مجله سلول و بافت
سال چهاردهم شماره 4 (زمستان 1402)

Low back pain (LBP) is one of the most common musculoskeletal diseases in the world. Apoptosis of nucleus pulposus (NP) cells and structural changes in the intervertebral disc (IVD) matrix cause its degeneration and are directly related to LBP. Because of exosomes derived from mesenchymal stem cells (MSC-Exo) are high therapeutic potential and may be valuable options for decreasing intervertebral disc degeneration (IDD). 
The present study evaluates changes in the expression level of genes responsible for repairing NP cells (collagen and aggrecan) and tumor necrosis factor-alpha (TNF-α) gene and the effectiveness of exosomes in inhibiting excessive apoptosis of NP cells under inflammation.

Exosomes were separated by ultracentrifuge. They were identified with the help of transmission electron microscopy (TEM), atomic force microscope (AFM), and dynamic light scattering (DLS). Cell viability was evaluated by MTT assay and DAPI staining. Real-time PCR measured the expression of collagen, aggrecan, and TNF-α genes.

Exosomes are vesicles with a diameter of about 35.5 to 100 nm. Based on the findings of the MTT assay, the survival rate of the inflammatory cells treated with exosomes was significantly different from the inflammation group without treatment (*P<0.05 at the dose of 100 μg/ml, **P<0.01 at the doses of 25 and 50 μg/ml). DAPI results showed a decrease in cell death in the exosome-treated group. Real-time PCR results showed increased expression of collagen (*P<0.05) and aggrecan (***P<0.001) genes and decreased the TNF-α (**P<0.01) gene in inflammatory cells treated with exosomes.

Exosomes derived from mesenchymal stem cells can increase collagen and aggrecan gene expression and decrease TNF-α gene expression in treated cells.

 As the second leading cause of death worldwide, cancer has been a long-standing and rapidly evolving focus of biomedical research and practice. Ovarian cancer is one of the deadly malignancies of women, which is known as the "silent killer". Because its symptoms usually appear when the disease has reached advanced stages and is mostly incurable. On the other hand, currently common treatment methods are associated with various limitations, failures and side effects, which have made researchers pay more attention to the compounds extracted from plants as anti-tumor and anti-cancer agents in the last two decades. The studies conducted on the various properties of Ashwagandha (Withanaia somnifera) show that this plant has therapeutic effects and anti-cancer properties that can even be effective on the process of apoptosis mediated by different genes, including P53. Today, P53 is known as a gene It has a key role in all types of cancers and mutations in this gene have been observed in 60% of ovarian cancers. Therefore, the aim of this study is to investigate the cytotoxic effects of the hydroalcoholic extract of Ashwagandha (Withanaia somnifera) and the changes in P53 gene expression in response to this substance in ovarian cancer cells (cell line A2780).

For this purpose, extraction of Ashwagandha plant was done using soaking method. Then, the compounds present in the extract were determined using standard phytochemical tests. Then A2780 cells were treated with different concentrations of Ashwagandha extract for 24, 48 and 72 hours. The effects of this extract on cell survival were evaluated using the MTT test, and IC50 was calculated. Then, A2780 cells were exposed to concentrations of 250 and 500 µg/ml for 24 and 48 hours, and the level of P53 gene expression was investigated by Real-Time PCR. Finally, statistical analysis and data interpretation was done using GraphPad Prism software.

 MTT results showed that Ashwagandha extract at concentrations of 62.5, 125, 250, 500, and 750 µg/ml significantly decreased cell viability in a time- and concentration-dependent manner. The lowest percentage of survival rate is related to the concentration of 750 μg/ml and the time of 72 hours, while the highest percentage is related to the concentration of 62.5 μg/ml and the time of 24 hours. After 24, 48, and 72 hours, respectively, IC50 was obtained at concentrations of 512.6, 339, and 226.6 μg/ml, and Real-Time PCR results showed that the expression of the P53 gene during 24 hours of treatment with concentrations of 250 and 500 μg/ml of extract had increased significantly. Also, increasing the concentration of the extract had a significant effect on the expression of this gene.

 The results of this study indicate the cytotoxic effect of Ashwagandha extract on ovarian cancer cells, and by increasing the expression of the P53 gene, it can induce anti-cancer effects. It is hoped that by knowing the mechanisms of action of this medicinal plant and designing new drugs, it will be possible to stop the progression of ovarian cancer and thus help to increase the life span of these patients against this silent killer.

