Effect of Goreisan, a Traditional Japanese Medicine, on Rat Hindlimb Lymphedema

Abstract

Secondary lymphedema occurs after cancer surgery involving lymph node dissection owing to the lymphatic system dysfunction. However, the pathophysiology of lymphedema and the molecular pathways involved remain unknown. This study aimed to develop a rat hindlimb lymphedema model and investigate the mechanisms that drive pathophysiology and the effects of the traditional Japanese medicine goreisan on lymphedema. The rat lymphedema model was induced by combination surgeries of popliteal lymph node dissection, skin cautery incision, and fascial ablation coagulation in the right hindlimb using male Wistar rats. The foot volume was significantly increased, and recovery was delayed by combination surgeries. Dermal thickness and dilated lymphatic vessels of the hindlimb were observed on postoperative day 2. The number of infiltrating leukocytes (CD45⁺ cells), including CD4⁺ T-cells, increased in the lymphedema group compared with that in the sham group. The relative mRNA expression and protein levels of interleukin-6 (IL-6), CC chemokine ligand 2 (CCL2), transforming growth factor β1 (TGF-β1), and Fms-related receptor tyrosine kinase 4 (FLT4) were significantly higher in the lymphedema group than in the sham group. Foot volume was decreased by goreisan, furosemide, and prednisolone treatments. Goreisan diminished the increase in CD4⁺ T-cells, and the same trend was observed for CCL2 and FLT4 expression. In conclusion, the rat hindlimb lymphedema model in this study exhibited increased foot volume, skin-infiltrating cells, and pathological changes accompanied by inflammatory and fibrotic responses, suggesting that the model presented significant clinical features of lymphedema. Goreisan may exert a therapeutic effect on lymphedema by inhibiting CD4⁺ T-cell infiltration.

INTRODUCTION

Secondary lymphedema (SLE) is a major complication of cancer management in patients with surgical lymph node dissection.¹⁾ It is developed in one-third of patients with breast cancer and one-sixth of patients with other solid tumors. Lower limb lymphedema is induced in 75% of patients with some gynecologic cancer.²⁾ SLE is a progressive disorder with swelling, fibrosis, inflammation, and recurrent infection of the affected area.^3–5) Based on the clinical characteristics of lymphedema, surgery decreases lymphatic flow, stimulates a feed-forward cycle of inflammation, and leads to tissue fibrosis, which further decreases lymph flow by inhibiting lymphatic vessel function.⁶⁾ Several studies on animal models of lymphedema have been reported,^7–10) but the mechanism underlying lymphedema remains poorly understood, and beneficial pharmacological therapies have not yet been established.¹¹⁾ Hence, despite the growing demand for lymphedema management, medical treatment options are limited.

The herbal medicine goreisan is used as a typical prescription for edema, nausea, and migraine that related to imbalances in interstitial water retention in Japan and East Asia.¹²⁾ There are two clinical reports of goreisan on lymphedema in patients who have undergone lymph node dissection. In patients with lower abdominal lymphedema after retroperitoneal lymph node dissection without radiotherapy despite performing complex decongestive therapy (CDT), goreisan administration was effective in 78% of patients, with a 2.1 cm decrease in abdominal circumference.¹³⁾ In patients with lymphedema of the lower extremities, the ratio of extracellular water to total body water was significantly decreased by combined treatment with goreisan compared to CDT alone.¹⁴⁾ While an animal study has demonstrated that goreisan could augment the pumping function of lymphatic vessels through the elevated expression of vascular endothelial growth factor receptor-3 in rat mesenteric lymphatic vessels,¹⁵⁾ the effect of goreisan on lymphedema remains unknown.

Therefore, in this study, we aimed to develop an experimental animal model of hindlimb lymphedema using a simple method without affecting radiation and investigate the pathological changes and the molecular mechanisms underlying the disease. Furthermore, we also applied goreisan treatment in this model and assessed its mechanism of action against experimental lymphedema.

MATERIALS AND METHODS

Materials

Goreisan extract powder (lot: 2170017010) used was manufactured by Tsumura & Co. (Tokyo, Japan). A mixture of the following components: Alismatis rhizoma (4 g, rhizomes of Alisma orientale (Sam.) Juz.), Atractylodis lanceae rhizoma (3 g, rhizomes of Atractylodes lancea DC.), Polyporus (3 g, sclerotium of Polyporus umbellatus Fries), Hoelen (3 g, sclerotium of Poria cocos Wolf), and Cinnamomi Cortex (1.5 g, cortex of Cinnamomum cassia Blume) was extracted with hot water and spray-dried to obtain 2 g of dry extract powder. Supplementary Fig. 1 shows the different ingredients in goreisan, which were identified via three-dimensional HPLC. It was approved by the Japanese Ministry of Health, Labor, and Welfare for clinical use. Furosemide (FRS) was purchased from Sanofi-Aventis (Tokyo, Japan). Prednisolone sodium succinate (PDN) was purchased from Shionogi Pharmaceutical Co., Ltd. (Osaka, Japan).

Animals

Seven-week-old male Wistar rats were purchased from Jackson Laboratory Japan (Yokohama, Japan). Rats were individually housed in cages under environmentally controlled conditions; 12 h light/dark cycle (lights on at 7 a.m.), temperature (24 ± 1 °C), and humidity (55 ± 5%). The rats were allowed free access to water and the standard laboratory chow MF (Oriental Yeast Company, Tokyo, Japan), and acclimated to the environment for six days prior to their participation into the experiment. All experimental procedures were conducted in accordance with the “Guidelines for the Care and Use of Laboratory Animals” approved by the Tsumura Animal Committee (Approval No. 18-029, 19-003, 21-022, 21-041, 22-008). The ethical standards required by the laws and guidelines for laboratory animals in Japan were met.

