Effects of Lujingyiqishengxue Pill on Iron Metabolism in Rats with Iron Deficiency Anemia

Xianmei XU Lida CHEN Renhui YANG Hai ZHAO Yan WU

Abstract ［Objectives］This study was conducted to observe the effect of Lujingyiqishengxue Pill on iron metabolism in rats with iron deficiency anemia.

［Methods］ The iron-deficiency anemia rat model was established by feeding low-iron diet. Meanwhile, the rats were given oral gavage of ferrous succinate (0.036 g/kg, positive drug group) and Lujingyiqishengxue Pill (4.4, 2.2, 1.1 g/kg, high, middle and low dose groups), once daily for 42 consecutive days. The body weight of the rats was observed every week, and the peripheral blood［red blood cells (RBC), hemoglobin (HGB), and hematocrit (HCT)］and the iron contents in tissues (the liver, spleen, small intestine, kidney) of the rats were detected after modeling; and serum iron (SI), serum total iron binding capacity (TIBC), transferrin saturation (TSAT), serum ferritin (SF) and serum transferrin receptor 1 (TFR1) and other iron metabolism indexes were determined.

［Results］ Compared with the model group, the high-dose Lujingyiqishengxue Pill significantly reversed the peripheral blood (HGB, HCT) and iron contents of various tissues (the liver, spleen, small intestine, kidney) in rats (P<0.01), and significantly increased SI, TSAT, SF (P<0.01), while the contents of TIBC and TFR1 were significantly decreased (P<0.01).

［Conclusions］ Lujingyiqishengxue Pill can significantly improve anemia and regulate iron metabolism in rats with iron-deficiency anemia, which provides a pharmacological reference for the clinical application of Lujingyiqishengxue Pill.

Key words Lujingyiqishengxue Pill; Iron deficiency anemia; Rat; Iron metabolism

Received: June 10, 2022  Accepted: August 12, 2022

Supported by Science and Technology Planning Project (ZKHT［2020］-18-4).

Xianmei XU (1975-), female, P. R. China, associate chief pharmacist, devoted to research about hospital preparation technology and quality.

*Corresponding author. E-mail: 231689494@qq.com.

Iron deficiency anemia (IDA) is a kind of anemia due to decreased red blood cell production in human blood resulted from the lack of iron in the body caused by insufficient iron intake, malabsorption, impaired transport, excessive loss, impaired utilization, and increased demand［1］. The most common treatment for iron deficiency anemia is oral iron supplements. However, in recent years, many adverse reactions of oral iron supplements have been reported, such as poor water solubility and low bioavailability, leading to excessive free iron entering the body, stimulating the gastrointestinal tract, causing vomiting, nausea, abdominal discomfort, etc. Moreover, excessive iron supplementation causes iron overload, which can lead to cardiovascular, liver damage and neurodegeneration［2］. Therefore, in recent years, traditional Chinese medicine iron supplements with low toxic and side effects, mild action and multi-components have highlighted their advantages in clinical application.

Lujingyiqishengxue Pill is a prescription of the Hematology Department of the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, which has a significant clinical effect in the treatment of iron deficiency anemia. In order to facilitate patients to take and carry it, the research group has developed it into a compound traditional Chinese medicine preparation water-bindered pill in the early stage. The whole prescription is composed of 14 traditional Chinese medicines including Radix Astragali, Paeoniae Radix Alba, Codonopsis Radix, Radix Glycyrrhizae Preparata, and fried Ziziphi Spinosae Semen. Radix Astragali in the prescription is sweet in taste, slightly warm in nature, and attributive to the lung and spleen meridians, and has the functions of tonifying qi and lifting yang, promoting the secretion of saliva or body fluid and nourishing blood［3］. Paeoniae Radix Alba is bitter and sour in taste, slightly cold in nature, and attributive to the liver and spleen meridians, and has the functions of nourishing blood and regulating menstruation, retaining yin with astringent and stopping sweating［4］. The combination of the two is the monarch medicine for reinforcing qi and nourishing blood. Codonopsis Radix is sweet in taste, neutral in nature, and attributive to the spleen and lung meridians, and has the effects of strengthening the spleen and benefiting the lungs, nourishing blood and promoting the secretion of saliva or body fluid［5］. Radix Glycyrrhizae Preparata is sweet in taste, neutral in nature, and attributive to the heart, lung, spleen, and stomach meridians, and has the effects of nourishing the spleen and harmonizing stomach, tonifying qi and restoring the pulse［6］. Fried Ziziphi Spinosae Semen is sweet and sour in taste, neutral in nature, and attributive to the liver, gallbladder, and heart meridians, and has the effects of calming the heart and tranquilizing the mind［7］. The use of the three medicines together not only assists the monarch medicine to reinforce qi and nourish blood, but also has the functions of nourishing blood and tranquilizing the mind, so they serve as the ministerial medicine. The prescription has the effect of benefiting qi and nourishing blood, calming the heart and tranquilizing the mind. It is mainly used for treating qi deficiency and hemophthisis, pattern/syndrome of dual deficiency, and suitable for iron deficiency anemia. Therefore, in this study, a rat model of iron-deficiency anemia induced by low-iron diet was established to explore the therapeutic effect of Lujingyiqishengxue Pill on rats with iron-deficiency anemia and its regulating effect on iron metabolism, in order to provide a pharmacological reference for the clinical application of Lujingyiqishengxue Pill.

