grade of pancreatic intraepithelial neoplasia than the KC mice, indicating a tumor-suppressor activity (protective effect) of Tff2 in the murine pancreas [Tff2-deficient . reviews: , Helicobastritis , and Yerreviews: ,25). Macpancreas .In the past, we discovered that the TFF2 forms in the porcine stomach and pancreas differ . GastricA murine gastric extract was separated by SEC, and the Tff2 immunoreactivities were tested . The TffPorcine gastric TFF2 was previously shown to bind strongly to mucin, even after boiling in SDS  . To testAs a next step, a murine pancreatic extract was analyzed in a similar manner . Here, TIn order to verify that the low-molecular-mass peak actually contains Tff2, fraction D6  was sepaFinally, a duodenal extract (complete duodenum) was analyzed . Here, p125I-labeled porcine pancreatic TFF2 (pTFF2) binds to the same regions .→4Galβ carbohydrate moiety of Muc6 are missing in the pancreas so that lectin-mediated Tff2/Muc complexes cannot form. A comprehensive expression profiling was performed including transcripts encoding TFF peptides  and polypeptides/proteins known to be associated with TFF peptides . Furthermore, expression of the mucins Muc2 and Muc5ac was included, as well as the enzyme responsible for the synthesis of the terminal GlcNAcα1 sugar moiety in Muc6 (A4gnt). In addition, and as controls, expression of Dmbt1 , the hormone gastrin (Gast) typical of the gastric antrum, the transcription factor Pdx1 typical of the gastric antrum and pancreas, and the ependymin-related protein  is the typical TFF gene expressed in the murine pancreas. (ii) Neither Muc6 nor A4gnt expression was detectable in the pancreas within this study. (iii) In contrast, Muc6 and A4gnt transcripts were easily detected in the stomach. (iv) Fcgbp and Gast are typically expressed in the gastric antrum.Here, using expression analysis, we investigate the possible reason for the different Tff2 forms in the stomach and pancreas, respectively . We evenr1/UCC1; ,65) was Tff2, Muc2, Muc6 and A4gnt  but also faintly to a region exclusively in the gastric antrum . In agrFurthermore, murine gastric Tff2 can be released from the Tff2/Muc6 complex by boiling in SDS B. Of notThe low-molecular-mass form of Tff2 observed in the pancreas A probablMuc6 and A4gnt transcripts were not detectable in the pancreas in this study  of gastric and pancreatic specimens  in orderis study . Thus, t125I-labeled TFF2 is predominantly bound to the ultrahigh-molecular-mass region of the stomach, which is typical of binding to Muc6. Furthermore, Fcgbp could be detected in the gastric antrum only, which again is in agreement with the RT-PCR analysis , as well as water. Thus, maintaining the integrity of this epithelium is extremely important. Monomeric Tff2 from the acini would be perfectly suited to protect the murine pancreatic ductal system , the latter being one of the precursor lesions of pancreatic ductal adenocarcinoma [arcinoma . These darcinoma ,25,73,74Remarkably, the situation in humans might be somewhat different. Here, only minute amounts of TFF2 were detectable ,20,75, bIn the future, it will be an ambitious aim to unambiguously identify the various TFF2 receptors, including their carbohydrate moieties. Of note, the latter probably differ in various cell types, as glycosylation patterns are dynamic and also cell type-specific . The chaThe animal care and experimental procedures were conducted in compliance with Directive 2010/63/EU of the European Parliament and the Council of 22 September 2010 on the protection of animals used for scientific purposes, the German Animal Welfare Act, and the regulations on the welfare of animals used for experiments or for other scientific purposes . In the course of these studies, adult wild type animals with a mixed 129/Sv and C57BL/6 background were used as described, i.e., they were fed ad libitum and non-fasted .w/v)  of buffer  in a Precellys 24 lyser/homogenizer, similarly as described previously in detail (aqueous extracts) [Total stomachs, pancreata, and duodena, respectively, from 3–9 animals were collected and extracted with a 5- to 15-fold amount (xtracts) ,78. Furtxtracts) .The aqueous extracts (5 mL each) were fractionated by SEC with the ÄKTA FPLC system  as described  using the following columns : HiLoad Denaturing SDS-PAGE under reducing and non-reducing conditions, respectively, native AgGE, and Western blot analysis were described in detail previously ,66,78,79G. simplicifolia or WFA from Wisteria floribunda, respectively, as reported [The murine Tff1 and Tff2 were detected with the affinity-purified polyclonal antisera anti-mTff1-1 , anti-TFreported ,61,63. Treported .For the protein identification, 150 or 200 µL of fraction D6  was conc125I was as described previously [The labeling of the porcine pancreatic TFF2 (pTFF2) with eviously . The pTFeviously .The isolation and purification of total gastric and duodenal RNA, respectively, , pancreatic RNA , as well as RT-PCR , were as described in detail previously ,83,84.Actb, MB2658/MB2659; Epdr1 MB1890/1891; Gast MB2450/MB2451; Muc2 MB2660/MB2661; Pdx1 MB2464/MB2465; Tff1 MD7/MD8; and Tff2 MB2306/2307) [A4gnt, Dmbt1, Fcgbp, Gkn2, Muc5ac, Muc6, Rps26, and Tff3). All the primer pairs used are intron spanning.The specific primer pairs used for the RT-PCR have been published previously (06/2307) ,84,85 an


Schistosoma mansoni eggs retained in host tissues induce innate cytokine release, contributing to the induction of Type-2 immune responses and granuloma formation, important to restrain cytotoxic antigens, but leading to fibrosis. Interleukin(IL)-33 participates in experimental models of inflammation and chemically induced fibrosis, but its role in S. mansoni-induced fibrosis is still unknown. To explore the role of the IL-33/suppressor of the tumorigenicity 2 (ST2) pathway, serum and liver cytokine levels, liver histopathology, and collagen deposition were comparatively evaluated in S. mansoni-infected wild-type (WT) and IL-33-receptor knockout (ST2−/−) BALB/c mice. Our data show similar egg counts and hydroxyproline in the livers of infected WT and ST2−/− mice; however, the extracellular matrix in ST2−/− granulomas was loose and disorganised. Pro-fibrotic cytokines, such as IL-13 and IL-17, and the tissue-repairing IL-22 were significantly lower in ST2−/− mice, especially in chronic schistosomiasis. ST2−/− mice also showed decreased α-smooth muscle actin (α-SMA) expression in granuloma cells, in addition to reduced Col III and Col VI mRNA levels and reticular fibres. Therefore, IL-33/ST2 signalling is essential for tissue repairing and myofibroblast activation during S. mansoni infection. Its disruption results in inappropriate granuloma organisation, partly due to the reduced type III and VI collagen and reticular fibre formation. Fibrotic diseases, such as liver cirrhosis, pulmonary fibrosis, chronic kidney disease, and cardiovascular disease, are responsible for approximately 45% of deaths in industrialised nations, showing that fibrosis is a major global health problem and should be better understood ,2. FibroSchistosoma spp., is fibrosis, and the liver is the main affected organ in this case [A wide variety of stimuli from different etiological origins, such as autoimmune reactions, allergic responses, tissue damage, and persistent infections, including parasitic diseases, can trigger fibrotic processes ,4,5. Onehis case ,7,8,9.Schistosoma spp., and approximately 780 million people live in areas at risk of infection [Schistosoma mansoni is the most prevalent in the human population and is the only species transmitted in the Americas [It is estimated that more than 250 million individuals are infected by nfection . Among aAmericas ,12. The Americas ,14,15. TAmericas ,15.S. mansoni infection is predominantly a type-1 response, induced by larval and adult worm antigens [The initial immune response to antigens ,17. Whenantigens ,21,22,23antigens ,25,26. Tantigens ,28.S. mansoni egg antigens during granuloma formation, can also activate HSCs, triggering their differentiation into myofibroblasts, thereby contributing to Schistosoma-induced fibrosis [As many parasite eggs are swept through the hepatic portal system and become trapped in the liver parenchyma, the induction and modulation of the immune response in this organ are essential for the outcome of schistosomiasis mansoni ,30. In tfibrosis .Schistosoma eggs injure the host’s tissues [+ T cells, macrophages, and eosinophils, besides activating tissue-resident cells, such as fibroblasts [Therefore, fibrosis is preceded by inflammation, and elements of the innate and adaptive immune responses are fundamental for the induction and regulation of fibrotic processes . IL-33 i tissues . The bio tissues ,36,37. I tissues ,39. This tissues ,40. More tissues . The IL3roblasts ,42,43,444 administration or Helicobacter hepaticus infection also showed the essential role of the IL-33/ST2 signalling pathway in the pathological alterations [The production of IL-33 has been associated with cellular recruitment, activation, and ECM deposition in several experimental models of fibrosis. Bleomycin-induced lung fibrosis has demonstrated that IL-33 promotes macrophage differentiation and activation towards an M2 phenotype, collaborating with fibrosis ,46. Liveerations . HoweverS. japonicum, exogenous administration of IL-33 leads to larger liver granulomas and a stronger Th2 response, whereas inhibition of the IL-33/ST2 pathway hinders fibrosis, collagen deposition, and differentiation of HSCs into myofibroblasts [S. mansoni infections, Vanella et al. [In experimental infections with roblasts ,43,48,49a et al.  reporteda et al.  indicateS. mansoni infection require further elucidation. In the current study, we provide data