Experimental Procedure

Deep lymphatic flow suppression by popliteal lymph node (PLN) dissection, and superficial and intermediate lymphatic flow suppression by skin and fascial cauterization were performed at 6-d intervals. Sham-operated rats served as controls. During surgical procedures, the rats were anesthetized with sevoflurane (induction dose, 1 L/min; maintenance dose, 0.5 L/min; Viatris, Canonsburg, PA, U.S.A.). Butorphanol tartrate (Meiji Seika Pharma, Tokyo, Japan) was intramuscularly administered to the rats for postoperative pain management. Povidone iodine (Meiji Seika Pharma) was applied to the cauterized area of the rats to prevent postoperative infection. Details are provided in Supplementary Fig. 2.

The foot volume was measured using a TK-101 CMP Plethysmometer (Muromachi Machine, Tokyo, Japan) and expressed as the volume ratio (%) of the operated foot (right) to the unoperated foot (left). Rats were orally administered the test drugs (goreisan, 1 g/kg; FRS, 5 mg/kg; and PDN, 10 mg/kg) or distilled water (10 mL/kg) once a day starting 6 d after PLN dissection. The doses of goreisan,^15,16) FRS,^17,18) and PDN^19,20) were estimated according to previous animal studies, respectively.

Histopathology

Two days after the second surgery (day 8), the hindlimb was amputated 1 cm from the sole, washed with saline, and fixed in 15% formalin solution for 3 d. The specimens were decalcified with ethylenediaminetetraacetic acid (EDTA) for 3 d, embedded in paraffin, and stained with hematoxylin and eosin.

Flow Cytometry (FCM) Analysis

Skin sampling was performed on day 8. The skin of the hindlimbs was removed 1 cm distal to the suture site at a size of 2 × 2 cm. Skin infiltrating cells were collected with reference to the method of Broggi.²¹⁾ Briefly, skin was digested for 90 min at 37 °C in RPMI 1640 solution containing 50 U/mL deoxyribonuclease (DNase) I (Merck, Darmstadt, Germany), 300 µg/mL Liberase TM (Merck), and 5% fetal bovine serum. The tissue digest was filtered and suspended in phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) after hemolysis of red blood cells.

The evaluation of skin-infiltrating cells by flow cytometry was based on the method of Zampell²²⁾ and Barnett-Vanes.²³⁾ Cells were blocked at 4 °C with Fc block (anti-rat CD32, BD Biosciences, Franklin Lakes, NJ, U.S.A.). Cell suspensions isolated from the skin were stained with antibodies against cell surface markers and analyzed using a FACSAria II flow cytometer (BD Biosciences) and the BD FACSDiva software. Data analysis was performed using the FlowJo software (Tree Star, Ashland, OR, U.S.A.). The gating strategies are illustrated in Supplementary Fig. 3.

Enzyme-Linked Immunosorbent Assay (ELISA)

Left and right hindlimb skin samples were collected in Cell Extraction Buffer (FNN0011, Thermo Fisher Scientific, Waltham, MA, U.S.A.) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, St. Louis, MO, U.S.A.) and Protease Inhibitor Cocktail (Sigma-Aldrich). Skin samples were homogenized using a Physcotron Micro Homogenizer (NS-310E; Microtec, Funabashi, Japan), and total protein was determined using the DC Protein Assay (BIO-RAD, Hercules, CA, U.S.A.). The protein levels were determined using ELISA kits (R&D Systems, Minneapolis, MN, U.S.A.).

Real-Time Quantitative PCR (RT-qPCR)

Skin samples were first incubated in RNAlater solution (Thermo Fisher Scientific) overnight at 4 °C. They were homogenized using a Micro Smash TM (MS-100R; Tomy Seiko, Tokyo, Japan), after which total RNA was isolated using the RNeasy fibrous tissue mini kit (Qiagen, Hilden, Germany) and quantified using a SpectraDrop Micro-Volume Microplate (Molecular Devices, San Jose, CA, U.S.A.). RNA (100 ng/µL) was denatured at 70 °C for 15 min, immediately cooled to 4 °C, reverse transcribed to cDNA using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific).

mRNA expression was analyzed using Gene Expression Assays with gene-specific primers and TaqMan Fast Advanced Master Mix on a QuantStudio 7 Flex Real-Time qPCR System (Thermo Fisher Scientific), and normalized to that of the reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (assay ID: Rn01775763_g1).

Statistical Analysis

Data are presented as mean ± standard error (S.E.). The statistical significance of differences was assessed using Student’s t-test. For multiple-group comparisons, one-way ANOVA followed by Tukey’s test, 2-way ANOVA followed by Sidak’s test, or Kruskal–Wallis test followed by Dunn’s test (when normal distribution was not met) was used. p-Values <0.05 were considered statistically significant.

