Siegesbeckiae herba (SH) with a cold nature exerts therapeutic effects including dispelling wind-dampness, benefiting joints. This study aims to explore the effects of SH and its fractionated components on neuroendocrine metabolism functions in heat syndrome rats and to clarify the property attribution of each component. The main components of SH were isolated via modern separation techniques. A heat syndrome rat model was established by administration of levothyroxine sodium. Metabolomics was employed to screen differential expressed metabolites (DEMs) and metabolic pathways, and indicators related to neuroendocrine metabolism were determined. A total of 107 DEMs involving 17 pathways were screened out. It was found that SH and its fractions could inhibit the abnormally activated metabolic pathways in hot syndrome rats. Among them, the terpenoids fraction showed the optimal regulatory effect on neurotransmitter imbalance, excessive activation of the hypothalamic-pituitary-thyroid axis, and energy/substance metabolism disorders and was confirmed as the main material basis for the cold nature of SH. This study demonstrates that SH and its fractionated components exert therapeutic effects on heat syndromes by regulating the neuroendocrine energy metabolism and related pathways. This provides scientific support for the clinical syndrome differentiation application and further development of SH.

The present study aimed to evaluate the therapeutic potential of the Unani formulation Ark-e-Kasni in alleviating the symptoms of polycystic ovary syndrome (PCOS). In the present study, letrozole and a high-fat diet (HFD) were used to induce PCOS-like symptoms in rats. After induction, different groups were treated with varying doses of Ark-e-Kasni (5, 10, and 20 mL/kg), metformin, and finasteride. At the end of treatment, blood and ovaries were collected for biochemical and histological analysis. A significant weight gain and prolonged diestrous phase were observed in PCOS rats, accompanied by a considerable elevation in LH (42.52 ± 0.90 mIU/mL) and testosterone (8370 ± 122.18 pg/mL), and a low level of FSH (11.86 ± 0.43 mIU/mL). Moreover, an increase in insulin (14.31 ± 0.35 mIU/mL), TNF-α (358.81 ± 9.81 pg/mL), IL-1β (143.74 ± 3.39 pg/mL), and IL-6 (154.04 ± 2.04 pg/mL), and a reduction in SOD, CAT, and GSH were noticed. While the Ark-e-Kasni restored the levels of LH (30.97 ± 1.04 mIU/mL), testosterone (5651.83 ± 182.69 pg/mL), FSH (15.66 ± 0.56 mIU/mL), insulin (5.48 ± 0.23 mIU/mL), TNF-α (212.16 ± 8.66 pg/mL), IL-1β (78.79 ± 1.46 pg/mL), and IL-6 (74.53 ± 1.60 pg/mL), and lipid markers. Histological and microscopic analysis showed reduced cystic follicles and enhanced corpus luteum formation, improving ovarian morphology. The findings suggest that Ark-e-Kasni ameliorates PCOS through diverse molecular mechanisms. Ark-e-Kasni may modulate steroidogenic enzyme activity to reduce hyperandrogenism, enhance insulin sensitivity through the PI3K/Akt pathway, and inhibit the downstream inflammatory pathway of NF-κB.