demonstrating that IL-33/ST2 signalling is an essential step for HSC activation during experimental schistosomiasis, and disruptions in this pathway result in inappropriate granuloma organisation with altered extracellular matrix composition.Therefore, the effects of the IL-33/ST2 pathway on hepatic fibrosis and HSC activation and differentiation during S. mansoni infection, we quantified serum levels of IL-12p70, IL-13, IL-33, TGF-β, CCL24, and IL-22  liver slides . Histopa−/− D. Infilt−/− BALB/c groups were characterised by multifocal to coalescing areas with loss of parenchyma and replacement by well-formed granulomas associated or not with schistosome eggs. These areas replaced approximately 50–60% of the parenchyma. Inflammatory cell infiltrate was comprised of macrophages, multinucleated giant cells, lymphocytes, neutrophils, and eosinophils in different proportions in the different stages of the granulomas  of recombinant IL-33, could not be activated, nor could they activate the c-Jun N-terminal kinase (JNK)/ERK/p38 transcription factors of the mitogen-activated protein kinase (MAPK) pathway, which are associated with the functionality of the IL-33/ST2 pathway, thereby indicating that the biological effect of IL-33 is dependent on the activation of the MAPK pathway and ST2 signalling in HSCs. By inhibiting the JNK/ERK/p38-MAPK pathway in HSCs isolated from C57BL/6 mice, a drastic decrease in collagen production was observed, indicating that IL-33/ST2 signalling through this pathway is essential for the activation and functionality of HSCs.The weak activation of HSCs in ST2n et al.  in an ex−/− mice, while systemic and local TGF-β levels remain similar between both strains infected with S. mansoni [−/− mice infected with S. mansoni presented an almost complete abrogation of the fibrosis process, despite the continuous and unaltered production of TGF-β. The same study also demonstrated that, by inhibiting a part of the TGF-β signalling cascade during experimental schistosomiasis mansoni, there was no change in the development of fibrosis or the production of IL-13, suggesting that the process of liver fibrosis induced by S. mansoni infections, while dependent on IL-13, is TGF-β-independent. As demonstrated in other models of liver fibrosis, IL-13 can be activated via the IL-33 pathway, implying that the liver fibrosis mechanisms during schistosomiasis are mainly driven by a balance between IL-33 and IL-13 instead of TGF-β [S. mansoni infections when the IL-33/ST2 pathway is not active. However, the lower levels of IL-13 during the chronic phase may have prevented appropriate extracellular matrix deposition, thus affecting granuloma formation in the absence of IL-33/ST2 signalling.Furthermore, a previous study from our research group using the same experimental model showed that the concentration of IL-13, which is one of the major factors involved in the activation of HSCs and fibrosis, was also lower in the liver homogenate of ST2 mansoni ,50. Kavi mansoni  demonstrof TGF-β . Our datS. mansoni soluble egg antigen (SEA) significantly decreased in the presence of IL-22 and IL-17 together, while in the presence of IL-17 alone, there is granuloma growth due to the inflammatory role of this cytokine.Other than that, a Th17 response has also been associated with granuloma formation and fibrosis ,68, whosS. mansoni infection in ST2−/− mice, the hydroxyproline content in the livers of these animals, an indirect measure of tissue collagen deposition, was similar to that of infected WT mice. Corroborating our data, Vannella et al. [−/− mice infected with S. mansoni. Although our data showed that the tissue content of hydroxyproline was similar between the two strains of mice analysed, the differences in the deposition of collagen in the granulomas between WT and ST2−/− animals, as portrayed by Masson’s trichrome staining, were markedly significant. ST2-deficient mice deposited collagen in a completely disorganised and exudative manner. Consistent with the increase in hydroxyproline in infected mice, there was an increase in Col I mRNA expression in the livers of infected WT and ST2−/− BALB/c mice, whereas collagens III and VI increased only in the WT mice. Type I and III collagen production and deposition are commonly found in schistosomiasis granulomas, with collagen I being prominent during the chronic phase of schistosomiasis [Despite the compromised differentiation of HSC-derived myofibroblasts during a et al.  also obssomiasis ,73,74.Col I expression was maintained in the absence of the IL-33/ST2 pathway, but the lack of the ST2 receptor significantly reduced Col III and Col VI expression in the acute phase of schistosomiasis, which was confirmed by histological analysis with Picrosirius staining under polarised light. The induction of Col I expression in infected ST2−/− animals may be related to the initial increase in the liver levels of IL-17, as this cytokine is described as a potent inducer of type I collagen production [Schistosoma-induced granulomas formed in ST2−/− mice, these structures may not correctly confine the antigens released by S. mansoni eggs, developing greater microscopic liver lesions and, consequently, higher mortality [Our data showed that oduction ,75,76. Moduction . The lowoduction , and thioduction ,53. As aortality .−/− and WT C57BL/6 mice subjected to inoculation with bleomycin. In these animals, although bleomycin stimulated type I collagen expression in both mouse strains, there was a significant reduction in Col III expression in ST2−/− mice, which was associated with early fibrosis repair. When WT mice received mIL-33 together with bleomycin, an increase in inflammatory cells, such as alternatively activated macrophages (M2) and ILC2, was observed, which increased the production of IL-13 and TGF-β, soluble collagen, and the expression of Col III, consequently increasing the fibrosis score.This difference in the production of collagen types was observed in another experimental model, as demonstrated by Li et al.  in a modS. japonicum infection [S. mansoni infection, which may be the key point for understanding the development of this process.In addition to HSCs, alternatively activated macrophages play a special role in the fibrosis process. Alternatively activated macrophages typically express low levels of inflammatory cytokines and high levels of TGF-β, which promote collagen expression and fibrosis . Experimnfection . However−/− BALB/c mice were used in the experiments. WT BALB/c mice were acquired from an established colony at the university mouse facility. ST2−/− mice, deficient in the receptor that is activated by IL-33 binding [Six-to-eight-week-old female WT and ST2 binding , were orS. mansoni from the LE strain, originally isolated from a human patient in Belo Horizonte, Brazil, has been maintained by successive passages through Biomphalaria glabrata snails and Mesocricetus auratus hamsters at the Laboratory of Schistosomiasis and Helminth Immunology of the Department of Parasitology , and it was used for the experimental infections performed in this study. S. mansoni cercariae were harvested from infected B. glabrata snails, washed, counted, and subcutaneously injected into each mouse , as previously described by Pellegrino and Macedo [d Macedo . To confd Macedo .mansoni infection, WT and ST2−/− BALB/c mice were infected with 25 or 50 cercariae and followed for up to 14 weeks. During this period, animals showing severe clinical signs of schistosomiasis, such as accentuated weight loss and apathy, were euthanised. At 0, 4, 8, 10, and 12 weeks after S. mansoni infection, 6 mice from each experimental group were immobilised, and blood was collected from the tail of each individual to quantify cytokines and chemokines. At weeks 8 and 12 or 8 and 14 post infection , 6–10 mice from each experimental group (WT and ST2−/−) were euthanised by exsanguination via the brachial plexus under deep anaesthesia by intraperitoneal injection of ketamine  and xylazine . The left liver lobes of mice infected with 50 cercariae were immediately removed and processed to quantify the gene expression of collagen types I, III, and VI using real-time quantitative polymerase chain reaction (qPCR). Pathological evaluation was carried out in experiments with 25 cercariae, and the chronic phase was analysed at 14 weeks. Due to the higher mortality rate of mice infected with a 50 cercariae inoculum, they were analysed at the early chronic phase, i.e., 12 weeks post infection. The left liver lobe was processed to estimate the number of parasite eggs retained in the parenchyma, the median liver lobe was harvested to estimate the hydroxyproline content, and the right liver lobe was promptly fixed and processed for histopathological analysis. Liver sections were subjected to immunohistochemical procedures or were stained using either haematoxylin-eosin, Masson’s trichrome, Picrosirius red, or Gomori’s ammoniacal silver.To evaluate the role of IL-33/ST2 activation on the liver pathology and fibrosis associated with S. −/− BALB/c mice by ELISA using commercially available kits , following the manufacturer’s instructions. In serum samples collected at weeks 0, 4, 8, 10, and 12 post infection, the concentrations of IL-13, IL-12p70, IL-33, TGF-β, CCL24, and IL-22 were measured. To measure TGF-β, each serum sample was activated with a HCl solution and diluted, as described by the manufacturer. IL-4, IL-13, IL-33, IL-17, and IL-22 cytokine levels were quantified in liver homogenate samples of all experimental groups, as previously described [The concentrations of cytokines and chemokines were quantified in serum and liver homogenate of WT and ST2escribed .As an indirect determination of collagen deposition, the hydroxyproline content was measured, as