RESULTS

Preparation of Animal Model for Lymphedema

We investigated methods to create experimental animal models of hindlimb lymphedema. First, PLN dissection was performed to inhibit the deep lymphatic flow in the rat hindlimb. The foot volume after the procedure was slightly higher than that in the sham group, but there was no significant change in foot volume after day 6 (Fig. 1A). Evans blue dye reflux in the femoral skin was observed on day 1 (Supplementary Fig. 2B), suggesting that superficial lymphatic flow was maintained. Next, a skin cautery incision was made 8 d after PLN dissection to inhibit both superficial and deep lymphatic flow. However, it had little effect on foot volume (Fig. 1B). Intermediate lymphatic flow in the hindlimb may be responsible for early recovery. Thus, fascial cautery coagulation to inhibit middle layer lymphatic flow was added concomitantly with the skin cautery incision 6 d after PLN dissection. A significant increase in foot volume and delayed recovery were observed, suggesting lymphedema induction in the hindlimbs (Fig. 1C).

Fig. 1. Foot Volume Increase in Hindlimb Lymphedema Model

Deep lymphatic flow is inhibited by popliteal lymph node dissection (popliteal d). On postoperative day 6 or 8, as indicated by the arrow, superficial lymph flow was inhibited by skin cautery incision (Skin i), and intermediate lymph flow was inhibited by fascial ablation coagulation (Fascial c). A: Foot volume was increased by popliteal d compared to sham operation (Sham); B: Foot volume increase caused by popliteal d and skin i compared to skin i alone. C: Foot volume was significantly increased by the combination of popliteal d, skin i, and fascial c, which was higher than that of fascial c alone. Data are shown as foot volume increase (%) in the operated limb (right) versus the unoperated limb (left) and represent mean ± S.E. (n = 3 animals in each group). Significance was determined using 2-way ANOVA followed by SIDAK’S multiple comparison test (A, B) or Tukey’s multiple comparison test (C). p-Value (C) indicates the statistical value for sham.

Effect of Goreisan on Hindlimb Lymphedema

Using a rat hindlimb lymphedema model, we evaluated the effects of goreisan, diuretics, and steroids on foot volume. Compared to the sham group (control), the lymphedema group (edema) showed a significantly increased foot volume from the day after the cautery procedure (day 7) to day 10, which was attenuated by daily oral administration of goreisan from day 6 (edema + goreisan (GRS)), as shown in Fig. 2A. FRS (edema + FRS) and PDN (edema + PDN) treatment also suppressed foot volume increase from day 8 to day 10 (Fig. 2B).

Fig. 2. Effect of Test Drugs on Foot Volume Increase of a Rat Lymphedema Model

Lymphedema was induced by popliteal lymph node dissection (day 0) and inhibition of lymphatic flow in the superficial and middle layers by skin cautery incision and fascial ablation coagulation on day 6. Rats received oral distilled water or test drugs (goreisan (GRS), 1 g/kg; FRS, furosemide 5 mg/kg; PDN, prednisolone 10 mg/kg) at 10 mL/kg for 4 d from day 6. Data are shown as foot volume increase (%) in the operated limb (right) versus the unoperated limb (left) and represent mean ± S.E. (n = 6 animals in each group). Significance was determined using 2-way ANOVA followed by Tukey’s multiple comparison test.

Pathological Evaluation of Hindlimb Lymphedema

Compared with the sham group (control) (Fig. 3A), thickening and edema of the dermis were observed in the lymphedema group (Fig. 3B). The lymphatic vessels were flat in the sham group (Fig. 3D arrow), whereas dilation of the lymphatic vessels (Fig. 3E arrow) and increased infiltration of immune cells were observed in the lymphedema group (Fig. 3E). Additionally, GRS-treatment exhibited a trend to suppress these changes in the lymphedema group (Figs. 3C, F).

Fig. 3. Pathological Evaluation of Hindlimb Lymphedema Model

Hindlimbs of the lymphedema groups (control, edema, edema + goreisan (GRS)) were excised and formalin-fixed 2 d after the second surgery. H&E staining was performed after demineralization treatment. Image acquisition of the pathological specimens was performed with 2× and 40 × magnification. Scale bars represent 100 µm for D–F.

FCM Analysis of Skin Infiltrating Cells in the Lymphedema Model

We investigated the infiltrating cells in the skin of the hindlimbs in three groups (control, edema, edema + GRS) using FCM. There was no difference among the groups in the abundance of CD45 (leukocyte common antigen)-positive cells in the unoperated foot (left) (Fig. 4A). On the contrary, in the operated foot (right), the number of CD45⁺ cells significantly increased in the lymphedema group but not in the GRS-treated group compared to that in the sham group (Fig. 4A).

Fig. 4. Comparison of Skin Infiltrating Cell Density

A: Skin-infiltrating cells collected from unoperated (left) and operated (right) limbs were stained with CD45. The number of samples measured was up to 36 (9 animals × 4 antibody sets) in each group. Data represent mean ± S.E. (n = 27–36). Significance was determined using one-way ANOVA and Tukey’s multiple comparison test. B–I: Skin-infiltrating cells were stained with four different antibody sets (#1, #2, #3, and #4), and cell types were identified according to the gating procedure shown in Supplementary Fig. 3. The cell density of infiltrating cell types was compared among the three groups. Data represent mean ± S.E. (n = 6–9 animals). Significance was determined using Kruskal–Wallis and Dunn’s multiple comparison tests.

Next, we identified the cell types infiltrated edematous tissue. A significant increase in CD4⁺ T-cells was observed in the operated foot of the lymphedema group compared to that in the sham group (control), which was attenuated in the GRS-treated group (Fig. 4B). In contrast, there was no difference in the number of CD8⁺ T-cells among the three groups (Fig. 4C). A trend toward an increase in B cells, NK cells, and NKT cells was observed in the operated foot of the lymphedema and GRS-treated groups compared to the sham group (Figs. 4D–F). No significant differences in myeloid cells, mast cells, or neutrophils were observed among the three groups (Figs. 4G–I).