Inflammation is a protective physiologic response against pathogens, trauma and injury (1, 2). Inflammation pathway are different in human body and some main pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α have key role in pathogenesis of inflammation (3). Current anti-inflammatory drugs including corticosteroids and non-steroid anti-inflammatory drugs have serious side effects (6), so, research on new compounds with anti-inflammatory property is necessary. Astaxanthin is a natural carotenoid with various pharmacological effects. Haematococcus Algae is one of the marine sources of astaxanthin. Anti-inflammatory and antioxidant effects of astaxanthin were reported in previous studies (8-10), but the molecular mechanism of the anti-inflammatory effect of astaxanthin on the peripheral cells of the immune system is not clear. The aim of this study was to evaluate anti-inflammatory effects of astaxanthin on TLR4 gene expression and secretion of key inflammatory cytokines including IL-1, IL-6, and TNFα in RAW264.7 macrophage cells.

Methods and Material: 

The RAW264.7 macrophage cells was used in this study. Astaxanthin was extracted from Haematococcus Algae, then different doses of 25, 50, 100 μM of astaxanthin were used to treat the cells. Cells divided to three groups: one group received LPS (1µg/ml), Treatment group received LPS (1µg/ml) in combination to different dose of astaxanthin (25, 50, 100 μM) and control group without LPS astaxanthin. After 24 hour incubation, cell viability was assay with MMT test.TLR4 gene expression was measured by RT-PCR at 24 and 48 hours after induction with LPS. β-actin was considered as housekeeping gene. The levels of IL-1β, IL-6, and TNFα cytokines were measured with a commercial sandwich ELISA kit. The optic absorbance was read at 45 nm. Descriptive statistics and one-way ANOVA test were used to analyze the data. To analysis the fold change of gene expression, formula 2 -ΔΔCT   was used.

None of different doses of astaxanthin (25, 50, 100 μM) and LPS (1µg/ml) had no toxicity effect on macrophages cell. (P>0.05). LPS increased TLR 4 gene expression at both 24 and 48 hours after induction, (P <0.001) in compare to control group. 

 For many years, medicinal plants have been used to treat many diseases, including diabetes and its side effects, due to their natural and antioxidant compounds. One of these systems that suffers from the complications of diabetes is the reproductive system. Many researches have been conducted to investigate the effectiveness of different medicinal plants on the reproductive system. In this study, the effect of Ferula assa-foetida L. gum extract on the spermatogenesis indices and testicular tissue structure of diabetic male Wistar rats was investigated.

In our study, 42 male Wistar rats were divided into 6 groups. Diabetes induced by a single dose of streptozotocin (55 mg/kg body weight). Evaluation of the effect of Ferula assa-foetida L. oleo gum extract was done by treating animals with gum extract (150 and 250 mg/kg b. w, gavage) for 42 days. After this period, the animals were anesthetized and sacrificed. After this period, the animals were anesthetized and sacrificed. Then the testicular tissue sampling was done and the samples were fixed in 10% formalin, then the preparation steps of the microscopic sections were performed included 1-dehydration, 2-clarification, 3-smearing, 5-paraffin molding, 6- Cutting with a microtome machine, 7- sticking the samples on a slide, 8- staining with hematoxylin and eosin, 9- mounting. Finally, the samples were prepared for microscopic study. The number of germinal epithelium cells of spermatogenic tubules was counted and Spermiogenesis Index (SI), Tubular Differentiation Index (TDI), Meiosis Index (MI) and Sertoli Cell Index (SCI) were measured.

 Results showed that Ferula assa-foetida L. oleo gum extract improves the tissue damages of diabetes on testicular tissue and spermatogenesis indices.

 Ferula assa-foetida L. oleo gum extract reduces testicular tissue damage caused by diabetes and has an improving effect on spermatogenesis and fertility disorders related to diabetes.

 Cannabis sativa L. contains valuable cannabinoids necessary for industrial, nutraceutical  and pharmaceutical applications. To ensure the availability of cannabis plant materials in vitro culture approaches guarantee rapid and mass production to meet the growing demand. On the other hand, callogenesis can also guarantee the production of secondary metabolites in vitro through suspension cell culture. The goal of the current study was to compare different basal culture media and plant growth regulator combinations to offer the best conditions for inducing callus formation in medical cannabis.

 Leaf explants were placed on MS and DKW basal culture media were used along with B5 vitamins, 30 g/l sucrose, and 7 g/l of Agar, together with various concentrations of plant growth regulators, including 2,4-D (2, 5, and 8 µmol/l) and BA (0.5 and 2.5 µmol/l).