described previously . A samplHistopathological evaluation of hepatic granuloma and collagen deposition was performed in liver sections from infected mice. Liver samples from each mouse were collected, fixed in 10% buffered formalin, embedded in paraffin, and cut into 5 μm thick sections. The liver sections were stained with haematoxylin-eosin to evaluate the overall inflammation and tissue damage. Masson’s trichrome and Picrosirius red staining were performed to illustrate extracellular collagen deposition around parasite eggs, and Gomori’s ammoniacal silver staining to characterise the reticular fibre organisation ,90,91,92+ Dual Link System-horseradish peroxidase (HRP) kit , in which the secondary antibody is associated with HRP-labelled polymers, and peroxidase activity was revealed by the chromogen 3,3′-diaminobenzidine . All slides were counterstained with Harris modified haematoxylin  for 2 min and with bluing reagent  for 1 min. Subsequently, the samples were analysed under an optical microscope, and α-SMA was quantitatively estimated in the livers of infected ST2−/− and WT mice using the ImageJ software (version 1.43). The amount of α-SMA in granuloma cells was expressed as a percentage of the α-SMA-stained area per field, analysing exudative granulomas containing viable miracidia from chronically infected mice of both strains (3 mice/group), using an Olympus Bx41TF microscope coupled to a DP12-camera 2 .Immunohistochemistry (IHC) was performed on slides with 5 μm liver sections of mice from the different experimental groups to evaluate the expression of α-SMA. Each section was deparaffinised, rehydrated, and subjected to antigen retrieval using a 0.01 M sodium citrate solution (pH 6.0) by microwave heating at 80% power for 10 min. The slides were then incubated with anti-α-SMA antibody  at a dilution of 1:800 for 24 h at 4 °C. After incubation with the primary antibody, the slides were washed, and the presence of α-SMA in the liver parenchyma was revealed using the EnVisionΔCT method [The quantification of gene expression of collagen types I, III, and VI was performed in liver samples using quantitative reverse transcription polymerase chain reaction (qPCR). Liver fragments of mice from different experimental groups were collected, stored in RNalater™ Stabilisation Solution , and processed for RNA extraction using a tissue homogeniser (Omni TH) and TRIzol kit (Invitrogen), according to the manufacturer’s instructions. After the quantification of RNA in a spectrophotometer , the samples were subjected to complete digestion using a DNAse Turbo DNA-free kit , followed by cDNA synthesis using a commercially available High-Capacity cDNA reverse transcription kit . For quantification of gene expression, each PCR experiment was performed in triplicate in a total volume of 10 µL, combining 5 µL of SYBR Green Master Mix , 0.3 µL of each primer (forward and reverse) for the targeted gene, 2.4 µL of Milli-Q water, and 2.0 µL of cDNA. The specific primers for the collagen type I, collagen type III, and collagen type VI genes are listed in T method . Data wet-test. For non-parametric datasets, comparisons between multiple groups were performed by the Kruskal–Wallis test, while comparisons between only two groups were carried out by the Mann–Whitney U test. After being assigned, p-values ≤ 0.05 were considered significant.Statistical analysis was performed, and graphs were generated using GraphPad Prism 8.0.2. Data were subjected to ROUT tests for the identification and removal of outliers, and their normal distribution was verified using the Kolmogorov–Smirnov test. Comparisons between the experimental groups at different time points of infection were carried out by two-way ANOVA, followed by Bonferroni’s post-test, and comparisons between only one phase of infection were carried out by Student’s S. mansoni-infected mice, the IL-33/ST2 activation pathway did not significantly change the quantity of collagen deposited around the parasite eggs retained in the host’s liver. However, activation of this pathway participates in the induction of some pro-fibrotic cytokines, such as IL-13 and IL-17, and the tissue-repairing IL-22 in the livers of chronically infected ST2−/− mice. Moreover, IL-33/ST2 signalling is essential for the differentiation of HSCs into myofibroblasts and, consequently, for the production of type III and VI collagen. This results in the proper organisation of collagen reticular fibres within the granulomatous reaction, enabling the adequate development of hepatic granulomas and antigen retention during schistosomiasis, protecting the liver parenchyma. Thus, the current study provides evidence that supports the role of the IL-33/ST2 pathway in liver-repairing mechanisms and the appropriate organisation of collagen around parasite eggs, participating in S. mansoni-induced liver fibrosis and controlling disease severity.The severity of schistosomiasis has been associated with the intensity and extent of tissue damage and fibrosis induced by parasite eggs trapped in the host’s tissues. This study shows that, in


Tryphosidae, Orchomenellacompressasp. nov., is described from hydrothermal vents in the Okinawa Trough. This is the first known Orchomenella species found in vent fields. Important morphological characters that differentiate O.compressasp. nov. from its congeners are the absence of eyes, the compressed distal three articles of gnathopod 2, the shape of the posterior margin of epimerons 2 and 3, and the number of dorsal spines on the telson. The genetic divergence of the analyzed COI gene clearly supports this new taxon.A new species of the family  Tryphosidae was established as a subfamily in family Lysianassidae Dana, 1849 by Lysianassinae based on following characters: molar of mandible asetose, apical margin of the outer plate of the maxilliped bearing robust setae, gnathopod 1 subchelate, and telson cleft. The genus Orchomenella Sars, 1890 contains 34 species  in 2014. The specimens are deposited in the Marine Biological Museum, Chinese Academy of Sciences (MBMCAS), Qingdao, China. Specimens were examined and dissected using a dissecting microscope (Zeiss Discovery V20). Line drawings were made with a tablet (Wacom Intuos Pro PTH-851) and Adobe Photoshop CS6 (v. 13). Length measurement is made along the outline of the animal, beginning from the anterior margin of head to the end of the urosomite 3.The present materials were collected by ROV Faxian suction sampler, together with Orchomenellacompressa sp. nov. (657 bp) was obtained from its mitochondrial genome by homologous alignment, and the mitochondrial genome of the new species was obtained by Illumina HiSeq sequencing. For Illumina pair-end sequencing of each strain, at least 3 μg of genomic DNA was used for sequencing library construction. Paired-end libraries with insert sizes of ~400 bp were prepared following Illumina’s standard genomic DNA library preparation procedure. Purified genomic DNA was sheared into smaller fragments with a desired size by Covaris, and blunt ends were generated by using T4 DNA polymerase. After adding an “A” base to the 3’ end of the blunt phosphorylated DNA fragments, adapters were ligated to the ends of the DNA fragments. The desired fragments were purified through gel-electrophoresis, then selectively enriched and amplified by PCR. The index tag was introduced into the adapter at the PCR stage, as appropriate, and we did a library quality test. Finally, the qualified Illumina pair-end library was used for Illumina NovaSeq 6000 sequencing . The raw paired end reads were trimmed and quality controlled by Trimmomatic with parameters (SLIDINGWINDOW:4:15 MINLEN:75) (v. 0.36 http://www.usadellab.org/cms/uploads/supplementary/Trimmomatic). Clean data obtained by above quality control processes were used to do further analysis.The COI sequence of Orchomenellacompressa sp. nov. sequenced in this study, the other 17 sequences from nine described species and the outgroup Orchomenyx , dissected, Okinawa Trough, 27°33'N, 126°58'E, RY0067, ROV-3, depth 1243 m, 16 April 2014. Paratype: MBM 286555, ♂ (5.6 mm), same collection data as holotype.27°33'N, 126°58'E, RY0069, ROV-3, depth 1243 m, 16 Apr. 2014.MBM 286566, 4♀♂, Okinawa Trough, Body smooth; epimerons 1–3 smooth, posteroventral margin rounded; urosomite 1 bearing dorsal notch. Head: head deeper than long; lateral cephalic lobe large, subtriangular, subacute apically. Eyes: no trace visible in ethanol-preserved materials. Antenna 1 with peduncular article 1 longest, length ~1.5× width, without dorsodistal protrusion, article 3 subequal to article 2; flagellum 9-articulate, with callynophore, without calceoli; accessory flagellum long, 5-articulate, article 1 longest, not forming cap. Antenna 2 longer than antenna 1; peduncle without brush setae, article 4 longer than article 5; flagellum 7-articulate, shorter than peduncle, without calceoli.Mouthparts. Upper and lower lip typical for the genus. Mandible incisors symmetrical, smooth, subtriangular; left lacinia mobilis a long, slender peg; accessory setal row without distal setal tuft, both left and right with 3 short, slender, simple setae; molar well developed, columnar; palp 3-articulate, attached lower than molar, article 1 shortest, article 3 blade-like, shorter than article 2, fringed with long simple setae. Maxilla 1 with inner plate narrow with 2 pappose apical setae; outer plate with 11 setal-teeth in 7/4 crown arrangement; palp large, 2-articulate, with 9 terminal short robust setae and 1 long, plumose seta. Maxilla 2 inner plate narrower and slightly shorter than outer plate. Maxilliped with inner plate rectangular, distal margin with teeth and 1 or 2 robust setae; outer plate does not extend to distal margin of palp article 3, with 2 