Materials and Methods

Animals and feed

Seventy two SD rats, SPF grade, weighing 180-220 g, half male and half female (not pregnant), were purchased from Changsha Tianqin Biotechnology Co., Ltd. AIN-93G standard formula feed (iron content about 70 mg/kg, batch number: 22031503) and AIN-93G low-iron feed (iron content <10 mg/kg, batch number: 22030216), were purchased from Ready Dietech (Shenzhen) Co. Ltd.

Medicines and reagents

Lujingyiqishengxue Pill (batch number: 20220201), provided by the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine; Sulifei tablets (ferrous succinate tablets, batch number 220104), provided by Nanjing Jinling Pharmaceutical Factory of Jinling Pharmaceutical Co., Ltd.; tissue iron test kit (batch number: 20210912), serum iron (SI) test kit (batch number: 20210923), serum total iron binding capacity (TIBC) test kit (batch number: 20210923), purchased from Nanjing Jiancheng Bioengineering Institute; Mouse TFR1 ELISA Kit (batch number: N4MX9A3MYN), purchased from Wuhan Elabscience Biotechnology Co., Ltd.

Main instruments

AUW220D analytical balance (Shimadzu Corporation); V4800 high-throughput tissue homogenizer, DHS Life Science & Technology Co., Ltd.; ST16R high-speed refrigerated centrifuge, Varioska multi-function microplate reader, American Thermo Company; 2000i fully automatic blood analyzer, Japan Sysmex Corporation.

Grouping, model replication, and dosing

After adaptive feeding for 7 d, the 72 SD rats were randomly divided into blank group, model group, ferrous succinate group (36 mg/kg, positive drug group) and Lujingyiqishengxue Pill high-dose, middle-dose and low-dose groups (4.4, 2.2, 1.1 g/kg), according to the principle of body weight and hemoglobin (HGB) balance, 12 animals in each group, half male and half male, and they were kept in separate cages. The body weight was weighed once a week, and the dosage was adjusted according to the body weight. After grouping, the rats in the blank group were fed with standard feed, while the rats in the model group and each administration group were fed with low-iron feed for 42 consecutive days. The rats in each group were fed in stainless steel cages and had free access to food and deionized water. When replicating the model, the blank group and the model group were given deionized water by gavage, and the ferrous succinate group and the Lujingyiqishengxue Pill high, middle and low dose groups were given corresponding doses of drugs, respectively. The gavage volume was 20 ml/kg, 1 time daily, and the rats were administrated for concessive 42 consecutive days.

Sample collection and treatment

After the last administration, 2 ml of rat whole blood was collected, stood at room temperature and centrifuged (centrifugation radius 10 cm, 3 000 r/min, 4 ℃) for 15 min, and the upper serum was taken and stored at -20 ℃. The rat liver, spleen, proximal small intestine and kidney were taken, accurately weighed with an analytical balance, and then added with normal saline at 4 ℃ at a ratio of m∶V=1∶9. The samples were placed in a high-throughput tissue grinder for homogenization, and then centrifuged for 15 min with a centrifugal radius of 10 cm and 3 000 r/min in a high-speed refrigerated centrifuge. The supernatants were collected and stored at -20 ℃ for later use.

