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Langchuang Recipes on Lupus Erythematosus

Regulatory Effect of Langchuang Serial Recipes on T-Lymphocyte Subsets Th and Tc in Patients with Systemic Lupus Erythematosus*
      TAO Xiao-juan , ZHENG Hong-xia , YU Jian-ning  MA Ji-lin , and ZHANG Wen 
   
ABSTRACT  Objective: To study the principle of clearing Fei , cooling blood, and detoxification as well as nourishing yin and moisening Fei (abbr. as CCD-NM) in regulating the levels of peripheral T-lymphocyte subsets Th and Tc cells to explore its mechanism for lowering the incidence of infection in patients with systemic lupus erythematosus (SLE). Methods: Sixty SLE patients without complicated infection were assigned to the treatment group and the control group, 30 in each group. The control group was treated with Western medicine alone, while the treatment group was treated with the same program of Western medicine, but additionally administered with either Langchuang No.1  or 2 , serial concerted Chinese recipes, applied respectively in patients in the active stage or in the resting stage. The total time of treatment for both groups was 1 year. Further, a healthy control group was set up with 20 healthy subjects. The expressions of Thl, Th2, and Tcl and Tc2 cells in peripheral blood were detected and compared with those in the healthy control group. Results: (1) As compared with the healthy control group, ratios of Thl/Th2 and Tcl/Tc2 in SLE patients, whether complicated with infection or not, were significantly lower (P<0.05 or P<0.01). (2) Comparison between patients with complications and those uncomplicated with infection showed that the two ratios and Thl expression were lower and Tc2 was higher in the former than those in the latter (all P<0.05). (3) Ratios of Thl/Th2 and Tcl/Tc2 increased after treatment in patients of both the treatment group and the control group (P<0.05 and P<0.01), but the changes in the treatment group were more significant (P<0.05).
Conclusion: The principle of CCD-NM could regulate the Th and Tc subsets toward equilibrium in SLE patients, which might be one of the mechanisms of action for alleviating complicated infection.
KEY WORDS  Langchuang No.l, Langchuang No.2, systemic lupus erythematosus, infection, Th and Tc subsets of T-lymphocyte
    
Systemic lupus erythematosus (SLE) is a kind of autoimmune disease involving multiple systems of the whole body. Along with the wide application of glucocorticoids and immunosuppressants, the prognosis of SLE patients has been obviously improving, but the infection is still an important factor that affects prognosis. Repeated clinical studies have shown that the complicated infection and its evoked serious complications remain the most common cause for a long time(1,2). From the point of view of TCM theory, the authors have used the principle of clearing Fei (?), cooling blood, and detoxification as well as nourishing yin and moistening Fei (abbr. as CCD-NM) in clinical practice, and have found that it shows an advantage in alleviating the complications of SLE(3), and could regulate the levels of the Th and Tc cell subsets, suggesting that it is possibly one of the mechanisms of action for preventing infection in SLE patients. The study is reported as follows.

METHODS
Diagnostic Standard
The SLE diagnostic standard issued by the American Association of Rheumatism (revised edition, 1982)(4) was adopted; the condition of illness was judged depending on the SLE activity index (AI) and calculated according to literature(5), AI ? 5 was regarded as in active stage, ? 4 as in resting stage.
    
The diagnosis of a complicated infection depended on: (1) confirmable signs and symptoms; (2) chest film showing infiltrative changes in the lung parenchyma, and cardiac pulmonary edema could be clinically excluded; (3) positive bacterial  culture of throat swab, sputum, mid-stream urine (count of bacterial colonies over 1 x 106/L), stool, or fluid of hydrothorax and ascites. Patients could be judged as with infection when they fitted condition (1) and (2) or (1) and (3).
     