Lymphedema-Related Protein and Gene Levels in Skin Tissue of Hindlimb

Skin tissues were collected on postoperative day 2, and cytokine protein levels were measured. There was no significant difference between each group in interleukin (IL)-1β, IL-4 and IL-6 levels in both tissues (left and right foot) (Figs. 5A–C). Transforming growth factor β1 (TGF-β1) levels in the operated hindlimb (right) of the lymphedema group significantly increased compared to that of the sham group, which was not inhibited by GRS treatment (Fig. 5D). The tumor necrosis factor α (TNF-α) levels were undetectable below the measurement limit (data not shown).

Fig. 5. Cytokine Levels in Lymphedema-Induced Skin

Two days after the second surgery, the skin was collected from unoperated (left) and operated (right) limbs, and proteins were extracted. IL-1β, IL-4, IL-6, and TGF-β1 cytokine levels were quantified using ELISA kits. Data represent mean ± S.E. (n = 3–6 animals; B: IL-4 in edema/left; n = 2). Significance was determined using Kruskal–Wallis and Dunn’s multiple comparison tests.

Subsequently, we measured the mRNA levels of cytokines (IL-1β, IL-6, CC chemokine ligand 2 (CCL2), TNF-α), fibrosis-related genes (TGF-β1, collagen type I alpha 1 chain (COL1A1), COL3A1), lymphangiogenesis-related genes (vascular endothelial growth factor C (VEGF-C), Fms-related receptor tyrosine kinase 4 (FLT4)), and the water channel aquaporins (AQP1, AQP2, AQP4) as described in the literature.^24–31) AQP2 could not be detected. IL-6, CCL2, TGF-β1, and FLT4 mRNA levels were significantly increased in the lymphedema group compared with that in sham group (Fig. 6). However, GRS treatment induced a trend toward lower levels of these factors and no significant difference was detected between the sham group and the GRS-treated groups (Fig. 6).

Fig. 6. mRNA Expression in Lymphedema-Induced Skin

Two days after the second surgery, the skin was collected from the unoperated (left) and operated (right) limbs and mRNA was extracted. Changes in the mRNA expression of IL-1β, IL-6, CCL2, TNF-α, TGF-β1, COL1A1, COL3A1, VEGFC, FLT4, AQP1, AQP2, and AQP4 were assessed by real-time quantitative PCR assay and the delta-delta Ct method. The following TaqMan assays from Thermo Fisher Scientific were used: Interleukin-1 beta (IL-1β) (assay ID: Rn00580432_m1), Il-6 (assay ID: Rn01410330_m1), Ccl2 (assay ID: Rn00580555_m1), Tnf-a (assay ID: Rn01525859_g1), Tgf-b1 (assay ID: Rn00572010_m1), Collagen type I alpha 1 (COL1A1) (assay ID: Rn01463848_m1), Collagen type III alpha 1 (COL3A1) (assay ID: Rn01437681_m1), Vascular endothelial growth factor C (VEGFC) (assay ID: Rn01488076_m1), fms-related tyrosine kinase 4 (Flt4) (assay ID: Rn00677893_m1), aquaporin 1 (Aqp1) (assay ID: Rn00562834_m1), aquaporin 2 (Aqp2) (assay ID: Rn00563755_m1), and aquaporin 4 (Aqp4) (assay ID: Rn01401327_s1). Data represent mean ± S.E. (n = 4–6 animals). Significance was determined using Kruskal–Wallis and Dunn’s multiple comparison tests.

DISCUSSION

Several lymphedema models have been developed to date. A combination of lymph node removal and irradiation can form severe and stable lymphedema in rat hindlimbs because radiation induces tissue fibrosis and inhibits lymphatic reconstruction.⁷⁾ In clinical practice, postoperative radiotherapy is a risk factor for the development and progression of lower limb lymphedema.^2,32) However, animal models established this way are not suitable for studying lymphedema developed in lymphadenectomy patients without receiving radiotherapy.¹⁰⁾ Recently, some researchers have successfully established a rat lymphedema model without affecting radiation. Sano reported a rat hindlimb SLE model that mimicked early to late human SLE in terms of cell proliferation, lymphatic fluid accumulation, and dermal fibrosis.²⁷⁾ Ogata showed that the mouse lymphedema model exhibited initial edema and later histological changes, such as dermal thickening, fibrosis, and lipogenesis associated with chronic lymphedema, which was suppressed by interference with early excessive lymphangiogenesis.²⁸⁾ However, these models are accompanied by difficulties owing to the need for advanced microsurgical techniques. Recently, Huang et al. reported a rat lymphedema model that could be created by extensive lymph node dissection combined with a complete circumferential soft-tissue defect on the hindlimb. The pathogenic mechanism of the model was related to T-cell-mediated inflammation; however, the fibrogenic process is still unclear.¹⁰⁾

In the present study, we created a rat hindlimb lymphedema model by combining PLN dissection, skin cautery incision, and fascial ablation coagulation, which may be similar to the model reported by Huang et al.¹⁰⁾ Our model exhibited a significant increase in hindlimb volume, which was accompanied by the thickening and edema of the dermis, dilated lymphatic vessels, infiltrating lymphocytes, and increased inflammatory and fibrotic mediators. These findings suggest that our model presents significant clinical features of lymphedema and can be helpful for evaluating the effects of medicines and elucidating the pharmacological mechanisms.