Blood routine analysis

On the day of grouping and after the last administration, 20 μl of blood was collected from the orbit of the rats, immediately added to an EP tube containing 2 ml of diluent for hematology analyzer. After mixing by blowing and beating, an automatic hematology analyzer was used to detect each group within 20 min for rat red blood cells (RBC), HGB, and hematocrit (HCT) in each group.

Serum biochemical index detection

SI kit, TIBC kits, serum ferritin (SF) kits and serum transferrin receptor 1 (TFR1) kits were used to detect serum SI, TIBC, transferrin saturation (TSAT), SF and TFR1 content of rats in each group according to the instructions.

Tissue biochemical index detection

Tissue iron kits were used to detect iron contents in the liver, spleen, small intestine and kidney tissues of rats in each group according to the instructions.

Statistical processing method

SPSS 21.0 statistical software was used for data analysis, and the measurement data were expressed as mean±standard deviation (x±s). One-way ANOVA was used for comparison between multiple groups, and LSD test was used for pairwise comparison. P<0.05 indicated a statistically significant difference.

Results

Effects of Lujingyiqishengxue Pill on peripheral blood routine in rats with iron deficiency anemia induced by low iron diet

The results are shown in Table 1. Before modeling, there were no significant differences in the blood routine indexes of rats in each group (P>0.05). After being given low-iron diet for 42 d, compared with the blank group, the rats in the model group showed significantly decreased RBC, HGB and HCT (P<0.05, P<0.01), and the HGB of rats in the model group was 63.34 g/L (<100 g/L), indicating that the iron-deficiency anemia model was successfully established［8］. After drug intervention, compared with the model group, the HGB and HCT of rats in each dose group of Lujingyiqishengxue Pill increased significantly (P<0.05, P<0.01), and the RBC had a certain upward trend, but the differences were not statistically significant (P>0.05); and the HGB of rats in the ferrous succinate group also significantly increased (P<0.05).

Effects of Lujingyiqishengxue Pill on tissue iron in rats with iron deficiency anemia induced by low iron diet

The results are shown in Table 2. After being given low-iron diet for 42 d, compared with the blank group, the rats in the model group showed significantly decreased iron contents in the liver, spleen, kidney and small intestines (P<0.01). After drug intervention, compared with the model group, the iron contents of the liver, spleen and small intestine of rats in the high-, middle- and low-dose Lujingyiqishengxue Pill groups and the ferrous succinate group significantly increased (P<0.01), and the iron contents in the kidney of rats in the high-dose Lujingyiqishengxue Pill group and the ferrous succinate group also increased significantly (P<0.01).

Effects of Lujingyiqishengxue Pill on iron metabolism in rats with iron deficiency anemia induced by low iron diet

The results are shown in Table 3. After being given low iron diet for 42 d, compared with the blank group, the rats of the model group exhibited significantly decreased SI, SF and TSAT (P<0.01), and their TIBC and TFR1 significantly increased (P<0.01). After drug intervention, compared with the model group, the rats in each administration group showed significantly reversed TSAT, TIBC and TFR1 (P<0.05, P<0.01); the SI and SF of rats in the high-dose Lujingyiqishengxue Pill group and the ferrous succinate group increased significantly (P<0.01).

Discussion and Conclusions

Iron is an essential trace element in the human body. Generally, the total amount of iron in normal adults is about 3 to 5 g, most of which is stored in hemoglobin, which is an essential substance for erythrocytes to synthesize heme［9］. However, iron supplements have problems such as low absorption and utilization rate and gastrointestinal side effects［10］. In this study, a rat model of iron-deficiency anemia induced by low-iron diet was used to observe the effects of Lujingyiqishengxue Pill on iron-deficiency anemia. The results showed that Lujingyiqishengxue Pill could significantly increase hemoglobin (HGB) and hematocrit (HCT) of the iron deficiency anemia rat model, increase the iron contents of the tissues including the liver, spleen, kidney and small intestine, and reverse serum iron (SI), serum total iron binding capacity (TIBC), serum ferritin (SF), serum transferrin receptor 1 (TFR1) and transferrin saturation (TSAT) and other iron metabolism indexes. The results of this study showed that Lujingyiqishengxue Pill improved iron deficiency anemia in rats with iron deficiency anemia, and it had a good iron supplementation effect and also regulated iron metabolism. Its mechanism of action in the treatment of iron deficiency anemia deserves attention and further research.

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