Only patients with no clinical or laboratory evidence of infection in the 1-week observation period after enrollment were included formally in the study, and their age had to be below 50 years old. Patients fitting the following criteria were excluded: patient's diagnosis was not confirmed by renal biopsy; female patients in pregnancy or lactation, or intending to have a baby in the near future; patients with mental diseases; and patients with the complication of multiple organ dysfunction.
Subjects
All the 60 SLE patients, inpatients or outpatients, were selected from the authors' hospital, the Department of Rheumatology and Immunology, from 2004 to 2006. They were equally assigned to two groups according to selective sequencing. The 30 patients in the treatment group were 2 males and 28 females, 15-50 years old, average 34.2 ± 7.1 years; 7 patients had their course of disease ranging from 1 month to 1 year, 8 between 1-5 years, 10 between 5-10 years and 5 between 10-30 years; their AI was 12.4±2.3 on average. The 30 patients in the control group included 1 male and 29 females, 13-50 years old, average 36.5 ± 8.2 years; 9 patients had their course of disease ranging from 1 month to 1 year, 7 between 1-5 years, 10 between 5-10 years and 4 between 10-30 years; their AI was 11.2 ± 3.6 on average. The two groups were not significantly different in terms of sex, age, and course and condition of disease (P>0.05).
     
Further, a healthy control group was set up, which consisted of 20 healthy subjects selected from the staff of the hospital, 2 males and 18 females, aged between 15-50 years, 35.9± 5.4 years on average. The difference in terms of sex and age between the healthy control group and the SLE patient groups was insignificant (P>0.05).

Treatment
The Western medical treatment protocol was as follows: For patients with obvious general symptoms but without organ damage, 30-40 mg of prednisone was administered per day for 6-8 successive weeks, and then the dosage was reduced gradually to the maintenance dose of 10-15 mg per day. Patients with lupus nephritis type I were treated with the same protocol as above. For patients with lupus nephritis type I1 a, and with only some mild mesangial change, the same protocol was applied as above; when they were type ? b and accompanied with proteinuria of over 1.0 g/d, high titers of serum anti-dsDNA antibody and hypocomplementemia, tripterygium glycoside tablets were given additionally besides the daily administration of 30-40 mg of prednisone.
     
For patients with lupus nephritis type ?, 1V and V, a complex of prednisone and cyclophosphamide (CsA) was administered. (1) Prednisone, at a dosage of 0.8-1.0 mg/kg per day, was given through oral administration for 8 weeks, and then the dose was reduced gradually to 10-20 mg daily for maintenance. For patients with active type IVa or type IV and complicated with severe leucopenia, encephalopathy, pneumonia or myocarditis, etc, concussion therapy with methylprednisolone (MP) was applied by intravenous dripping of MP 0.5 g per day for 3 successive days as one course, and a second course could be given as necessary after an interval of 10 days. No more than two courses could be given. Prednisone 30 mg was then administered daily and the dose would be reduced gradually to the maintenance dose of 10 mg/d. (2) CsA, at a dosage of 1.0 g/m2, was administered by adding it to normal saline and given via intravenous drip slowly for over 1 h, once a month, for 6 successive months, and then it was reduced to once every 3 months.
     
Corresponding anti-bacterial drugs were administered respectively to patients with various infections occurring during the therapeutic process according to the outcome of drug sensitivity tests. The above-mentioned Western medical therapeutic protocol was carried out in all patients for one year.
     
Patients in the treatment group were additionally medicated with different TCM recipes, which are serial concerted Chinese recipes arranged in accordance with the stage of SLE. Namely, for patients in the active stage, the recipe was Langchuang No.1 ,mainly for clearing Fei, cooling blood, and detoxification, which consisted of mulberry bark 30 g, wolfberry root-bark 30 g, licorice root 3 g, white peony root 30 g, figwort root 30 g, southernwood 15 g, and wild weed 15g. For patients in the resting stage, the recipe, mainly with the action of nourishing yin and moistening Fei, was Langchuang No.2 ,and consisted of lily bulb 30g, asparagus root 15 g, lilyturf root 15 g, white peony root 15 g, Chinese angelica root 15 g, figwort root 30 g, fritillary bulb 10 g, balloonflower root 10 g, licorice root 3 g, and bamboo leaf 10g.
     