CD4⁺ T-cells are known to play a central role in lymphedema.²²⁾ The number of infiltrating CD4⁺ T-cells increases in human lymphedema biopsy samples and is linearly and positively correlated with disease severity.⁶⁾ In the early process of lymphedema, CD4⁺ T-cells activate macrophages to produce VEGF-C, resulting in excessive lymphangiogenesis that is thought to induce lymphedema.²⁷⁾ The FCM analysis in this study revealed an increase in skin-infiltrating cells (CD45⁺) in the lymphedema group, among which CD4⁺ T-cells were significantly increased. These findings are consistent with the phenotype characterization of a human lymphedema.

Furthermore, we found that several lymphedema-related genes and proteins including cytokines and chemokines were upregulated in the model rats. In patients with breast cancer-related lymphedema, CCL2 and total protein concentrations in the aspirate blister fluid induced on the medial upper arm were increased compared to those in the non-edema arm.⁵⁾ IL-6 expression was increased in lymphedema tissue and serum of lymphedema patients.³³⁾ Low levels of LTB4 detected in lymph fluid from patients with early lymphedema stimulated FLT4 expression in cultured human lymphatic endothelial cells.³⁴⁾ Several studies have shown that TGF-β1 plays a central role in tissue fibrosis of the liver, lung, kidney, skin, and myocardium.^35,36) In this model, TGF-β1 mRNA and protein were increased, and mRNA expression levels of COL1A1 and COL3A1 showed a decreasing trend, which is consistent with the initial state of the rat hindlimb SLE model reported by Sano.²⁷⁾ In the initial process of lymphedema, TGF-β1 produced by macrophages promotes the transition from fibroblasts to myofibroblasts and causes tissue fibrosis by promoting collagen synthesis.²⁷⁾ Taken together, we believe that our model can be used to evaluate inflammatory and fibrotic responses, which are the early symptoms of lymphedema.

Goreisan is used to treat various edematous disorders, including water distribution imbalance.¹²⁾ The present study showed that the increase in foot volume of hindlimb lymphedema models was restored following goreisan treatment. To elucidate the mechanism of action of goreisan, we performed FCM of skin infiltrating immune system cells and measured cytokines and proteins involved in inflammation and fibrosis. Our study revealed that goreisan treatment attenuated the increased CD4⁺ T-cell infiltration in the skin of the hindlimb after surgery. Moreover, the same trend was observed for the gene expression of CCL2, VEGFC, and FLT4 upon goreisan treatment. A landmark study using a mouse tail lymphedema model reported that lymphedema was associated with T-helper 2 (Th2) cytokine production, and the removal of CD4⁺ T-cells prevented inflammation, fibrosis, and swelling.^6,8) These findings suggest that goreisan can ameliorate lymphedema after surgery by inhibiting CD4⁺ T-cell infiltration.

Although no appropriate medications for lymphedema exist, a few case studies reported that the diuretic FRS and the common anti-inflammatory corticosteroid PDN improved lymphedema.^37,38) In this study, we observed that FRS and PDN inhibited the increase in foot volume in hindlimb lymphedema rats. In support of the important role of inflammation, ketoprofen, a nonsteroidal anti-inflammatory drug (NSAID), restored edema and fibrosis in experimental lymphedema in mice due to the inhibition of 5-lipoxygenase-dependent leukotriene B4 (LTB4) production.⁹⁾ The use of diuretics and corticosteroids for pure lymphedema is not physiologically sound but may be beneficial in the management of mixed edema and in palliative care settings.^39,40) Concerning other traditional Japanese medicine, saireito, which contains both goreisan and shosaikoto, is known to be effective for edematous disorders, including inflammation, and reportedly improves lymphedema caused by radiotherapy.⁴¹⁾ Concomitant saireito and goreisan therapy was more effective than goreisan monotherapy for lower abdominal lymphedema after retroperitoneal lymphadenectomy in patients with gynecologic cancer.¹³⁾ However, the mechanisms and effects have not been fully elucidated.

Goreisan is composed of five herbal medicines, each containing several components. In a pharmacokinetic study of goreisan extract, alisols, atractylodin derivatives, cinnamic acid, 2-methoxy cinnamic acid, pachyimic acid, and tumulosic acid were detected as components transferred into the body.⁴²⁾ These components have also been reported to have anti-inflammatory and fibrosis-inhibiting effects. More specifically, alisol A, alisol B, and alisol B O-23 acetate, components of Alismatis rhizoma, have been reported to exhibit binding inhibitory activity on the receptors of angiotensin II and arginine vasopressin,⁴³⁾ and to alleviate hapten-induced dermatitis symptoms in NC/Nga mice, a model of atopic dermatitis.⁴⁴⁾ Alisol B 23-acetate treatment also attenuated renal fibrosis in unilateral ureteral obstructed rat model by inhibiting TGF-β/Smad3 activation.⁴⁵⁾ Furthermore, atractylodin, a component of Atractylodis lanceae rhizoma, decreased NO production and mRNA levels of iNOS, TNF-α, CCL2, and LCN2 in IL-1β-treated hepatocytes,⁴⁶⁾ inhibited the increase in mRNA levels of type I and type III collagen by TGF-β1 treatment in A549 cells, and reduced bleomycin-induced pulmonary fibrosis in mice.⁴⁷⁾ Cinnamic acid, a component of Cinnamomi Cortex, downregulated NOX2 gene expression in PMA-stimulated THP-1 cells, subsequently reducing the expression levels of inflammatory mediators, such as IL-1β, IL-8, CCL5, and cyclooxygenase (COX)-2, suggesting that it has antioxidant and anti-inflammatory effects.⁴⁸⁾ Dehydrotumulosic acid and pachymic acid, components of Hoelen, have been shown to inhibit LTB4 release from human leukocytes.⁴⁹⁾