To observe the effect of warming needle moxibustion (WNM) on the expression of AMP-activated protein kinase (AMPK)/Unc-51 like autophagy activating kinase 1 (ULK1) signaling pathway in chronic fatigue syndrome (CFS) rats, so as to explore its mechanism underlying improvement of CFS. Male SD rats were randomly divided into control, model, WNM and coenzyme groups, with 10 rats in each group. The CFS model was established by multi-factor compound stress stimulation method (exhaustive swimming + chronic restraint + alternate-day fasting). The rats of the WNM group received warming needle moxibustion stimulation at bilateral "Zusanli" (ST36), "Guanyuan" (CV4) and "Zhongwan" (CV12)for 15 min, once daily for 14 days. The rats of the coenzyme group were administered coenzyme Q10 (1 mg/kg) by gavage once daily for 14 days. Body weight and swimming time of the rats were recorded before and after modeling and after intervention. Behavioral changes were assessed using the open-field test. Histological changes in skeletal muscle were observed via HE staining. The structure of mitochondria and autophagosomes in skeletal muscle were observed using transmission electron microscopy. The protein expression levels of AMPK, phosphorylated (p)-AMPK, ULK1, p-ULK1, microtubule-associated protein 1 light chain 3 (LC3)-Ⅰ and LC3-Ⅱ in skeletal muscle were detected by Western blot, and the ratios of p-AMPK/AMPK, p-ULK1/ULK1 and LC3-Ⅱ/LC3-Ⅰ were calculated. The mRNA expression levels of AMPK, ULK1 and LC3 in skeletal muscle were detected by real-time PCR. Compared with the control group, the body weight, swimming time, and the duration of standing and the number of grid crossing were significantly reduced (P<0.05), while the mRNA expression levels of ULK1 and LC3, and the ratio of p-ULK1/ULK1 were significantly increased in the model group (P<0.05). Compared with the model group, the body weight, swimming time, and the duration of standing and the number of grid crossing were significantly increased (P<0.05), and the mRNA expression levels of AMPK, ULK1 and LC3, as well as the ratios of p-ULK1/ULK1 and LC3-Ⅱ/LC3-Ⅰ, were significantly up-regulated in both the WNM and coenzyme groups (P<0.05). Warming needle moxibustion can alleviate chronic fatigue syndrome by activating the AMPK/ULK1 pathway, upregulating the expression of AMPK/ULK1/LC3, and promoting mitochondrial autophagy, thereby enhancing mitochondrial morphology.

Polycystic ovary syndrome (PCOS) has been associated with conflicting effects on bone mass, underscoring the need for deeper investigation into its impact on skeletal health. This study aimed to assess the expression of osteoporosis-related genes in femoral bone, alongside hormonal profile (bone-related hormones) alterations, across aging in a rat model of PCOS compared to controls. Femoral bone RNA was extracted from rat model of PCOS and controls (n = 10-13 per group) at 3, 10, and 18months of age. The expression of IL-11, DKK1, RANKL, AKT1, IGF-1, EphB4, STAT3, and CTNNB1 genes was quantified via Real-Time PCR. Concurrently, serum levels of Anti-Mullerian Hormone (AMH), calcitonin, cortisol, total testosterone (TT), and vitamin D3 were measured using ELISA. A significantly elevated expression of IL-11, DKK1, RANKL, AKT1, and IGF-1 genes, accompanied by a decreased expression of EphB4 and STAT3 in the femoral bone of rat model of PCOS compared to controls, was observed. Additionally, rat model of PCOS exhibited higher serum levels of AMH, cortisol, and TT, whereas calcitonin and vitamin D3 levels were decreased. These molecular and hormonal alterations highlight a dysregulation in bone metabolism associated with PCOS and suggest that PCOS may represent a substantial risk factor for osteoporosis later in life. The employed rat model thus offers a valuable platform for elucidating the cellular and molecular mechanisms contributing to bone mass disorders in PCOS, facilitating the development of targeted therapeutic strategies.

Polycystic Ovary Syndrome (PCOS) is a condition marked by hormonal imbalances, metabolic dysfunctions & genetic predispositions; presenting with symptoms such as anovulation, hyperandrogenism, irregular menstruation, hirsutism, acne, alopecia and infertility. This study assessed the therapeutic effects of date palm pollen (DPP) extract on a rat PCOS model. Twenty-four prepubertal female rats were divided into four groups: control, received saline; PCOS, was given letrozole (1mg/kg/day for 21 days) to induce PCOS; DPP, received extract (200mg/kg/day for 21 days)& Group 4 was first induced with letrozole, followed by DPP extract treatment. Polycystic Ovary Syndrome induction resulted a significant decline in follicle-stimulating hormone (FSH), progesterone& estrogen (E2) levels, with a concurrent increase in luteinizing hormone (LH), testosterone& insulin levels. Date palm pollen treatment restored hormonal balance, increasing FSH, progesterone, and E2 while decreasing LH, testosterone, and insulin. Polycystic Ovary Syndrome was also associated with oxidative stress, evidenced by lower catalase (CAT) activity and glutathione (GSH) levels, and higher malondialdehyde (MDA) levels, which were improved by DPP treatment. Apoptosis markers (caspase-3 and annexin V) and inflammatory mediators (TNF-alpha and IL-6) were elevated in PCOS but were reduced with DPP treatment. Histologically, DPP-treated PCOS rats exhibited fewer cystic follicles, a higher proportion of healthy follicle types, and improved corpus luteum counts. Additionally, the DPP-treated females showed higher significantly pregnancy rate than the untreated PCOS rats. In conclusion DPP extract alleviates the hormonal and tissue abnormalities in PCOS, offering potential as a therapeutic agent for managing PCOS symptoms and improving fertility outcomes.