Both Langchuang No.1 and No.2 were prepared by water decoction by the pharmaceutical unit of the authors' hospital and given as medication at one dose per day, taken in two portions, one in the morning and one in the evening, for one year, in synchrony with the whole course of Western medical treatment.

Indexes and Methods of Observation
Expressions of Thl, Th2, Tcl and Tc2 in the peripheral blood of all patients were determined before and after the therapeutic course, and they were also determined during the course of the infection. The method of determination was: 1 mL of venous blood was withdrawn and anti-coagulated with heparin, then 100 µ L of the blood sample was diluted with RPMI 1640 medium at a ratio of 1:1 (volume), mixed evenly, placed in a culture plate with stimulant phorbol 12-myristate 13-acetate (PMA, purchased from Sigma Co., USA) and ionomycin; protein transporting inhibitor monensin working fluid (purchased from Pharmingen Co., USA) was added as well. The ingredients were mixed evenly and incubated for 4 h at 37? in an incubator containing 5% CO2. After being stained with 4-color fluorescent staining, the plate was examined using a flow cytometer (Epics XL.MCL, Backman Coulter, USA) according to the instructions for the reagent.

Statistical Analysis
Using SPSS 11.5 software, comparisons of the measurement data between the two groups of patients was conducted using the t-test, and that of the mean values among the three groups was carried out by variance analysis; while paired comparison between the mean values in the two groups was conducted by adopting the q-test.

RESULTS
Comparison of T-Lymphocyte Subsets among Groups
During the observation period, 21 patients had complications with their infection (SLE-C) and 39 did not (SLE-N). The comparisons of Th and Tc subsets showed that the ratios Thl/Th2 and Tcl/Tc2 were lower in the patients than those in the healthy subjects (P<0.05 or P<0.01); the Thl expression was lower but Tc2 was higher in SLE-C as compared to healthy subjects (P<0.01); and Thl expression was lower while Tc2 was higher and the ratios Thl/Th2 and Tcl/Tc2 were also lower in SLE-C than those in SLE-N (P<0.05, Table 1, Figure 1).

Comparison of T-Lymphocyte Subsets in Patients before and after Treatment
The ratios Thl/Th2 and Tcl/Tc2 increased after treatment, showing a significant difference (P<0.05 or P<0.01), but the increments were more significant in the treatment group than in the control group (P<0.05, Table 2).

DISCUSSION
Studies in recent years have shown that SLE is a kind of auto-immune disease, and its immune function disorder is closely related to the disequilibrium of T-lymphocyte subsets, which could lead to deviation of cytokines with important regulatory effects synthesized or secreted by T-lymphocytes, thus producing a series of abnormal immune effects and pathological injuries(6). Therefore, studies on the levels of T-lymphocyte subsets and the cytokines secreted by T-lymphocytes will be helpful for exploring the immune regulatory mechanism at the level of the T-lymphocyte, and is of important value for immune intervention of the disease.
    
It was shown in this study that, compared with healthy subjects, the Thl/Th2 and Tcl/Tc2 ratios were lower in SLE patients (P<0.05 or P<0.01). As compared with SLE-N, the level of Thl was lower and Tc2 was higher in SLE-C (P<0.05), thus resulting in the two ratios lowering more significantly in SLE-C (P<0.01). These results suggested the existence of a de-equilibrium condition in the T-lymphocyte subsets, and it appeared in SLE-C mainly as a decrease in Thl and an increase in Tc2.
     
On the basis of TCM theory, the principle of CCD-NM was put forward and applied for the treatment of SLE by the authors, and its advantage in lowering the incidence of the infection rate has been proven by a clinical study(3). In this study, it was found that the Thl/Th2 and Tcl/Tc2 ratios increased more significantly after treatment in the treatment group compared to the control group (P<0.05), suggesting that the regulatory effect of the combined therapy on Th and Tc cell equilibrium was superior to that of Western therapy alone. It indicated that TCM treatment could enhance the equilibrium regulation to a certain extent, and it might be one of the mechanisms of action for reducing the infection rate in SLE.
     