Herbal medicines, which are mixtures of several medicinal plants, contain numerous compounds with diverse mechanisms of action. Given that herbal medicines are extracts of plant material, it is not surprising that they contain modulators of both activation and inhibition of biological activity. Therefore, interactions between the components and their multifaceted actions must be considered.⁵⁰⁾

Because the cellular analysis (FCM, ELISA, qPCR) in this study was only a single measurement 2 d after lymphedema induction, it was not possible to clearly demonstrate the long-lasting effect of goreisan. Future analysis over time and evaluation in a chronic model are also important to clarify the mechanism of the inhibitory effect of goreisan. In addition, the issue regarding the elucidation of the mechanisms of FRS and PDN needs to be addressed in future studies. Further studies are needed to clarify the dose-response of each drug in order to compare drug efficacy.

In the rat hindlimb lymphedema model, we observed an increase in foot volume, skin-infiltrating cells, and pathological changes, which were accompanied by increased inflammatory and fibrotic mediators. Furthermore, the increased foot volume was attenuated by goreisan treatment in the model, which could be mediated by decreased CD4⁺ T-cell infiltration. Thus, goreisan administration may have a beneficial effect on hindlimb lymphedema.

Acknowledgments

We would like to thank Dr. Atsushi Kaneko (Tsumura & Co.) for valuable and constructive suggestions during the planning of this research.

Funding

The research was funded by Tsumura & Co.

Author Contributions

ZK: study conception and design, performing experiments and data analysis, manuscript writing. YM: study conception and design, performing experiments and data analysis, manuscript writing. KO: performing experiments and data analysis. SI (Sachiko Imamura): performing experiments and data analysis. SI (Seiichi Iizuka): performing experiments and data analysis. NZ: performing experiments and data analysis. NF: manuscript writing, experiments supervision. CM: experiment supervision All authors commented on the previous versions of the manuscript. All authors read and approved the submitted manuscript.

Conflict of Interest

All authors would like to declare that they are employees at Tsumura & Co.

Supplementary Materials

This article contains supplementary materials.