apical, robust setae; palp 4-articulate, dactylus nearly as long as article 3, unguis present.Coxae 1–4 longer than broad, overlapping. Gills present on coxae 2–7; pereopod 5 and 6 bearing accessory gill. Oostegites slender, present on coxae 2–5.Pereopods. Gnathopod 1 subchelate; coxa anterior margin slightly concave, posterior margin nearly straight; basis slender, anterior margin fringed with thin, simple setae; ischium shorter than merus; merus shorter than carpus, with distal margin bearing 4 long setae; carpus slightly shorter than propodus; propodus subrectangular, posterior margin straight, posterodistal corner with a group of simple setae, without robust setae; dactylus simple, as long as palm. Gnathopod 2 minutely chelate, with distal 3 articles distinctly compressed compared to other articles; coxa large, subequal in size to coxa 3; ischium 1.8× longer than merus; carpus compressed, 1.6× longer than propodus, posterior margin strongly convex, anterior margin nearly straight; propodus subrectangular; dactylus reaching corner of palm, tufts of setae covering most of distal part of propodus. Pereopod 3 coxa large; merus with posterior margin bearing simple, long setae; propodus with posterior margin bearing small, robust setae; dactylus slender. Pereopod 4 coxa large, with large posteroventral lobe, anterior margin convex; ischium to carpus with posterior margin bearing simple, long setae; propodus nearly as long as merus, posterior margin with small robust setae; dactylus slender. Pereopod 5 coxa slightly deeper than wide, posterior lobe slightly larger than anterior one; basis broadly expanded with posterior and anterior margin bearing small robust setae; merus longer than carpus, slightly expanded posteriorly; propodus with anterior margin bearing 4 small, robust setae; dactylus slender. Pereopod 6 coxa posterior lobate; basis expanded, posterior and anterior margin bearing small, robust setae; merus subequal in length to carpus, anterior margin with robust setae, posterior margin bearing 1 distal and 1 subdistal seta; propodus with small, robust setae along anterior and posterior margins; dactylus slender. Pereopod 7 with coxa small; basis wider than that of pereopod 6, posterior margin broadly rounded; distal 5 articles similar to that of pereopod 6.Uropods and telson. Uropod 1 peduncle longer than rami, with 4 dorsomedial and 1 apicomedial robust setae; outer ramus slightly longer than inner ramus; inner ramus with 2 dorsal robust setae. Uropod 2 peduncle subequal in length to rami, with 1 apicolateral, 1 dorsolateral, and 1 apicomedial robust seta; outer ramus slightly longer than inner ramus, with 3 dorsal, robust setae; inner ramus with 2 dorsal, robust setae. Uropod 3 peduncle slightly shorter than outer ramus, with 6 robust setae; outer ramus 2-articulate, article 1 with 2 lateral and 2 distal robust setae, article 2 about 0.5× length of article 1; inner ramus not reaching to base of article 2 of outer ramus, with 2 lateral robust setae. Telson cleft, about 68%, bearing 3 dorsal and 1 apical robust setae on each side.compressa (= compressed), referring to the compressed distal 3 articles of the gnathopod 2.From the Latin Presently known only from Okinawa Trough, at a depth of 1243 m.Orchomenella has been reported from hydrothermal vents. The new species can be distinguished from other Orchomenella species in lacking of eyes and having the distal three articles of gnathopod 2 compressed. The new species is morphologically most similar to O.tabasco , which was collected from the Cedros Trench at 1720–1728 m. However, O.compressa sp. nov. differs from O.tabasco by following characters: carpus of the gnathopod 1 shorter than propodus in the new species, rather than subequal in length in O.tabasco, and telson cleft more than 50% in the new species, rather than cleft only 40% in O.tabasco  and O.minuta  . Interspecific genetic divergence (K2P) among these 10 species is summarized in Table O.pinguis and O.minuta genetically , although morphological characters do not support this close relationship. It is a pity that the COI sequence of O.tabasco, which is morphologically most similar to O.compressa sp. nov., is still unavailable. Of the species analyzed, O.rotundifrons , is most genetically divergent from the new species (0.215)  .


Immune checkpoint inhibitors (ICIs) have made significant breakthroughs in the treatment of a variety of malignancies. As its use increases, the unique immune-mediated toxicity profile of ICls are becoming apparent. We report a case of immune-related endocrine adverse events (irAE) in a patient with hepatocellular carcinoma treated with anti-programmed cell death protein 1 (PD-1) (tislelizumab). Although many irAEs have been reported, few cases of severe thyrotoxicosis have been described after immunotherapy in the literature. We present the case of a 49-year-old male who experienced a Grade 3 tislelizumab-related adverse reaction according to Common Terminology Criteria for Adverse Events (CTCAE5.0) and received methylprednisolone, thiamazole, and levothyroxine sodium tablets. Early identification of irAEs, risk factors, regular monitoring, use of steroids and/or immunoglobulins, and adjuvant supportive care are critical to the clinical prognosis of patients. It should be underlined that the tumor benefits of ICI therapy outweigh the risks associated with ICI-induced endocrine disorders, and ICI treatment should not be stopped or delayed except in rare cases . The familiarity of healthcare professionals with irAEs of the thyroid when thyrotoxicosis occurs is important to facilitate an effective diagnosis and appropriate treatment of this increasingly common thyroid disorder. Immunotherapy has made a significant breakthrough and has evolved to be a standard treatment regimen in cancer treatment since the 1990s –4. PD-1 Tislelizumab is a new humanized IgG4 PD-1 inhibitor that was approved by the National Medical Products Administration (NMPA) in China for the treatment of classical relapsed or refractory Hodgkin lymphoma after at least second-line systemic chemotherapy, locally advanced or metastatic urothelial cancer, non-small cell lung cancer, and hepatocellular carcinoma in December 2019. Several clinical trials involving multiple indications for tislelizumab are ongoing Table 1.The main side effects related to treatment are irAEs, including rash, pruritus, thyroiditis, diarrhea, hepatitis, and pneumonitis. Grade 3 and higher adverse effects caused by tislelizumab include severe skin reactions, anemia, pneumonitis, hypertension, and adrenocortical insufficiency –10. Howe. Laboratory tests showed hepatitis B virus surface antigen(+) and hepatitis B virus core antibody(+). During this period, the patient had no abdominal pain, abdominal distension, nausea, vomiting, or other discomfort. He had a history of hypertension of more than 3 years, regular oral administration of perindopril tert-butylamine and bisoprolol fumarate, and a history of coronary heart disease of 3 years. He underwent a coronary stent implantation at another institution 3 years prior and is currently taking aspirin, ezetimibe tablets, and rosuvastatin regularly. He had no prior history of infectious diseases or other surgical history. With regard to family history, the patient’s mother died due to esophageal cancer. The father was alive and denied any history of hepatitis-related or genetic diseases in the family. Diagnosis at admission included (i) primary liver cancer; (ii) posthepatitic cirrhosis; (iii) chronic active viral hepatitis B; (iv) coronary heart disease, coronary stent immediately after the postoperative period; and (iv) very high risk of Grade 1 hypertension. The electrocardiogram on admission showed a ventricular rate of 66 bpm, sinus rhythm, QA pattern in V1 and V2, and Q waves in III and aVF. Imaging studies revealed a huge mass, approximately 13 cm in size. Potential for surgical examination was evaluated based the patient’s physical condition and residual liver volume. Considering that the patient’s liver cancer lesion was located in the right liver and the volume was huge, it was considered that if surgical treatment was performed, the residual liver volume would be less than 40%. Thus, the risk of surgical treatment was high, and the patient was at increased risk of postoperative liver failure. Hepatic arterial infusion chemotherapy (HAIC) combined with tislelizumab was recommended. Baseline thyroid function tests included free triiodothyronine (FT3) 3.15 pmol/L (3.1–6.8), free thyroxine (FT4) 11.09 pmol/L . Laboratory results revealed alpha-fetoprotein 1.56 ng/mL, carcinoembryonic antigen 2.56 ng/mL, alanine aminotransferase 35 U/L, aspartate aminotransferase 147 U/L, direct bilirubin 34 μmol/L, and indirect bilirubin 11 μmol/L. Abdominal computed tomography (CT) revealed space-occupying lesions that involved the right lobe of the liver, with a small amount of bleeding. A subsequent upper abdominal plain scan and enhanced CT performed in our hospital (11 October) confirmed space-occupying lesions in the right lobe of the liver, about 13.5×13.9 cm in size, suggestive of liver cancer On 27 December 2021, the patient experienced systemic pruritus, poor appetite with trembling of the hands, irritability, and weight loss, and severe difficulty falling asleep. Physical examination did not show yellowing of the skin mucosa or sclera of the whole body, multiple scratches on the upper limbs of the trunk and skin damage, mild bulging of the eyeballs, and no maculopapule on the skin. Thyroid function tests showed FT3 20.95 pmol/L, FT4 67.17 pmol/L, serum TSH 0.01 uIU/mL, TgAb 819 IU/mL, and TPOAB 8.24 IU/mL. Thyroid ultrasound revealed a thickness of 1.8 cm in the right thyroid lobe, 0.4 cm in the isthmus, and 1.8 cm in the