The paper describes the results of studying the effect of an aqueous solution of a chelated complex of micronutrients for intramuscular injection. Chronic radiation syndrome of moderate severity was simulated in rats using fractionated total irradiation according to the following scheme: 2 times a week with an exposure of 4 minutes at a power of 0.1 Gy/min for eight weeks, with a total dose of 6.4 Gy. Three groups of animals were formed during the experiment: Group 1 consisted of intact animals, Group 2 consisted of control animals, and Group 3 consisted of experimental animals with chronic radiation syndrome. Intramuscular administration of an aqueous solution of a chelated complex of microelements at a dose of 0.1 ml/100 g had a positive effect on the dynamics of body weight gain, led to an increase in immune homeostasis, while stabilizing the leukocyte count in the blood of the experimental animals, and also stimulated bone marrow hematopoiesis and regulated the functional activity of platelets.

This study investigated how aerobic exercise improves metabolic disorders in rats with metabolic syndrome (MS) and explored the role of miR-27a in this process. MS was induced by a high-fat and high-sugar diet. After eight weeks of treadmill training, aerobic exercise was found to reduce metabolic abnormalities and decrease elevated miR-27a levels, while increasing the expression of PPARγ and key downstream molecules involved in glycolipid metabolism. Downregulating miR-27a via a viral vector produced benefits similar to those of exercise, and combining miR-27a inhibition with exercise led to further improvement. These results suggested that aerobic exercise alleviates MS-related metabolic disorders partly through suppressing miR-27a and promoting PPARγ signaling, revealing a potential therapeutic target.

This study investigated how aerobic exercise improves metabolic disorders in rats with metabolic syndrome (MS) and explored the role of miR-27a in this process. MS was induced by a high-fat and high-sugar diet. After eight weeks of treadmill training, aerobic exercise was found to reduce metabolic abnormalities and decrease elevated miR-27a levels, while increasing the expression of PPARγ and key downstream molecules involved in glycolipid metabolism. Downregulating miR-27a via a viral vector produced benefits similar to those of exercise, and combining miR-27a inhibition with exercise led to further improvement. These results suggested that aerobic exercise alleviates MS-related metabolic disorders partly through suppressing miR-27a and promoting PPARγ signaling, revealing a potential therapeutic target.

The article "Preclinical study of vitamin D deficiency in the pathogenesis of metabolic syndrome in rats" by S.K. Mahjoub, M.A.A. Sattar Ahmad, F.O. Kamel, M. Alseini, L.M. Khan published in Eur Rev Med Pharmacol Sci 2022; 26 (23): 9001-9014-DOI: 10.26355/eurrev_202212_30575-PMID: 36524519 has been retracted in accordance with the Publisher and the Editor in Chief. Following the publication of the above-mentioned article, concerns were raised by a whistleblower and on PubPeer regarding multiple concerns affecting the reliability of the reported data. The journal contacted the corresponding author on multiple occasions and formally requested clarification and access to the original raw data supporting the published results. Despite repeated attempts, no response or supporting documentation was received. In accordance with COPE guidance, the journal conducted an independent assessment of the published material. This evaluation identified major methodological and analytical deficiencies and confirmed the concerns raised. On the basis of the available information, the reliability of the findings could not be verified and the conclusions of the study cannot be confirmed. Therefore, the article is retracted. The authors have been informed of this decision and have not responded. This article has been retracted. The Publisher apologizes for any further inconvenience this may cause.