 The calli appeared between 5 to 7 days after establishment on various basal media supplemented with different types and concentrations of hormones. The presence of plant growth regulators is essential for callus formation in this plant, as the cultured explants in control treatments without hormones did not produce any calli. Callus formation was observed in all media and hormonal treatments, but produced calli were different from each other in terms of size, color and texture. Our study showed that texture of calli was compact or friable. The texture of calli in DKW was the most compact with the least amount of cytokinin at any concentration of auxin. However, in MS, it was the most friable with the highest amount of cytokinin at any concentration of auxin. Also, in this medium, the texture of calli was the most friable with the highest concentration of auxin. The color of calli in hormonal treatments present in MS culture medium was white and light cream, but in most hormonal treatments in DKW culture medium, it was white. The results of this study indicate that the best medium was MS medium supplemented with 2 (µmol/l) 2,4-D with 2.5 (µmol/l) BA (friable callus). Furthermore, the most inappropriate medium for callus formation from cannabis leaves in terms of all the characteristics examined was the DKW culture medium along with 5 (µmol/l) 2,4-D in combination with 2.5 (µmol/l) BA. The compression of calli can be caused by the reduction of proliferation in the cells that are dividing, this reaction can be influenced by the auxin inside the explant. Adding high auxin to endogenous auxin, in addition to adding cytokinin with a low concentration can affect the formation of compact texture. The formation of friable callus requires a balanced combination of auxin and cytokinin. Another important factor in plant tissue culture is the color of the calli, which is a sign of tissue health and vitality. For example, a healthy calli under light conditions is usually green in color, but a dark brown or black color is usually a sign of the death of the calli, which is due to pollution, stress or the production of substances such as phenols. Maximizing callus formation in the shortest possible time is one of the important objectives in tissue culture techniques, which not only saves time and cost but also prevents possible somaclonal variations. In general, according to the results, one of the suitable mediums for callus production in cannabis is the DKW,so the results of this study confirm the introduction of  DKW as the best culture medium in past studies. However, the MS culture medium is also the best medium in the available study and the least suitable result of this study is related to one of the hormonal combinations in the DKW culture medium. This difference with the mentioned study may be due to differences in genotype, laboratory materials, hormone amount, vitamins or other conditions affecting the tissue culture of this plant.

 Calli provides the opportunity to reproduce plants on a large scale and produce disease-free plants. Generally according to the plant, friable calli have a more suitable tissue for somatic embryogenesis and suspension culture, whereas compact calli have a higher potential for regeneration. This medium is recommended for future studies on somatic embryogenesis and cell suspension cultures due to the calli being friable in it. 

 Cartilage defects of the knee such as osteoarthritis disease (OA) are one of the most debilitating and public diseases that are related to high individual and socioeconomic problems. Many recent studies have applied mesenchymal stem cells (MSCs) incorporated with tissue engineering to repair articular cartilage defects or regeneration of OA.  So, the choice of the best cell type in this regard is one of the challenging issues of tissue engineering and OA cell therapy. This study aims to find the best cell source for the regeneration of critical size defects of cartilage knee using autologous chondrocytes and bone marrow-derived MSCs (Bm-MSCs), and adipose-derived MSCs (Ad-MSCs) that were isolated, cultured, and expanded in similar in vitro conditions.

 In the current study, Najdi sheep were used at 12 months of age. After standard anesthesia, cartilage was isolated from the hyaline cartilage at the end of the ribs. The Bm-MSCs and Ad-MSCs were isolated from bone marrow and tails' adipose tissue, respectively. Following the enzymatic digestion of cartilage and adipose tissues, using collagenase I enzymes, the chondrocyte, Ad-MSCs, and Bm-MSCs were cultured in growth media at 37º C with similar conditions. Then, MSCs were identified by morphology analysis and also osteogenic/adipose/chondrogenic differentiation, in vitro. In addition, chondrocytes were identified by morphology and analysis of cartilage-related gene expression such as Aggrecan, Col II, and SOX9 genes by Real-time PCR technique. After that, an amount of 5×106 cells/ml from each cell source was seeded in the type I collagen gel and transplanted into an experimentally created articular cartilage defect in the knee’s sheep model. Two months after transplantation, the animals were sacrificed in the standard ways and the implanted tissue was removed. The range of regeneration was investigated by macroscopic scoring and histological staining such as H&E and safranin o/fast green.

 MSCs showed spindle shape of morphology, and skeletal differentiation were confirmed the identity of MSCs and chondrocytes. The macroscopic observation showed that the defects in cell-treated groups of chondrocytes, Bm-MSCs, and Ad-MSCs were filled with hyaline cartilage-like tissue in contrast to the control groups of untreated and sham (without cell) groups. In addition, the surface of new cartilage formed in Bm-MSCs and chondrocyte groups appeared to be smoother than in the Ad-MSCs group and the hyaline cartilage of Bm-MSCs is more clearly than that of the Ad-MSCs group. Although histological scores (ranging from 1 to 4) were evaluated, there was no significant difference among the three experimental groups regarding newly formed cartilage repair tissues. Furthermore, the histological analysis of H&E and safranin O revealed that all defects were filled by chondrocyte-like cells that were enclosed in the secreted matrix (*P<0.05).

 We used three prevalent and main autologous cell sources such as chondrocyte, BM-MSCs, and AD-MSCs cells in exactly equal conditions to find the most significant cell sources for critical size defect of cartilage in sheep’s knees. The results demonstrated that three cell sources are suitable for this purpose; Although the Ad-MSCs due to ease and more accessibility are further recommended.