left lobe. The thyroid echoes were ancestral, reduced and heterogeneous, and the diagnosis indicated diffuse thyroid lesions Figure 2Thyroid function tests on 7 January 2022 revealed FT3 2.52 pmol/L, FT4 56.4 pmol/L, and serum TSH 0.006 uIU/mL. The patient had normal diet, normal sleep patterns, no special discomfort, and hyperthyroidism improved significantly. The electrocardiogram showed a ventricular rate of 61 bpm and sinus rhythm. The third cycle of HAIC combined with tislelizumab was administered on 14 January. On 12 February, thyroid function tests showed free FT3 1.67 pmol/L, free FT4 4.58 pmol/L, serum TSH 54.97 uIU/mL, TGAB 408.6 IU/mL, and TPOAB 7.51 IU/mL. Physical examination revealed no facial edema, no tenderness in the neck, and no palpable enlargement of the thyroid gland. A supplementary diagnosis was made of immune-related thyroiditis, hypothyroidism. Levothyroxine sodium tablets 12.5 μg once daily were administered orally for thyroiditis. The CT performed on 14 February 2022 showed a large mixed density shadow in the right lobe of the liver that measures approximately 12.7x12.1 cm. Considering that the lesion was not significantly smaller than before, despite the general improvement, stable disease (SD) was evaluated for 4 months.).The patient had normal diet and physical strength, increased sleep, unresponsiveness, or other discomfort. Physical examination showed no facial edema, no tenderness in the neck, and no palpable enlargement of the thyroid gland. The dose of levothyroxine sodium tablets was adjusted to 100 μg orally once daily. Follow-up examinations on 28 April 2022, 23 July 2022, and 26 August 2022, showed a large mass in the right lobe, with no significant volume changes and no significant enhancement of injury. The complete tumor evaluation was SD for 6 months, and oral lenvatinib mesylate is being continued to date. A timeline describing diagnosis, treatment, and irAEs is shown in Subsequent treatment was changed to transcatheter arterial chemoembolization (TACE) combined with lenvatinib mesylate. Three cycles of TACE combined with lenvatinib mesylate were performed from 17 February 2022 to 29 April 2022, and then treatment was changed to 8 mg of lenvatinib mesylate once daily as maintenance therapy. On 27 April 2022, reexamination of thyroid function showed free FT3 2.48 pmol/L, free FT4 5.26 pmol/L, serum TSH 62.47 uIU/mL, TGAB 575.6 IU/mL, and TPOAB 8.69 IU/mL. The clinical time course of the development of thyroid disease in the present patient is shown based on several clinical parameters  in Tislelizumab binds to human PD-1 with high specificity and affinity   using keImmunotherapy with ICIs uses the body’s own immune system to attack cancer cells but causes unwanted autoimmune side effects in up to 60% of patients. These irAEs may lead to treatment interruption, permanent organ dysfunction, hospitalization, and premature death. Thyroiditis is one of the most common irAEs. Studies have confirmed that thyroiditis caused by ICIs is composed of T-cell-predominant but varied immune infiltrates, mainly including γδT17 cells, CD41 Th17, and CD81 Tc17 cells. The γδT17 cells are a major subset of interleukin (IL)-17A1-producing cells and early expansion may contribute to the activation and recruitment of Th17, CD81, and other T cell populations. Following treatment with ICIs, multilineage IL-17T cells expand in the thyroid tissue, and IL-17-producing innate-like γδT17 cells increase in the thyroid gland, and adaptive Th17 cells also increase significantly in the thyroid gland. Targeting Th17 and γδT17 cell function via the IL-17A axis may therefore be a generalizable strategy for addressing type 3 immune-mediated irAEs in the future. Several hypotheses for thyroiditis include genetic susceptibility associated with the human leukocyte antigen (HLA) haplotype associated with autoimmune thyroid disease, CTLA-4 or PD-1 polymorphisms, underlying autoimmune susceptibility, disinhibited regulatory T cell function, and/or cytokine -mediated thyroiditis , 14.Endocrine dysfunction is one of the most common irAEs reported in ICI clinical trials and includes hypothyroidism, hyperthyroidism, hypophysitis, primary adrenal insufficiency (PAI), and insulin-deficient diabetes mellitus (IDD) , 16. ICIThe incidence of ICI-related thyroid dysfunction depends on two factors, one is the type of ICI and the other is the form of treatment (monotherapy or immune combination therapy). It has been reported that CTLA-4 inhibitors result in ICI-related thyroid dysfunction in 1% to 5%, and in 5% to 10% following PD-1 or PD-L1 inhibitor treatment, whereas the incidence of thyroid dysfunction is as high as 10% to 20% during immune combination therapy , 20. TheThe reported incidence of tislelizumab related thyroid dysfunction has varied widely across different clinical trials involved lung cancer, esophageal cancer, gastric/esophageal junction cancer, urothelial cancer, and classical Hodgkin’s lymphoma –28. In tP = 0.02) and overall survival (OS) ; however, there was no association between hypothyroidism and cancer outcomes . A retrospective analysis of the US Food and Drug Administration adverse event reporting system reported that 9.3% (171/1842) of patients presented with hyperthyroidism before hypothyroidism . Lee et outcomes . A systeoutcomes –34.P = 0.05) and a significantly higher risk of TD in patients with baseline TSH levels >2.19 mIU/mL   .Another clinical study reported that thyrotrophin receptor antibodies (TRAbs) tested in 6 of 7 cases of irAE-associated TD, were negative, which makes Graves’ disease caused by thyroid autoantibody TRAbs unlikely to be the main cause of irAEs-associated TD .Multivariate analysis showed that disease duration (≥1a), thyroid color ultrasound revealing no nodules, and female sex were independent risk factors for the development of thyroid dysfunction, whereas patients with characteristics of female sex, disease duration (≥1 a), thyroid color ultrasound revealing no nodules, and elevated BMI were more likely to develop thyroid dysfunction –44. DrugOur patient presented with hyperthyroidism during treatment cycle 3 (Day 65) of HAIC combined with tislelizumab for advanced hepatocellular carcinoma, which progressed to hypothyroidism during Cycle 5. Tislelizumab is a PD-1 inhibitor that predisposes to thyroid dysfunction. At present, there has been no relevant report describing thyroid dysfunction associated with HAIC treatment. In addition, our patient had no previous history of underlying thyroid disease, only thyroglobulin antibody was high before treatment, and other thyroid examination indicators were normal. Therefore, the patient may have developed thyroid toxicity caused by tislelizumab. The patient was assessed as having Grade 3 thyrotoxicosis and Grade 1 hypothyroidism according to the CTCAE 5.0 evaluation criteria .In terms of immune-related hyperthyroidism, Chinese Society of Clinical Oncology/National Comprehensive Cancer Network (CSCO/NCCN) guidelines indicate that ICIs can be continued in patients exhibiting hyperthyroidism of Grade s 1 to 4 and β-blockers may treat symptoms, while further assessment of Graves’ disease is required if persistent thyrotoxicosis occurs , 48. ESMThe clinical presentation and impact of IrAE are diverse and complex, and the management of patients using ICI often requires a balance between efficacy, toxicity, and specific treatments and active multidisciplinary collaboration. Patients with mild to moderate irAEs had longer OS compared to patients without irAEs. That is, the occurrence of moderate to low grade immune-related adverse events is associated with improved survival. High-grade irAEs, on the other hand, can be life-threatening and may require discontinuation of therapy or suppression of systemic immunity, which may counteract the effects of ICIs . TherefoImmune-related hypothyroidism is usually permanent and requires thyroid hormone replacement therapy . The CSCThe patient’s perspective was as follows: after 2 cycles of tislelizumab, I experienced problems including poor appetite, shaking hands, atrial fibrillation, irritability, weight loss, and difficulty falling asleep. The clinician paid great attention to me throughout the treatment and found that it may be that I had had a significant adverse repose response to tislelizumab, which had improved after providing symptomatic treatment. At present, I am pleased to know that the cause of the problem was resolved by oral treatment with levothyroxine sodium tablets. I would like to thank the clinician for making this diagnosis, which improves my perception of anti-cancer therapy.In conclusion, we report for the first time a case of severe thyroiditis caused by tislelizumab and discuss the treatment of immune-related thyroiditis, which suggests that it is necessary to improve the clinical understanding of PD-1-inhibitor-induced thyroid dysfunction and strengthen multidisciplinary collaboration in the management of such patients, especially within 1 – 2 months of treatment initiation, to monitor thyroid-related parameters, and once a patient develops immune-related thyroiditis, the patient’s condition should be comprehensively assessed and appropriate treatment should be given for symptomatic treatment and treatment if necessary to improve the patient’s prognosis.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article. Written informed consent has been obtained from the participant/patient for the publication of this case report.PL, JH: Conceptualization, project administration, funding acquisition, writing–review and editing. LH, CW: Writing – review and editing. HD, XS: Supervision. RZ, BS: Writing – original draft, formal analysis. All authors contributed to the article and approved the submitted version.