Since SLE is an auto-immunological disease involving multiple systems of the whole body, always with a long illness course and an alternating repetition of the active stage and the resting stage, the treatment takes a long therapeutic course and a large dose of immunosuppressants, which is the main cause for the decreasing vital qi of the human body. TCM holds that when an external pathogen attacks, it intrudes into Fei first. Thus, the human body could only be protected from invasion of evil pathogens when one's Fei-qi is healthy.
     
SLE patients are prone to infectious complications during the therapeutic process, and investigations summarizing relevant Chinese literature in recent years show that the nosocomial infection rate in SLE patients reached 36.4%-60.6%(7-10). The authors' clinical study showed that the most commonly seen infection in SLE patients is respiratory infection, and secondary to that, the skin is another focus. Infection of the two sites accounts for 73.8% of all infections in SLE patients(3), which confirms the above-mentioned TCM theory on Fei and external invasion. It is therefore clear that, viewing the disease from either theoretical or practical clinical points, Fei is the most principal factor for the occurrence of infections in SLE patients.
    
On the basis of the above-mentioned consideration, the authors established the therapeutic principle of CCD-NM for preventing infections in SLE. Through another clinical study, the treatment has been confirmed to have the effect of reducing the infectious complication rate in SLE patients(3). In this study, it was found that Langchuang No.1 and No.2, the concerted recipes formulated according to this principle, are capable of correcting infections relevant to de-equilibrium of T-lymphocyte subsets, thus illustrating that there exists objective evidence supporting the principle of CCD-NM in reducing the infection rate in SLE patients. Since the equilibrium of T-lymphocyte subsets keeps the immune function of patients in a stable state, it is assumed that treatment depending on the principle could also play an active role in the rehabilitation of patients.

REFERENCES
1.  Cervera R, Khamashta MA, Font J, et al. Morbidity and mortality in systemic lupus erythematosus during a 5-year period. A multicenter prospective study of 1000 patients. European Working Party on Systemic Lupus Erythematosus. Medicine (Baltimore) 1999;78:167-175.
2. Mok CC, Lee KW, Ho CT, et al. A prospective study of survival and prognostic indicators of  systemic lupus erythematosus in a southern Chinese population. Rheumatology (Oxford) 2000;39(4):399-406.
3. Tao X J, Zheng HX, Yu JN, et al. Clinical research on effect of TCM treatment beginning with Fei in treating systemic lupus erythematosus for reducing incidence of complicated infection. Chin J Integr Tradit West Med (Chin) 2007;27(1 ):33-36.
4. Tan EM, Cohen AS, Fries JF, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus (SLE). Arthritis Rheum 1982;25:1271 - 1277.
5. Hay E, Cordon C, Emery P, et al. Assessment of lupus. Where are we now? Ann Rheum Dis 1993;52:169-172.
6. Varadhachary AS, Peter ME, Perdow SN, et al. Seletive up-regulation of phosphatidylinositol 3'-kinase activity in Th2 cells inhibits caspase-8 cleavage at the death-inducing complex: a mechanism for Th2 resistance from Fas-mediated apoptosis. J Immuno11999;163:4772-4779.
7. Li ZJ, Wang CL, Fan XY, et al.  Clinical study on the  patients with systemic lupus erythematosus complicated by infections. Chin J Rheumatol (Chin) 2000;4(3): 165-168.
8. Xu G, Liu ML, Yu KM. A prospective study  of nosocomial infection in patients with systemic lupus erythematosus. Chin J Rheumatol (Chin) 2003;7(4):216-219.
9. Xu G, Xu Q, Yu LM. An analysis of nosocomial infection in  patients with systemic lupus erythematosus. Chin J Nosocomiol (Chin) 2000;10(5):362-364.
10. Yu Y, Chen J, Xie FA, et al. Clinical investigation of nosocomial infection cases with lupus nephritis. Chin J Nosocomiol (Chin) 2000;10(3): 174-176.

                                                   

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