REFERENCES

• 1) Sakorafas GH, Peros G, Cataliotti L, Vlastos G. Lymphedema following axillary lymph node dissection for breast cancer. Surg. Oncol., 15, 153–165 (2006).
• 2) Kuroda K, Yamamoto Y, Yanagisawa M, Kawata A, Akiba N, Suzuki K, Naritaka K. Risk factors and a prediction model for lower limb lymphedema following lymphadenectomy in gynecologic cancer: a hospital-based retrospective cohort study. BMC Womens Health, 17, 50 (2017).
• 3) Rockson SG. Lymphedema after breast cancer treatment. N. Engl. J. Med., 379, 1937–1944 (2018).
• 4) Rockson SG, Keeley V, Kilbreath S, Szuba A, Towers A. Cancer-associated secondary lymphoedema. Nat. Rev. Dis. Primers, 5, 22 (2019).
• 5) Jensen MR, Simonsen L, Karlsmark T, Bülow J. Lymphoedema of the lower extremities—background, pathophysiology and diagnostic considerations. Clin. Physiol. Funct. Imaging, 30, 389–398 (2010).
• 6) Mehrara BJ, Park HJ, Kataru RP, Bromberg J, Coriddi M, Baik JE, Shin J, Li C, Cavalli MR, Encarnacion EM, Lee M, Van Zee KJ, Riedel E, Dayan JH. Pilot study of anti-Th2 immunotherapy for the treatment of breast cancer-related upper extremity lymphedema. Biology (Basel), 10, 934 (2021).
• 7) Avraham T, Zampell JC, Yan A, Elhadad S, Weitman ES, Rockson SG, Bromberg J, Mehrara BJ. Th2 differentiation is necessary for soft tissue fibrosis and lymphatic dysfunction resulting from lymphedema. FASEB J., 27, 1114–1126 (2013).
• 8) Nakamura K, Radhakrishnan K, Wong YM, Rockson SG. Anti-inflammatory pharmacotherapy with ketoprofen ameliorates experimental lymphatic vascular insufficiency in mice. PLoS One, 4, e8380 (2009).
• 9) Triacca V, Pisano M, Lessert C, Petit B, Bouzourene K, Nahimana A, Vozenin MC, Stergiopulos N, Swartz MA, Mazzolai L. Experimental drainage device to reduce lymphoedema in a rat model. Eur. J. Vasc. Endovasc. Surg., 57, 859–867 (2019).
• 10) Huang CW, Chang YC, Hsiao HY, Liu JW, Chang FC, Huang JJ. Creation of a rat lymphedema model using extensive lymph node dissection and circumferential soft tissue resection: is this a reliable model? Microsurgery, 41, 762–771 (2021).
• 11) Brown S, Dayan JH, Coriddi M, Campbell A, Kuonqui K, Shin J, Park HJ, Mehrara BJ, Kataru RP. Pharmacological treatment of secondary lymphedema. Front. Pharmacol., 13, 828513 (2022).
• 12) Ohnishi N, Nagasawa K, Yokoyama T. The verification of regulatory effects of Kampo formulation on body fluid using model mouse. J. Trad. Med., 17, 131–136 (2000).
• 13) Komiyama S, Takeya C, Takahashi R, Yamamoto Y, Kubushiro K. Feasibility study on the effectiveness of Goreisan-based Kampo therapy for lower abdominal lymphedema after retroperitoneal lymphadenectomy via extraperitoneal approach. J. Obstet. Gynaecol. Res., 41, 1449–1456 (2015).
• 14) Yoshikawa N, Kajiyama H, Otsuka N, Tamauchi S, Ikeda Y, Nishino K, Niimi K, Suzuki S, Utsumi F, Shibata K, Kikkawa F. The therapeutic effects of Goreisan, a traditional Japanese herbal medicine, on lower-limb lymphedema after lymphadenectomy in gynecologic malignancies: a case series study. Evid. Based Complement. Alternat. Med., 2020, 6298293 (2020).
• 15) Jo M, Trujillo AN, Shibahara N, Breslin JW. Impact of Goreisan components on rat mesenteric collecting lymphatic vessel pumping. Microcirculation, 30, e12788 (2023).
• 16) Shimizu T, Murakami K, Matsumoto C, Kido T, Isohama Y. Goreisan alleviates cerebral edema: possibility of its involvement in inhibiting aquaporin-4 function. Tradit. Kampo Med., 10, 168–176 (2023).
• 17) Ntchapda F, Bonabe C, Kemeta Azambou DR, Talla E, Dimo T. Diuretic and antioxidant activities of the aqueous extract of leaves of Vepris heterophylla (Engl.) R. Let (Rutaceae) in rats. BMC Complement. Altern. Med., 16, 516 (2016).
• 18) Uchida T, Yamanaga K, Nishikawa M, Ohtaki Y, Kido H, Watanabe M. Anti-aldosteronergic effect of torasemide. Eur. J. Pharmacol., 205, 145–150 (1991).
• 19) Kang SS, Pak SC, Choi SH. The effect of whole bee venom on arthritis. Am. J. Chin. Med., 30, 73–80 (2002).
• 20) Hurtenbach U, Boggemeyer E, Stehle T, Museteanu C, Del Pozo E, Simon MM. Prednisolone reduces experimental arthritis, and inflammatory tissue destruction in SCID mice infected with Borrelia burgdorferi. Int. J. Immunopharmacol., 18, 281–288 (1996).
• 21) Broggi A, Cigni C, Zanoni I, Granucci F. Preparation of single-cell suspensions for cytofluorimetric analysis from different mouse skin regions. J. Vis. Exp., 110, e52589 (2016).
• 22) Zampell JC, Yan A, Elhadad S, Avraham T, Weitman E, Mehrara BJ. CD4⁺ cells regulate fibrosis and lymphangiogenesis in response to lymphatic fluid stasis. PLOS ONE, 7, e49940 (2012).
• 23) Barnett-Vanes A, Sharrock A, Birrell MA, Rankin S. A single 9-colour flow cytometric method to characterise major leukocyte populations in the rat: validation in a model of LPS-induced pulmonary inflammation. PLOS ONE, 11, e0142520 (2016).
• 24) Ridner SH, Dietrich MS, Sonis ST, Murphy B. Biomarkers associated with lymphedema and fibrosis in patients with cancer of the head and neck. Lymphat. Res. Biol., 16, 516–524 (2018).
• 25) Jensen MR, Simonsen L, Karlsmark T, Lanng C, Bülow J. Higher vascular endothelial growth factor-C concentration in plasma is associated with increased forearm capillary filtration capacity in breast cancer-related lymphedema. Physiol. Rep., 3, e12403 (2015).