In this study, the physicochemical properties, gene expression level, and secondary metabolite of P. chinense Schneid seedlings under GA3 treatment were investigated. The results showed that GA3 significantly improved the plant height, ground diameter, fresh weight, chlorophyll content, soluble substance content, superoxide dismutase, and peroxidase activities. This was accompanied by elevated relative expression levels of Pc(S)-GA2ox, Pc(S)-DELLA, Pc(S)-SAUR50, Pc(S)-PsaD, Pc(S)-Psb 27, Pc(S)-PGK, Pc(S)-CER3, and Pc(S)-FBA unigenes. Conversely, a notable reduction was observed in the carotenoid content, catalase activity and the relative expression abundances of Pc(S)-KAO, Pc(S)-GID1/2, and Pc(S)-GH 3.6 unigenes in leaves of P. chinense Schneid seedlings (p < 0.05). Furthermore, GA3 evidently decreased the contents of pinocembrin, pinobanksin, isosakuranetin, naringin, naringenin, (−)-epicatechin, tricetin, luteolin, and vitexin belonged to flavonoid in stem bark of P. chinense Schneid seedlings (p < 0.05). These results indicated that exogenous GA3 promoted growth through improving chlorophyll content and gene expression in photosynthesis and phytohormone signal pathway and inhibited flavonoid synthesis in P. chinense Schneid seedlings.The phytohormone gibberellic acids (GAs) play a crucial role in the processes of growth, organ development, and secondary metabolism. However, the mechanism of exogenous GA Concurrently, it increases the fresh weight and plant height, subsequently promoting plant growth [Plant growth and secondary metabolism are influenced by various plant growth regulators, including abscisic acid (ABA), auxin, and gibberellic acids (GAs) ,2. GA3 in plants ,5,6. Pret growth ,9,10,11.3 regulates the biosynthesis and its signal transduction pathway of endogenous phytohormone, which controls the plant growth [3 influences the endogenous levels of phytohormones, such as GA, ABA, and auxin, which, in turn, regulates various aspects of plant growth and development. The synthesis and signal transduction of these phytohormones are controlled by specific genes, such as GA20 OXIDASE (GA20ox), PYR/PYL, and Gretchen Hagen 3 (GH3) [3 reduces the relative expression levels of ZmGA20 OXIDASE7 (GA20ox7) and ZmGA3 OXIDASE1/3 (GA3ox1/3) genes, and enhances the levels of ZmGA2 OXIDASE1/4 (GA2ox1/4) and ZmGA20 OXIDASE2 (GA20ox2) genes in GA biosynthesis in maize [3 elevates the relative expression levels of major genes in GA signal pathway while decreasing the levels of NcGA20ox and NcGA3ox genes in biosynthesis after 3 days, ultimately promoting the growth of Neolamarckia cadamba [3 impacts the biosynthesis and accumulation of other phytohormones, such as ABA, auxin, and cytokinin (CTKs), to balance the plant growth [Exogenous GAt growth ,13. The  3 (GH3) ,15,16. Iin maize . Meanwhit growth ,18.3 in plants [3 inhibited the biosynthesis of flavonoid and isoflavonoid through negatively regulating GA-mediated signal pathway and promoted the growth by improving nitrogen metabolism in Medicago truncatula [3 has been shown to decrease their content. This occurs through the downregulation of the relative expression levels of key genes involved in flavonoid biosynthesis and the suppression of enzyme activities [3 reduces the flavonoid content by inhibiting the expression levels of certain transcription factors related to flavonoid biosynthesis. Alternatively, it may indirectly depress the biosynthesis of endogenous phytohormones, ultimately promoting flavonoid accumulation [Secondary metabolites comprise a diverse group of compounds, including flavonoids, alkaloids, and terpenoids, where flavonoids play a protective role against biotic and abiotic stresses. The biosynthesis of these compounds is regulated by exogenous GAn plants ,20. Prevuncatula . The flativities ,23,24. Fmulation ,25.Phellodendron chinense Schneid (P. chinense Schneid) is a traditional woody medicinal plant in China that mainly contains alkaloid, flavonoid, and terpenoid compounds in stem bark [P. chinense Schneid determines the quality and benefit, and its biosynthesis and accumulation are affected by exogenous GA3. In a previous study, we demonstrated that exogenous GA3  treatment promotes the growth of P. chinense Schneid seedlings, and the promotion of 100 mg L−1 concentration was better compared with the control group (CK) , and differentially accumulated metabolites (DAMs) in leaves and stem bark and revealed the molecular mechanism of exogenous GA3 regulating the growth and flavonoid synthesis in P. chinense Schneid. These findings may help to provide a theoretical basis for the scientific planting and quality improvement of P. chinense Schneid.tem bark . These atem bark ,28,29. Toup (CK) . However3 led to significant enhancements in both plant height and ground diameter of P. chinense Schneid seedlings, showing an increase of approximately 1.19- and 1.25-fold, respectively, compared to the control group (CK)  a,b. Undefference c,d. Thes3 significantly increased the contents of chlorophyll a (Chl a), chlorophyll b (Chl b), and total chlorophyll (Chl) in leaves, with 1.72-, 2.20-, and 1.57-fold, while it observably decreased the carotenoid content in leaves, with 0.49-fold compared with that in CK group  and peroxidase (POD) in leaves, with 1.06-fold and 1.36-fold, whereas it remarkably reduced the catalase (CAT) activity and malondialdehyde (MDA) content in leaves, with 0.70-fold and 0.41-fold  were obtained, the G-C contents ranged from 43.41% to 44.68%, and Q30 ranged from 92.25% to 93.29% (−5) alignment in eight public databases . A total of 13,574 (5.88%), 31,747 (13.75%), 17,745 (7.69%), 25,104 (10.88%), 26,407 (11.44%), 27,444 (11.89%), 41,511 (17.98%), and 47,307 (20.49%) unigenes were annotated in the COG, GO, KEGG, KOG, Pfam, Swissprot, eggNOG, and Nr databases  ”, “genetic information processing (20)”, and “environmental information processing (14)” were the primary terms, the “Plant hormone signal transduction (six upregulated and seven downregulated)”, “carbohydrate metabolism (five upregulated and seven downregulated)”, and “amino acid biosynthesis (five upregulated and three downregulated)” were the main pathways. In the plant hormone signal pathways, the GA signal pathway had five DEGs (two upregulated and three downregulated), the IAA signal pathway contained four DEGs (two upregulated and two downregulated), the CTK signal pathway included one upregulated DEGs, the ABA signal pathway had two DEGs (one upregulated and one downregulated). Furthermore, the photosynthetic system contained four DEGs (three upregulated and one downregulated), and the carbon metabolism had nine DEGs (four upregulated and five downregulated)  showed that samples of GAanalysis a. The DEnge ≥ 2) b. GO enrnge ≥ 2) c. Furthegulated) d. 3 treatment were identified, among which the Pc(S)-Psb27 , Pc(S)-PsaD  and Pc(S)-PsaN  unigenes were upregulated, and the Pc(S)-Lhcb3  unigene was downregulated -CER3 , Pc(S)-PGK  and Pc(S)-FBA  unigenes were upregulated, whereas the Pc(S)-FBA1 (c63247.graph_c0) and Pc(S)-GAPDH  unigenes were downregulated. In the sugar metabolism pathway, the Pc(S)-GUS17/40  and Pc(S)-GP2  unigenes were downregulated, whereas the Pc(S)-EG8  unigene was upregulated -KAO (c65280.graph_c0), Pc(S)-GID1B/C , and Pc(S)-GID2 (c56133.graph_c0) unigenes were downregulated, whereas the Pc(S)-GA2ox (c56918.graph_c0) and Pc(S)-DELLA (c24239.graph_c0) unigenes were upregulated compared with that in CK group, indicating that exogenous GA3 inhibited biosynthesis and signal transduction of GA hormone in leaves of P. chinense Schneid seedlings (Pc(S)-SAUR50 (c55986.graph_c1) and Pc(S)-GH3.10 (c43034.graph_c0) unigenes were upregulated, while the Pc(S)-SAUR32 (c24304.graph_c0) and Pc(S)-GH3.6 (c69394.graph_c0) unigenes were downregulated, demonstrating that exogenous GA3 played a key role in IAA signal transduction  in GAeedlings . Under tsduction .3 treatment were identified, among which the Pc(S)-PYR/PYL (c60619.graph_c0) unigene was downregulated, and the Pc(S)-PP2C (c65623.graph_c0) unigene was upregulated compared with that in CK group, showing that exogenous GA3 inhibited ABA signal pathway in leaves of P. chinense Schneid seedlings. Moreover, the Pc(S)-A-ARR (c36315.graph_c0) involved in CTK signal pathway was upregulated in GA3 treatment sample, indicating that exogenous GA3 regulated CK signal pathway in the leaves of P. chinense Schneid seedlings  in leaves under GAeedlings .3 treatment samples were distinguishable by 2D PCA analysis. In the PCA score plot, two principal components (PC1 and PC2) were extracted to be 31.45% and 23.54%, and the three biological replicates of GA3 treatment group were significantly clustered, which obviously separated from CK group, indicating that the GA3 had an important impact on the metabolites in the stem bark of P. chinense Schneid seedlings , phenolic acid (62), lipid (59), amino acid and derivative (46), alkaloid (45), organic acid (31), nucleotide and derivative (30), and lignan and coumarin (20) (The differences in the metabolites in stem bark from CK and GAeedlings . Meanwhieedlings . In thisrin (20) .3 treatment group were clustered, which distinctly separated from CK group, demonstrating that this experiment was reproducible and reliable, and GA3 had an important effect on the DAMs in stem bark of P. chinense Schneid seedlings  were annotated in KEGG database, which primarily included flavonoid (1 upregulated and 30 downregulated), phenolic acid (one upregulated and six downregulated), alkaloid (six downregulated), lignan and coumarin (one upregulated and two downregulated), amino acid and derivative (three upregulated), nucleotide and derivative (two downregulated), terpenoid (one upregulated), and lipid (one upregulated)  showed that two principal components  were extracted to be 23.3% and 23.5%, and the three biological replicates of GAeedlings a. Based gulated) b–d. Accoeedlings .3 inhibited the flavonoid biosynthesis in stem bark of P. chinense Schneid seedlings (The DAMs obtained from stem bark metabolites mainly enriched metabolic pathways (10), flavonoid biosynthesis (9), biosynthesis of secondary metabolites (9), flavone and flavonoid biosynthesis (5), and phenylpropanoid biosynthesis (3). In the flavonoid biosynthesis pathway, a total of 9 DAMs were identified, among which the pinocembrin (pmp000562), isosakuranetin (mws1034), naringin (mws0046), naringenin (pme0376), pinobanksin (mws0914), tricetin (pmb0713), luteolin (pmb0566), vitexin (mws0048), and (−)-epicatechin (pme0460) were all downregulated, indicating that exogenous GAeedlings . 