Background: Aberrant protein glycosylation mediated by the UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase (GALNT) family has been implicated in the pathogenesis of metabolic syndrome (MetS), yet the roles of GALNT2 and GALNT3 remain poorly characterized. Objective: This study aimed to evaluate the modulatory effects of decaffeinated green tea and green coffee on GALNT2 and GALNT3 mRNA expression in MetS rats, compared with standard therapies, metformin and empagliflozin. Methods: Twenty-five male Sprague Dawley (SD) rats were divided into five groups randomly: (1) MetS rats (METS), (2) MetS rats treated with 300 mg/kgBW decaffeinated green tea and 200 mg/kgBW decaffeinated green coffee (TKD), (3) MetS rats treated with 500 mg/kgBW metformin (MFN), (4) MetS rats treated with 30 mg/kgBW empagliflozin (EMPA), and (5) negative control (NC). The MetS model was induced using a high-fat, high-sucrose (HFHS) diet followed by streptozotocin injection (30 mg/kgBW). After 10 weeks, the treatment groups received interventions for 9 weeks. GALNT2 and GALNT3 mRNA expression was investigated using reverse transcription polymerase chain reaction (RT-PCR).. Result: TKD and MFN tended to decrease GALNT2 and increase GALNT3 mRNA expression, although the differences were not significant statistically compared to the METS group (p&gt;0.05). EMPA decreased GALNT2 and increased GALNT3 mRNA expression significantly compared to the METS group (p&lt;0.05). Conclusion: The significant effect of EMPA on GALNT2 and GALNT3 mRNA expression suggests its therapeutic potential in targeting glycosylation pathways in MetS. Although TKD and MFN exhibited similar trends with EMPA, the lack of statistical significance suggests the need for further study. Keyword : GALNT2; GALNT3; Green Coffee; Green Tea; Metabolic Syndrome.

Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disorder characterized by hyperandrogenism, ovulatory dysfunction, and the formation of ovarian cysts. Key contributors to its pathophysiology include oxidative stress, inflammation, and altered intracellular signaling, especially within the PI3K/pAKT/PTEN pathway. Galangin, a dietary flavonoid derived from Alpinia galanga, exhibits antioxidant, anti-inflammatory, and estrogen- modulatory properties. This study investigated the protective effects of galangin in a PCOS rat model induced by letrozole and explored its underlying molecular mechanisms. Thirty-six adult female Wistar rats were divided into six groups: control, galangin (8mg/kg), letrozole (1mg/kg), letrozole + galangin (4 or 8mg/kg), and letrozole + metformin (20mg/kg). All treatments were administered orally for 21days. Serum hormones, oxidative stress biomarkers, inflammatory mediators, and key proteins in the PI3K/pAKT/PTEN pathway were assessed, along with histopathological and immunohistochemical analyses. Letrozole administration induced characteristic PCOS-like features, including cystic follicle formation, hormonal imblanaces, oxidative stress, inflammation, and suppression of PI3K/pAKT signaling, accompanied by an increase in PTEN levels. Galangin pretreatment improved ovarian morphology, restored hormonal balance, reduced oxidative and inflammatory responses, and reactivated PI3K/pAKT signaling while downregulating PTEN. These effects were comparable to those observed with metformin. Galangin provides multidimensional protection against letrozole-induced ovarian dysfunction by alleviating oxidative stress, inflammation, and dysregulation of the PI3K/pAKT/PTEN pathway. These findings support the potential of galangin as a safe, multitarget natural adjunct for managing PCOS.

This study investigated the therapeutic potential of aqueous fruit and seed extract of Momordica charantia L. (MC) as an alternative treatment for PCOS. Evaluations included ovarian histopathology, fasting blood sugar, lipid profiles, antioxidant markers and hormone levels. Female Wistar rats induced with PCOS via a single 4.5 mg/kg dose of estradiol valerate were treated with MC extracts at 500 and 1000 mg/kg/day for 60 days. The results showed that treatment with MC fruit and seed extract at the dose of 500 and 1000 mg/kg/day possesses potent anti-inflammatory and antioxidant activity in in-vitro models. MC fruit and seed extract showed no cytotoxicity in HeLa cell viability in the MTT assay and the highest antioxidant activity followed by DPPH compared with ascorbic acid (p < 0.01). The fasting blood sugar serum levels, total lipid profile, LH, FSH, estradiol and antioxidant enzyme levels were significantly restored after 60 days of treatment (p < 0.01). Further, MC extract significantly reduced body weight (p < 0.01) and ovarian weight (p < 0.05), restored the normal estrous cycle, resolved cysts in the ovaries and displayed positive effects on ovarian histopathology after 60 days of treatment. MC fruit and seed extracts is considered a potential treatment for PCOS in estradiol-induced female rats.