• 26) Sano M, Hirakawa S, Sasaki T, Inuzuka K, Katahashi K, Kayama T, Yamanaka Y, Tsuyuki H, Endo Y, Naruse E, Yokoyama Y, Sato K, Yamauchi K, Takeuchi H, Unno N. Role of subcutaneous adipose tissues in the pathophysiology of secondary lymphedema. Lymphat. Res. Biol., 20, 593–599 (2022).
• 27) Sano M, Hirakawa S, Suzuki M, et al. Potential role of transforming growth factor-beta 1/Smad signaling in secondary lymphedema after cancer surgery. Cancer Sci., 111, 2620–2634 (2020).
• 28) Ogata F, Fujiu K, Matsumoto S, Nakayama Y, Shibata M, Oike Y, Koshima I, Watabe T, Nagai R, Manabe I. Excess lymphangiogenesis cooperatively induced by macrophages and CD4⁺ T cells drives the pathogenesis of lymphedema. J. Invest. Dermatol., 136, 706–714 (2016).
• 29) Gousopoulos E, Proulx ST, Bachmann SB, Dieterich LC, Scholl J, Karaman S, Bianchi R, Detmar M. An important role of VEGF-C in promoting lymphedema development. J. Invest. Dermatol., 137, 1995–2004 (2017).
• 30) Ohtani O, Ohtani Y. Recent developments in morphology of lymphatic vessels and lymph nodes. Ann. Vasc. Dis., 5, 145–150 (2012).
• 31) Gannon BJ, Carati CJ. Endothelial distribution of the membrane water channel molecule aquaporin-1: implications for tissue and lymph fluid physiology? Lymphat. Res. Biol., 1, 55–66 (2003).
• 32) Yoshihara M, Shimono R, Tsuru S, Kitamura K, Sakuda H, Oguchi H, Hirota A. Risk factors for late-onset lower limb lymphedema after gynecological cancer treatment: a multi-institutional retrospective study. Eur. J. Surg. Oncol., 46, 1334–1338 (2020).
• 33) Cuzzone DA, Weitman ES, Albano NJ, Ghanta S, Savetsky IL, Gardenier JC, Joseph WJ, Torrisi JS, Bromberg JF, Olszewski WL, Rockson SG, Mehrara BJ. IL-6 regulates adipose deposition and homeostasis in lymphedema. Am. J. Physiol. Heart Circ. Physiol., 306, H1426–H1434 (2014).
• 34) Tian W, Rockson SG, Jiang X, Kim J, Begaye A, Shuffle EM, Tu AB, Cribb M, Nepiyushchikh Z, Feroze AH, Zamanian RT, Dhillon GS, Voelkel NF, Peters-Golden M, Kitajewski J, Dixon JB, Nicolls MR. Leukotriene B4 antagonism ameliorates experimental lymphedema. Sci. Transl. Med., 9, eaal3920 (2017).
• 35) Deng YL, Xiong XZ, Cheng NS. Organ fibrosis inhibited by blocking transforming growth factor-β signaling via peroxisome proliferator-activated receptor γ agonists. Hepatobiliary Pancreat. Dis. Int., 11, 467–478 (2012).
• 36) Meng XM, Nikolic-Paterson DJ, Lan HY. TGF-β: the master regulator of fibrosis. Nat. Rev. Nephrol., 12, 325–338 (2016).
• 37) Kim HS, Lee JY, Jung JW, Lee KH, Kim MJ, Park SB. Is mannitol combined with furosemide a new treatment for refractory lymphedema? A case report. World J. Clin. Cases, 9, 8804–8811 (2021).
• 38) Moreira S, Crespo J, Santos L. Lymphedema secondary to limited cutaneous systemic sclerosis. BMJ Case Rep., 2018, bcr2017224148 (2018).
• 39) Bruns F, Micke O, Bremer M. Current status of selenium and other treatments for secondary lymphedema. J. Support. Oncol., 1, 121–130 (2003).
• 40) Executive Committee of the International Society of Lymphology. The diagnosis and treatment of peripheral lymphedema: 2020 Consensus Document of the International Society of Lymphology. Lymphology, 53, 3–19 (2020).
• 41) Nagai A, Shibamoto Y, Ogawa K. Therapeutic effects of saireito (chai-ling-tang), a traditional Japanese herbal medicine, on lymphedema caused by radiotherapy: a case series study. Evid. Based Complement. Alternat. Med., 2013, 241629 (2013).
• 42) Hida M, Sadakane C, Mizuhara Y, Watanabe J, Isohama Y. Pharmacokinetics of ingredients after a single oral administration of Goreisan extraxt in rats. Jpn. Pharmacol. Ther., 49, 849–860 (2021).
• 43) Makino B, Kobayashi M, Kimura K, Ishimatsu M, Sakakibara I, Higuchi M, Kubo M, Sasaki H, Okada M. Local variation in the content of angiotensin II and arginine vasopressin receptor antagonistic terpenoids in the rhizomes of Alisma orientale. Planta Med., 68, 226–231 (2002).
• 44) Lee JH, Kwon OS, Jin HG, Woo ER, Kim YS, Kim HP. The rhizomes of Alisma orientale and alisol derivatives inhibit allergic response and experimental atopic dermatitis. Biol. Pharm. Bull., 35, 1581–1587 (2012).
• 45) Chen H, Wang MC, Chen YY, Chen L, Wang YN, Vaziri ND, Miao H, Zhao YY. Alisol B 23-acetate attenuates CKD progression by regulating the renin-angiotensin system and gut-kidney axis. Ther. Adv. Chronic Dis., 11, 2040622320920025 (2020).
• 46) Ishii T, Okuyama T, Noguchi N, Nishidono Y, Okumura T, Kaibori M, Tanaka K, Terabayashi S, Ikeya Y, Nishizawa M. Antiinflammatory constituents of Atractylodes chinensis rhizome improve glomerular lesions in immunoglobulin A nephropathy model mice. J. Nat. Med., 74, 51–64 (2020).
• 47) Chang KW, Zhang X, Lin SC, Lin YC, Li CH, Akhrymuk I, Lin SH, Lin CC. Atractylodin suppresses TGF-β-mediated epithelial-mesenchymal transition in alveolar epithelial cells and attenuates bleomycin-induced pulmonary fibrosis in mice. Int. J. Mol. Sci., 22, 11152 (2021).
• 48) Kim NY, Kim S, Park HM, Lim CM, Kim J, Park JY, Jeon KB, Poudel A, Lee HP, Oh SR, Ahn J, Yoon DY. Cinnamomum verum extract inhibits NOX2/ROS and PKCδ/JNK/AP-1/NF-κB pathway-mediated inflammatory response in PMA-stimulated THP-1 monocytes. Phytomedicine, 112, 154685 (2023).
• 49) Prieto JM, Recio MC, Giner RM, Máñez S, Giner-Larza EM, Ríos JL. Influence of traditional Chinese anti-inflammatory medicinal plants on leukocyte and platelet functions. J. Pharm. Pharmacol., 55, 1275–1282 (2003).
• 50) Yamamoto M. KAMPOmics: a framework for multidisciplinary and comprehensive research on Japanese traditional medicine. Gene, 831, 146555 (2022).