3 plays crucial roles in the processes of growth, seed germination, physicochemical properties, nutrient uptake, secondary metabolism, and stress response in plants [3 promoted the growth of Neolamarckia cadamba and Camellia sinensis seedlings and increased the biomass [3 enhanced the chlorophyll content but reduced carotenoid content in the leaves of Valencia oranges, suggesting that GA3 promoted growth by improving chlorophyll content in plants [3 application significantly improved chlorophyll content, increased plant height, ground diameter, and fresh weight, whereas it decreased carotenoid content in leaves, indicating that GA3 promoted growth through improving chlorophyll content in the leaves of P. chinense Schneid seedlings. The results were consistent with the findings of Valencia oranges and Camellia sinensis seedlings [3 treatment, the increase in chlorophyll content in leaves may facilitate light absorption and carbon assimilation for improving photosynthetic efficiency, ultimately promoting the growth of P. chinense Schneid seedlings [GAn plants ,31. The  biomass ,32. At tn plants . In the eedlings . Under Geedlings .3 treatment significantly increased the contents of soluble sugar and soluble protein, concurrently enhanced the activities of SOD and POD, but distinctly reduced MDA content and CAT activity in leaves, demonstrating that GA3 promoted the accumulation of soluble substance and improved antioxidant capacity of P. chinense Schneid seedlings, and were similar to the previous findings [3 enhances the antioxidase activity, which keeps the integrity of chloroplast structure for improving the photosynthetic efficiency in leaves, and then promotes the biosynthesis and accumulation of soluble sugar and soluble protein, providing precursor materials and energies for anabolism during the growth process. These results demonstrated that GA3 promoted the growth of P. chinense Schneid seedlings by improving chlorophyll content and antioxidant capacity.At the same time, GAfindings ,35. The 3 in plants [3 elevated the relative expression abundances of Pt-TUA6 and Pt-DRP4C genes, encoding important components of the chlorophyll division ring in leaves, concurrently improving the photosynthesis capacity and biomass of Populus tomentosa seedlings [3 treatment significantly enhanced the relative expression levels of Pc(S)-Psb27 and Pc(S)-PsaD/N unigenes in leaves, suggesting that exogenous GA3 promoted the growth through improving relative expression levels of the photosynthetic genes in leaves of P. chinense Schneid seedlings, these results were consistent with the chlorophyll contents and fresh weights. The Psb27 protein is a lumenal protein and auxiliary factor associated with PSII, which binds to the large extrinsic E-loop of CP43 at the lumenal side, maintains the stability of the D1 protein structure in PSII reaction center, and then improves the photosynthetic capacity and promotes plant growth [+, the latter combines with PsaG/F polypeptides to form trimer that regulating the plastocyanin function, subsequently improving photosynthetic efficiency and biomass in plants [3 promoted growth by improving the relative expression levels of key genes in photosynthesis in the leaves of P. chinense Schneid seedlings.The photosynthesis apparatus consists of two key components: photosystem I (PSI) and photosystem II (PSII) which are multisubunit pigment-protein complexes that catalyze the light-driven water oxidation, electron transport, energy conversion, and state transition, but these processes are controlled by genes and regulated by exogenous GAn plants ,37,38,39eedlings . In the t growth ,41,42. Tn plants ,45,46. T3 [3 decreased the relative expression levels of Nc-GA20ox and Nc-GA3ox genes but increased the expression abundances of key genes in the auxin pathway, thereby promoting the growth of Neolamarckia cadamba seedlings [3 evidently reduced the relative expression levels of Pc(S)-KAO, Pc(S)-GA2ox, and Pc(S)-GID1/2 unigenes, while notably increasing the transcript level of Pc(S)-DELLA unigene in leaves of P. chinense Schneid seedlings. The Pc(S)-KAO unigene participates in GA biosynthesis. The Pc(S)-GA2ox unigene is involved in the conversion of its inactive form, indicating that GA3 prevented GA biosynthesis and promoted its degradation in leaves of P. chinense Schneid seedlings [3 that combined with DELLA protein to form a trimer, subsequently labeled by SCFSLY1/GID2 complex and degraded through 26S protease, releasing the repression of DELLA protein and promoting cell elongation in plants [Pc(S)-GID1/2 (downregulation) and Pc(S)-DELLA (upregulation) unigenes demonstrated that exogenous GA3 inhibited the GA signal transduction in leaves of P. chinense Schneid seedlings, speculating that GA3 may promote its growth by other phytohormones. Plant growth is a sophisticated process regulated by endogenous phytohormones, such as auxin, GA, ABA, and CTK, where biosynthesis is controlled by the exogenous GA3 ,48,49. Teedlings . In the eedlings . The GIDn plants ,52. Thus3 treatment reduced the relative expression levels of Pc(S)-GH3.6 and Pc(S)-SAUR32 unigenes but elevated the transcript levels of Pc(S)-GH3.10 and Pc(S)-SAUR50 unigenes in auxin signal pathway in leaves of P. chinense Schneid seedlings. The GH3 acyl acid amido synthetase catalyzed the conjugation of auxin with amino acid that inhibited cell division and expansion and played a positive role in the processes of JA synthesis and its signal pathway, which, in turn, maintained the phytohormone homeostasis and regulated the growth and development in plants [Pc(S)-GH3.6 unigene facilitated the accumulation of free auxin in leaves, combined with Pc(S)-GH3.10 unigene to maintain its homeostasis and promoted the growth of P. chinense Schneid seedlings. In the auxin signal pathway, the SAUR32 as a response factor negatively influenced regulation during plant growth and development, but the SAUR50 was responsible for hypocotyl elongation, cotyledon expansion, and leaf development by activating plasma membrane H+-ATPase, resulting in cell wall acidification in plants [Pc(S)-SAUR32 unigene alleviated the inhibition of cell division and elongation, while the upregulation of Pc(S)-SAUR50 unigene promoted the growth and development by improving cell wall acidification, demonstrating that GA3 promoted the growth of P. chinense Schneid seedlings by regulating key gene expressions of auxin in its synthesis and signal pathway. These results were consistent with the findings of Neolamarckia cadamba seedlings [3 reduced the relative expression levels of Pc(S)-PYR/PYL unigene, whereas it increased the transcript levels of Pc(S)-PP2C and Pc(S)-A-ARR unigenes that played a negative role in CTK and ABA pathways, illuminated that GA3 repressed these signal pathways in leaves of P. chinense Schneid seedlings [3 promoted growth through regulating phytohormone homeostasis and auxin signal pathway in leaves of P. chinense Schneid seedlings.In addition, GAn plants ,54,55. Tn plants ,57,58,59eedlings ,61,62. T3 not only promotes growth, but also influences the biosynthesis and accumulation of secondary metabolites in plants [3 significantly increased the contents of total anthocyanin and artemisinin in Vitis vinifera (Syrah grapevine) and Artemisia annua, but decreased the flavonoid content in Medicago truncatula, showing that the GA3 had a distinct effect on secondary metabolites in different plants [3 significantly reduced the flavonoid contents in stem bark, including pinocembrin, isosakuranetin, naringin, naringenin, pinobanksin, tricetin, luteolin, vitexin, and (−)-epicatechin, indicating that GA3 blocked the flavonoid biosynthesis of P. chinense Schneid seedlings. These results were consistent with the findings of Medicago truncatula [3, but GA3 usually plays a negative role in these processes [PsaD, Psb27, PsaN, DELLA, GA2ox, SAUR50, GH3.10, A-ARR, FBA, CER3, PGK, and EG8 genes showed a significant positive correlation with the growth of P. chinense Schneid seedlings. However, naringin, pinobanksin, naringenin, and pinocembrin showed a significant negative correlation with the growth of P. chinense Schneid seedlings. Thus, our results suggested that GA3 decreased flavonoid content in stem bark by repressing the expression levels of key genes associated with biosynthesis, which also illuminated a competitive inhibition between the growth and flavonoid biosynthesis of P. chinense Schneid seedlings.GAn plants ,63,64. At plants ,64,65. Iuncatula . In planrocesses . Moreove3 regulating the growth and flavonoid biosynthesis of P. chinense Schneid seedlings (3 enhanced the antioxidase (SOD and POD) activity and chlorophyll content that independently or cooperatively regulated key unigenes