Background: Polycystic ovary syndrome (PCOS) is a metabolic condition that impacts females during their reproductive years. PCOS is marked by the presence of multiple cyst-like follicles on the ovaries, irregular or absent ovulation, and resistance to insulin. This research seeks to investigate the consequences of flaxseed extract on the ovary in the PCOS rat’s model by histological techniques and serum hormone level. Materials and Methods: Sixteen female albino rats (6–8 weeks old, estrous cycle in female rats repeats every 4–5 days) were assigned to four groups: the first group was the control, second group received 0.2 mg/kg body weight of letrozole by gavage, the third group was given 100 mg/kg body weight flaxseed extract intraperitoneally, and the mixed group received both letrozole and flaxseed daily for 21 days. The total period of the study was 2 months, including 15-20 days for preparation, 21 days for animal experimentation, and 20 days for histological and statistical analysis. Results: Histological examination of the ovary revealed letrozole-induced ovarian cysts and disruption of follicles. Flaxseed treatment significantly restored ovarian morphology, with a recovery of follicular development. Hormonal assay indicated that testosterone and AMH were elevated, while estrogen and progesterone were decreased in the letrozole group to other groups. Extraction of flaxseed supplementation restored hormonal equilibrium, lowering testosterone and increasing estrogen and progesterone.

Polycystic ovary syndrome (PCOS) accelerates ovarian follicular depletion through atresia. This study investigated the therapeutic potential of aqueous (AE) and hydroethanolic (HEE) extracts of Cymbopogon citratus in mitigating PCOS‐induced ovarian damage (specifically ferroptosis, apoptosis, and oxidative stress) in a rat model of PCOS. PCOS was induced by oral administration of letrozole (1 mg/kg/day for 21 days), followed by treatment with AE or HEE (100, 200, or 400 mg/kg/day for 20 days) with metformin (200 mg/kg) as a positive control. The results showed that HEE contains 5 times more alkaloids than AE. In experimental animals, C. citratus corrected ovarian damage caused by PCOS, compared to the disease control treated with distilled water. Indeed, C. citratus decreased serum testosterone levels [maximum decrease rate of 37.10% (p < 0.0001) obtained with HEE100] and increased estradiol levels [maximum increase rate of 56.15% (p < 0.0001) obtained with AE200]. An increase in serum ghrelin levels [maximum increase rate of 129.38% (p < 0.0001) obtained with AE400] and a decrease in leptin levels [maximum decrease rate of 43.85% (p < 0.0001) obtained with AE400] were observed. In the ovaries, C. citratus decreased Fe2+ [maximum decrease rate of 31.57% (p < 0.01) obtained with HEE200], caspase 3 [maximum decrease rate of 24.47% (p < 0.05) obtained with HEE400], oxidative stress markers [MDA: up to 23.28% decrease (p < 0.05) induced by AE100], and increased antioxidants [catalase: up to 29.99% increase (p < 0.05) induced by HEE100 and GSH: up to 42.37% increase (p < 0.01) induced by HEE200]. C. citratus also decreased cystic and atretic follicles and promoted follicle growth and ovulation. In conclusion, C. citratus extracts are capable of protecting the ovaries from the adverse effects of PCOS, primarily ferroptosis, and apoptosis of follicular cells. Of the two extracts, AE seems ideal due to its low alkaloid content.

White kidney bean extract improves letrozole-induced polycystic ovary syndrome in rats by regulating the Wnt signaling pathway.

Osteocytes function as central regulators of skeletal health by acting as mechanosensors that control bone remodelling mediated by osteoblasts and osteoclasts. Disrupted osteocyte function, often driven by oxidative stress and linked to ageing and osteoporosis, contributes to pathological bone remodelling. Tocotrienols (TTs), a family of vitamin E, are intensively investigated for their bone-protective effects, with mechanisms that involve reducing intracellular reactive oxygen species, enhancing antioxidant defences, and modulating signalling pathways of bone remodelling. Preliminary studies suggest that TTs exert protective and anabolic effects by influencing osteocytes, including shielding them from oxidative damage. In vivo models using ovariectomised or metabolic syndrome rats demonstrated that TT supplementation modulated key osteocyte-secreted factors, including sclerostin, dentin matrix protein 1, Dickkopf-related protein 1, fibroblast growth factor 23, and receptor activator of nuclear factor κB ligand. However, the current evidence is limited by the use of models that may not fully represent degenerative osteoporosis, restricted dose-dependent studies, and the challenge of real-time in vivo monitoring. This perspective summarises the reported effects of TTs on osteocytes' function and emphasises the critical need for future research to employ more representative animal models, advanced imaging techniques, and complex 3D co-culture or bone explant systems to accurately define the mechanism of action of TTs and their resulting functional outcomes on overall bone quality.