expression for improving photosynthetic capacity and carbon metabolism, subsequently promoting the growth of P. chinense Schneid seedlings. At the same time, GA3 mainly regulated the unigene expression in the signal pathways of GA, auxin, and ABA that synergistically promoted plant growth. On the other hand, exogenous GA3 inhibited flavonoid biosynthesis to alleviate the interference effect on the growth of P. chinense Schneid seedlings. However, the molecular mechanism of exogenous GA3 promoting growth through inhibiting flavonoid biosynthesis will require further investigation. Based on the obtained results, a conceptual model was developed to describe the mechanism of exogenous GAeedlings . BrieflyP. chinense Schneid were collected from Xiangxi Autonomous Prefector (Hunan Province), then seeds were separated and dried at room temperature. The seeds were placed in seedling discs containing peat substrate  for germination and transferred to an experimental field  when the seedling height was 20 cm. When the height reached 30 cm, these seedlings were sprayed using a 100 mg L−1 GA3 solution three times at 5:00~5:30 p.m. on 0, 10, and 20 days, and the distilled water treatment served as a control group (CK) [3 treatment, among which three seedlings were used for transcriptomic and metabolomic analysis, and the remaining seedlings were frozen in liquid nitrogen and stored at −80 °C for physicochemical property analysis. The study was conducted in a greenhouse at Central South University of Forestry and Technology, Changsha City, Hunan Province, China . The mellow fruits of oup (CK) . Each exn = 3).The plant heights of collected seedlings were measured using a tape measure. The ground diameters were measured using a sliding caliper. The aboveground tissue and root of the collected seedlings were separated with scissors and then determined the aboveground fresh weights and the stem fresh weights which removed leaves from aboveground tissues with an analytical balance. Each experiment was repeated three times , and the pigment contents (mg g−1) in leaves of P. chinense Schneid seedlings were calculated. Each experiment was repeated three times (n = 3).The pigment contents in the leaves of collected seedlings were measured according to the published literature . Briefly−1 anthrone sulfate solution) was reacted at 100 °C for 10 min. The soluble sugar content was measured at 625 nm after cooling to room temperature. Each experiment was repeated three times.The soluble sugar contents in the leaves of collected seedlings were measured according to the published literature . Briefly−1 phosphate buffer (pH 7.8), the grinding fluid was moved into a 10 mL centrifuge tube, centrifuged at 4000 rpm for 10 min, and the supernatant was collected. Next 1 mL of extract and 5 mL of Coomassie brilliant blue G250 solution were mixed and left to rest for 2 min. Soluble protein content was measured at 595 nm. Each experiment was repeated three times.Fresh leaves (0.5 g) were homogenized in a mortar containing 5 mL of 0.05 mol L2O2, and enzyme extract. The specific activity of the enzyme was expressed in terms of micromolar H2O2 in min per mg of soluble protein. Peroxidase (POD) activity was determined following the published literature [2O2 was read at 420 nm with a 30 s interval up to 2 min and used the absorbance change 0.01 as a POD activity. Each experiment was repeated three times (n = 3).The fresh leaves (0.2 g) were milled into pulp in precooled mortar containing precooled 5 mL 0.05 M phosphate buffer (harbored 0.2 M disodium hydrogen phosphate and 0.2 M sodium dihydrogen phosphate), then the pulp was transferred into a centrifuge tube and centrifuged at 4 °C, 11,000 rpm for 20 min, subsequently, the supernatant was collected for measuring the antioxidase activity. The activities of superoxide dismutase (SOD) and catalase (CAT) in leaves were determined according to the published literature . The SODterature . The abs3 treatment group were used to extract total RNA with an RNAprep Pure plant kit . The total RNA of each sample was quantified and qualified via Bioanalyzer  and spectrophotometer . Thereafter, a total of 3 μg RNA from each sample was used as the input material for RNA sample preparation. Sequencing libraries were generated using the RNA-seq library Prep Kit for Illumina . The generated sequencing libraries were qualified using a Qsep400 analyzer , then the eligible libraries were sequenced on Illumina platform , and paired-end reads were generated. Subsequently, clean data (clean reads) were obtained by removing reads containing adapters, reads including poly-N sequences, and low-quality reads  from raw data. Furthermore, the Q20, Q30, G-C contents and sequence duplication levels were calculated from the clean data. All downstream analyses were based on high-quality, clean data. Three independent biological replicates of leaves in CK and GA3 treatments was performed using the DESeq2 software. The obtained p-value was adjusted using Benjamini–Hochberg method [The clean reads were de novo assembled using Trinity software . Gene functions were annotated based on the following databases: Clusters of Orthologous Groups (COG), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), EuKaryotic Orthologous Groups (KOG), Protein family (Pfam), Swiss-Prot , evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG), and National Center for Biotechnology Information Non-Redundant protein sequences (Nr). Different expression analysis between CK and GAg method  to contrThe GO term annotation of the DEGs was produced according to biological processes (BPs), cellular components (CCs), and molecular functions (MFs) through Blast2GO software . The KEGG annotation of DEGs was conducted according to cellular processes, environmental information processing, genetic information processing, and metabolism and organismal systems. KOBAS software  was used to test the statistical enrichment of the DEGs in KEGG pathways. Each sample was placed in a lyophilizer  for vacuum freeze-drying and ground into powder  using a grinder . Weigh 50 mg powder of each sample, add 1200 μL of 70% methanol water internal standard extract precooled at −20 °C, and vortex every 30 min for 30 s 6 times. Finally, after centrifugation at 12,000 rpm for 3 min, the supernatant was absorbed, and the sample was filtered with a microporous filter (0.22 μm pore size) and stored in an injection vial for UPLC-MS/MS analysis.−1; Column temperature is 40 °C; The injection volume is 2 μL.The data acquisition instrument system mainly includes Ultra Performance Liquid Chromatography (UPLC)  and Tandem mass spectrometry (MS/MS) . Liquid phase conditions mainly include: the chromatographic column: Agilent SB-C18 ; Mobile phase: Phase A is ultrapure water containing 0.1% formic acid, Phase B is a solution of acetonitrile containing 0.1% formic acid; Elution gradient: 0.00 min B phase ratio of 5%, B phase proportion increased linearly to 95% within 9.00 min, and was maintained at 95% of 1 min, 10.00–11.10 min, Phase B ratio decreased to 5%, and balanced at 5% to 14 min; Flow rate: 0.35 mL min2) to medium. The decluster voltages  of each MRM ion pair were accomplished by optimizing the DP and CE. A specific set of MRM ion pairs was monitored during each period based on the metabolites eluted in each period.The MS conditions mainly include: electrospray ion source  temperature 550°C; ion spray voltage (IS) 5500 V (positive ion mode)/−4500 V (negative ion mode); ion source gas I (GSI), gas II (GSII), and air curtain gas (CUR) were set to 50, 60, and 25 psi, respectively, and collision-induced ionization parameters were set to high. The QQQ scan uses the MRM  mode and sets the collision gas  with 95% and a post hoc Tukey’s test. Pearson correlation analysis and the t-test were performed to integrate and visualize transcriptomics and metabonomics data sets. These graphs and random forest analysis were conducted using the R package “random Forest ”  and Origin software . Finally, DEGs and DAMs were annotated in the KEGG database for the enrichment of metabolic pathways and the mapping of related pathways.The data were analyzed using SPSS software  with a significant difference level. The significant differences in growth index, photosynthetic pigment content, soluble substance content, antioxidase activity, and MDA content in the leaves of 3 significantly enhanced the chlorophyll content, fresh weight, soluble substance content, SOD and POD activities, evidently reducing the carotenoid content and CAT activity, indicating that GA3 promoted the growth of P. chinense Schneid seedlings by improving the chlorophyll content and some antioxidase activities. At the same time, exogenous GA3 elevated the relative expression levels of Pc(S)-Psb27, Pc(S)-PsaD/N, Pc(S)-DELLA, Pc(S)-GH3.10, Pc(S)-SAUR50, Pc(S)-PP2C, and Pc(S)-A-ARR unigenes, concurrently reduced the relative expression abundances of Pc(S)-KAO, Pc(S)-GA2ox, Pc(S)-GID1/2, Pc(S)-GH3.6, Pc(S)-SAUR32, and Pc(S)-PYR/PYL unigenes in leaves, revealed that GA3 promoted the growth of P. chinense Schneid seedlings through improving the relative expression levels of key unigenes in photosynthetic system and GA and auxin signal pathways. Furthermore, exogenous GA3 observably decreased the flavonoid content in stem barks, demonstrating that GA3 inhibited the biosynthesis and accumulation of flavonoids in P. chinense Schneid seedlings. Our results revealed that exogenous GA3 promoted the growth and inhibited flavonoid synthesis in P. chinense Schneid seedlings by regulating the relative expression levels of unigenes in photosynthesis and phytohormone signal pathways.Exogenous GA