To evaluate the effects of chronic oral administration of commercial guava (Psidium guajava L.) leaf extract on biochemical markers and liver histology in a model of metabolic syndrome inrats. 35 Wistar rats were assigned to five independent groups (n=7 per group): the control group (CG), which received a diet with standard rodent food (Nutri-Cubos®) and water ad libitum; the metabolic syndrome group (MS), MS + P. guajava (Pg) low-dose (PgL; 100 mg/kg), MS + Pg medium-dose (PgM; 200 mg/kg), and MS + Pg high-dose (PgH; 300 mg/kg) groups. MS was induced with a rich diet of fat and carbohydrates, in addition to the intake of a 30 % sucrose solution ad libitum for 14weeks. From week 15 onward, guava leaf extract or vehicle was administered orally every 24 h for 30 consecutive days without changing the experimental diet. At the end of the treatment, biochemical tests, liver histopathological analysis and adipose tissue analysis were performed. The MS group presented levels above the reference ranges for biochemical tests, weight gain, waist circumference gain, arterial hypertension, atypical liver cells and aberrant appearance in adipose rat tissue. The PgM and PgH groups presented opposite effects of MS on biochemical parameters, such as increased blood pressure and liver damage. Chronic treatment with Pg leaf extract attenuated MS in Wistar rats, restoring the levels of biochemical parameters and exerting hepatoprotective effects. These findings suggest that guajava extract could be used in the development of therapeutic agents to improve the biochemical and histological alterations induced byMS.

The core bioactive components of Zhenwu Decoction (ZWD)—utilized for managing kidney diseases—and the associated anti-nephrotic syndrome (NS) edema mechanism remain incompletely characterized. We assessed the potential mechanism of ZWD against adriamycin (ADR)-induced NS. Sprague–Dawley rats (n = 6/group) received intravenous adriamycin (6 mg/kg) to establish NS models and were administered ZWD (4.2, 8.4, 16.8 g/kg/day) and prednisone (5 mg/kg/day) for 4 weeks. Renal function was assessed using the serum biomarkers: blood urea nitrogen (BUN), serum total protein (TP), creatinine (Scr), albumin (ALB), triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C). H&E and Masson’s trichrome staining were used to assess the pathological alterations and renal fibrosis status. Enzyme-linked immunosorbent assay was used to detect the serum levels of arginine vasopressin (AVP), renin (Ren), angiotensin II (AngII), and aldosterone (ALD). Immunohistochemistry, Western blot, and RT-qPCR were performed to examine the levels of relevant intracellular renal signaling molecules. ZWD treatment significantly reduced proteinuria by 58% in NS rats, along with marked improvements in renal pathology and fibrosis. ZWD also ameliorated serum levels of TP and LDL-C, and lowered the concentrations of AVP, Renin, AngII, and ALD. The expression of type 2 vasopressin receptor (V2R), mineralocorticoid receptor (MR), aquaporin 1 (AQP1), AQP2, and AQP3 was remarkably decreased at the protein and mRNA levels. Three components (benzoylpaeoniflorin, 6-gingerol, and paeoniflorin) of ZWD exhibited synergistic effects on AQP1, AQP2, AQP3, MR, and V2R. Our findings suggest that ZWD alleviates NS through renal repair and dual suppression of the AVP-V2R-AQP2 and RAAS-MR-AQP3 pathways.

Optimization strategy for modeling sick sinus syndrome in rats: Balancing effect and animal care.

Metformin-loaded Chitosan nanoparticles alleviate insulin resistance in the polycystic ovarian syndrome rat model through modulation of PI3K/AKT/ GLUT4 in ovarian tissue.